Background:Administration of propofol,an intravenous anesthetic with antioxidant property,immediately at the onset of post-ischemic reperfusion(propofol postconditioning,P-PostC) has been shown to confer cardioprotect...Background:Administration of propofol,an intravenous anesthetic with antioxidant property,immediately at the onset of post-ischemic reperfusion(propofol postconditioning,P-PostC) has been shown to confer cardioprotection against ischemia–reperfusion(I/R) injury,while the underlying mechanism remains incompletely understood.The forkhead box O(FoxO) transcription factors are reported to play critical roles in activating cardiomyocyte survival signaling throughout the process of cellular injuries induced by oxidative stress and are also involved in hypoxic postconditioning mediated neuroprotection,however,the role of FoxO in postconditioning mediated protection in the heart and in particular in high glucose condition is unknown.Methods:Rat heart-derived H9c2 cells were exposed to high glucose(HG) for 48 h,then subjected to hypoxia/reoxygenation(H/R,composed of 8 h of hypoxia followed by 12 h of reoxygenation) in the absence or presence of postconditioning with various concentrations of propofol(P-PostC) at the onset of reoxygenation.After having identified the optical concentration of propofol,H9c2 cells were subjected to H/R and P-PostC in the absence or presence of FoxO1 or FoxO3a gene silencing to explore their roles in P-PostC mediated protection against apoptotic and autophagic cell deaths under hyperglycemia.Results:The results showed that HG with or without H/R decreased cell viability,increased lactate dehydrogenase(LDH) leakage and the production of reactive oxygen species(ROS) in H9c2 cells,all of which were significantly reversed by propofol(P-PostC),especially at the concentration of 25 μmol/L(P25)(P<0.05,NC vs.HG;HG vs.HG+HR;HG+HR+P12.5 or HG+HR+P25 or HG+HR+P50 vs.HG+HR).Moreover,we found that propofol(P25) decreased H9c2 cells apoptosis and autophagy that were concomitant with increased FoxO1 and FoxO3a expression(P<0.05,HG+HR+P25 vs.HG+HR).The protective effects of propofol(P25) against H/R injury were reversed by silencing FoxO1 or FoxO3a(P<0.05,HG+HR+P25 vs.HG+HR+P25+siRNA-1 or HG+HR+P25+siRNA-5).Conclusions:It is concluded that propofol postconditioning attenuated H9c2 cardiac cells apoptosis and autophagy induced by H/R injury through upregulating FoxO1 and FoxO3a under hyperglycemia.展开更多
Rosmarinic acid,a common ester extracted from Rosemary,Perilla frutescens,and Salvia miltiorrhiza Bunge,has been shown to have protective effects against various diseases.This is an investigation into whether rosmarin...Rosmarinic acid,a common ester extracted from Rosemary,Perilla frutescens,and Salvia miltiorrhiza Bunge,has been shown to have protective effects against various diseases.This is an investigation into whether rosmarinic acid can also affect the changes of white matter fibers and cognitive deficits caused by hypoxic injury.The right common carotid artery of 3-day-old rats was ligated for 2 hours.The rats were then prewarmed in a plastic container with holes in the lid,which was placed in 37°C water bath for 30 minutes.Afterwards,the rats were exposed to an atmosphere with 8% O2 and 92% N2 for 30 minutes to establish the perinatal hypoxia/ischemia injury models.The rat models were intraperitoneally injected with rosmarinic acid 20 mg/kg for 5 consecutive days.At 22 days after birth,rosmarinic acid was found to improve motor,anxiety,learning and spatial memory impairments induced by hypoxia/ischemia injury.Furthermore,rosmarinic acid promoted the proliferation of oligodendrocyte progenitor cells in the subventricular zone.After hypoxia/ischemia injury,rosmarinic acid reversed to some extent the downregulation of myelin basic protein and the loss of myelin sheath in the corpus callosum of white matter structure.Rosmarinic acid partially slowed down the expression of oligodendrocyte marker Olig2 and myelin basic protein and the increase of oligodendrocyte apoptosis marker inhibitors of DNA binding 2.These data indicate that rosmarinic acid ameliorated the cognitive dysfunction after perinatal hypoxia/ischemia injury by improving remyelination in corpus callosum.This study was approved by the Animal Experimental Ethics Committee of Xuzhou Medical University,China (approval No.20161636721) on September 16,2017.展开更多
Objective: To establish the rat model with myocardial hypoxia/reoxygenation (H/R) injury, and investigate the protective effect of EPO pretreatment on the myocardium. Methods: Sixty male adult Wistar rats were randoml...Objective: To establish the rat model with myocardial hypoxia/reoxygenation (H/R) injury, and investigate the protective effect of EPO pretreatment on the myocardium. Methods: Sixty male adult Wistar rats were randomly divided into 3 groups: control group, H/R group, and EPO group, 20 in each group. The rats in EPO group accepted injection of 5 000 U/kg recombinant human erythropoietin (RHuEPO) through vein, and the other rats accepted the injection of the same volume of saline. Twenty-four hours after the injection, rats in the EPO and H/R groups were put into the hypoxia environment for 12 h and then returned to the normoxic environment for 2 h, and then the samples of blood and myocardium were collected. Serum myocardial enzyme activity, apoptosis, ultrastructure, myocardial MDA contents, EPO receptor (EPOR) expression in cardiac myocytes and cardiac functions were tested. Results: EPOR expression was positive in cardiac myocytes of adult rat according to the result of immunonistochemitry assaying. Compared to those in H/R group, rats in EPO group presented lighter injury of myocardial ultrastructure, the reduction of serum myocardial enzyme activity, inhibition of apoptosis, the better recovery of cardiac functions, and the less production of oxygen-derived free radicals. Conclusion: Adult rat cardiac myocytes could express EPOR, and EPO pretreatment produced protective effects on myocardium with H/R injury.展开更多
BACKGROUND:Disturbance of mitochondrial fi ssion and fusion(termed mitochondrial dynamics)is one of the leading causes of ischemia/reperfusion(I/R)-induced myocardial injury.Previous studies showed that mitochondrial ...BACKGROUND:Disturbance of mitochondrial fi ssion and fusion(termed mitochondrial dynamics)is one of the leading causes of ischemia/reperfusion(I/R)-induced myocardial injury.Previous studies showed that mitochondrial aldehyde dehydrogenase 2(ALDH2)conferred cardioprotective effect against myocardial I/R injury and suppressed I/R-induced excessive mitophagy in cardiomyocytes.However,whether ALDH2 participates in the regulation of mitochondrial dynamics during myocardial I/R injury remains unknown.METHODS:In the present study,we investigated the effect of ALDH2 on mitochondrial dynamics and the underlying mechanisms using the H9c2 cells exposed to hypoxia/reoxygenation(H/R)as an in vitro model of myocardial I/R injury.RESULTS:Cardiomyocyte apoptosis was significantly increased after oxygen-glucose deprivation and reoxygenation(OGD/R),and ALDH2 activation largely decreased the cardiomyocyte apoptosis.Additionally,we found that both ALDH2 activation and overexpression significantly inhibited the increased mitochondrial fission after OGD/R.Furthermore,we found that ALDH2 dominantly suppressed dynamin-related protein 1(Drp1)phosphorylation(Ser616)and adenosine monophosphate-activated protein kinase(AMPK)phosphorylation(Thr172)but not interfered with the expression levels of mitochondrial shaping proteins.CONCLUSIONS:We demonstrate the protective effect of ALDH2 against cardiomyocyte H/R injury with a novel mechanism on mitochondrial fission/fusion.展开更多
Morphological and functional abnormalities of vascular endothelial cells(VECs) are risk factors of ischemiareperfusion in skin flaps.Signaling pathway mediated by interleukin-1 receptor(IL-1 R) is essential to hypoxia...Morphological and functional abnormalities of vascular endothelial cells(VECs) are risk factors of ischemiareperfusion in skin flaps.Signaling pathway mediated by interleukin-1 receptor(IL-1 R) is essential to hypoxia/reoxygenation(H/R) injury of VECs.While the TIR/BB-loop mimetic(AS-1) disrupts the interaction between IL-1 R and myeloid differentiation primary-response protein 88(MyD88),its role in the VECs dysfunction under H/R is unclear.In this study,we first showed that there was an infiltration of inflammatory cells and the apoptosis of VECs by using a skin flap section from patients who received flap transplantation.We then showed that the H/R treatment induced apoptosis and loss of cell migration of endothelial cell line H926 were attenuated by AS-1.Furthermore,our data suggested that AS-1 inhibits the interaction between IL-1 R and MyD88,and subsequent phosphorylation of IκB and p38 pathway,as well as the nuclear localization of NF-κB subunit p65/p50.Thus,this study indicated that the protective role of AS-1 in H/R induced cellular injury may be due to the AS-1 mediated down-regulation of IL-1 R signaling pathway.展开更多
Astrocytes in an in vitro murine astrocyte model of oxygen and glucose deprivation/hypoxia and reoxygenation were treated with different concentrations of triptolide （250, 500, 1 000 ng/mL） in a broader attempt to e...Astrocytes in an in vitro murine astrocyte model of oxygen and glucose deprivation/hypoxia and reoxygenation were treated with different concentrations of triptolide （250, 500, 1 000 ng/mL） in a broader attempt to elucidate the protection and mechanism underlying triptolide treatment on astrocytes exposed to hypoxia/reoxygenation injury. The results showed that the matrix metalloproteinase-9, interleukin-1β, tumor necrosis factor α and interleukin-6 expressions were significantly decreased after triptolide treatment in the astrocytes exposed to hypoxia/ reoxygenation injury, while interleukin-10 expression was upregulated. In addition, the vitality of the injured astrocytes was enhanced, the triptolide＇s effect was apparent at 500 ng/mL. These experimental findings indicate that triptolide treatment could protect astrocytes against hypoxia/ reoxygenation injury through the inhibition of inflammatory response and the reduction of matrix metalloproteinase-9 expression.展开更多
BACKGROUND： Phycocyanin can relieve decrease of mitochondrial membrane potential through reducing production of active oxygen so as to protect neurons after hypoxia/reoxygenation. OBJECTIVE： To observe the effect of...BACKGROUND： Phycocyanin can relieve decrease of mitochondrial membrane potential through reducing production of active oxygen so as to protect neurons after hypoxia/reoxygenation. OBJECTIVE： To observe the effect of phycocyanin on activity of PC12 cells and mitochondrial membrane potential after hypoxia/reoxygenation. DESIGN： Randomized controlled study SETTING ： Cerebrovascular Disease Institute of Affiliated Hospital, Medical College of Qingdao University MATERIALS： The experiment was carried out at the Key Laboratory of Prevention and Cure for cerebropathia in Shandong Province from October to December 2005. PC12 cells, rat chromaffin tumor cells, were provided by Storage Center of Wuhan University; phycocyanin was provided by Ocean Institute of Academia Sinica; Thiazoyl blue tetrazolium bromide （MTT） and rhodamine 123 were purchased from Sigma Company, USA; RPMI-1640 medium, fetal bovine serum and equine serum were purchased from Gibco Company, USA. METHODS： ① Culture of PC12 cells： PC12 cells were put into RPMI-1640 medium which contained 100 g/L heat inactivation equine serum and 0.05 volume fraction of fetal bovine serum and incubated in CO2 incubator at 37℃. Number of cells was regulated to 4 × 10^5 L 1, and cells were inoculated at 96-well culture plate. The final volume was 100μL. ② Model establishing and grouping： Cultured PC12 cells were randomly divided into three groups： phycocyanin group, model control group and non-hypoxia group. At 24 hours before hypoxia, culture solution in phycocyanin group was added with phycocyanin so as to make sure the final concentration of 3 g/L , but cells in model control group did not add with phycocyanin. Cells in non-hypoxia group were also randomly divided into adding phycocyanin group （the final concentration of 3 g/L） and non-adding phycocyanin group. Cells in model control group and phycocyanin group were cultured with hypoxia for 1 hour and reoxygenation for 1, 2 and 3 hours; meanwhile, cells in non-hypoxia group were cultured with oxygen and were measured at 1 hour after hypoxia/reoxygenation. ③ Detecting items： At 1, 2 and 3 hours after reoxygenation, absorbance （A value） of PC12 cells was measured with MTT technique so as to observe activity and quantity of cells. Fluorescence intensity of PC12 cells marked by rhodamine 123 was measured with confocal microscope in order to observe changes of mitochondrial membrane potential. MAEN OUTCOME MEASURES： Comparisons between quantity and activity of PC12 cells and mitochondria membrane potential at 1, 2 and 3 hours after reoxygenation. RESULTS： ① Effect of phycocyanin on quantity and activity of PC12 cells： A value was 0.924±0.027 in adding phycocyanin group and 0.924±0.033 in non-adding phycocyanin group. A value was lower in model control group and phycocyanin group than that in non-hypoxia group at 1, 2 and 3 hours after reoxygenation （0.817±0.053, 0.838±0.037, 0.875±0.029; 0.842±0.029, 0.872±0.025, 0.906±0.023, P 〈 0.05）. A value was higher in phycocyanin group than that in model control group at 1, 2 and 3 after culture （P 〈 0.05）. With culture time being longer, A value was increased gradually in phycocyanin group and model control group after reoxygenation （P 〈 0.05）. ~ Effect of phycocyanin on mitochondrial membrane potential of PC12 cells： Fluorescence intensity was 2.967±0.253 in adding phycocyanin group and 2.962±0.294 in non-adding phycocyanin group. Fluorescence intensity was lower in model control group and phycocyanin group than that in non-hypoxia group at 1, 2 and 3 hours after hypoxia/reoxygenation （1.899±0.397, 2.119±0.414, 2.287±0.402; 2.191±0.377, 2.264±0.359, 2.436±0.471, P 〈 0.05）; but it was higher in phycocyanin group than that in model control group at 1, 2 and 3 after reoxygenation （P 〈 0.05）. With culture time being longer, fluorescence intensity was increased gradually in phycocyanin group and model control group after reoxygenation （P 〈 0.05）. CONCLUSION： Phycocyanin and reoxygenation can protect PC12 cells after hypoxia injury through increasing mitochondrial membrane potential and cellular activity, and the effect is improved gradually with prolonging time of reoxygenation.展开更多
Objective：Carbamylated EPO（CEPO） is a derivative of erythropoietin（EPO） by subjecting it to carbamylation. It does not stimulate erythropoiesis, but effectively protects tissue from injury. The present study was ...Objective：Carbamylated EPO（CEPO） is a derivative of erythropoietin（EPO） by subjecting it to carbamylation. It does not stimulate erythropoiesis, but effectively protects tissue from injury. The present study was to investigate the effect of CEPO treatment using in vitro models of hypoxia/reoxygenation（H/R）. Methods：Cardiomyocytes were exposed to hypoxia（95% N2 and 5% CO2） for 1 hour followed by 4 hours of reoxygenation（95% O2 and 5% CO2）. CEPO was administered after hypoxia, just before reoxygenation. The apoptotic cardiomyocytes were determined by flow cytometry. The level of protein was assessed by western blot analysis. Results： CEPO treatment significantly decreased the apoptotic cardiomyocytes by 54.20% compared with H/R group. Western blot analysis showed that CEPO administration increased the level of Bcl-2（an antiapoptotic protein） by 62.22% compared with H/R group. Conclusion： Acute administration of CEPO protected cardiomyocytes from H/R-induced apoptosis. CEPO protected cardiomyocytes with a concomitant upregulation of Bcl-2 after H/R injury.展开更多
[Objectives]To explore the protection mechanism of crocin against ischemia-reperfusion injury of myocardial cells.[Methods]Newborn male SD rats were selected,left ventricular cardiomyocytes(CMs)were isolated,and a hyp...[Objectives]To explore the protection mechanism of crocin against ischemia-reperfusion injury of myocardial cells.[Methods]Newborn male SD rats were selected,left ventricular cardiomyocytes(CMs)were isolated,and a hypoxia/reoxygenation model of CMs was established to simulate the process of ischemia/reperfusion injury.The cells were randomly divided into four groups:normal cell group(control group),crocin group),hypoxia/reoxygenation group(H/R group),hypoxia/reoxygenation+crocin group(H/R+crocin group).H/R+crocin group selected the concentration of crocin 1,10,and 100μmol/L,and determined the optimal concentration of crocin by detecting the cell proliferation ability.After the cells were pretreated using the optimal concentration of crocin,the levels of superoxide anion,cell proliferation,apoptosis and Nox2 levels in each group of cells were detected.[Results]Compared with the control group,the proliferation ability of CMs after hypoxia-reoxygenation injury was reduced(P<0.05),while cell apoptosis and intracellular superoxide anion levels were significantly increased(P<0.01);the CMs pretreated with crocin can reduce the level of Nox2(P<0.01),increase the cell proliferation ability of CMs,reduce cell apoptosis,and accordingly reduce the level of superoxide anion in the cell(P<0.05).[Conclusions]Crocin protects CMs from hypoxia/reoxygenation injury through down-regulating the level of Nox2 and reducing oxidative stress injury.展开更多
Objective：To observe the protective effects of erythropoietin （EPO） pretreatment on cardiac myocyte with hypoxia/reoxygenation （H/R） injury and the role of NF-κBin this effects. Methods：After the H/R model of c...Objective：To observe the protective effects of erythropoietin （EPO） pretreatment on cardiac myocyte with hypoxia/reoxygenation （H/R） injury and the role of NF-κBin this effects. Methods：After the H/R model of cardiac myocytes of neonatal rats was established, the cultured cardiac myocytes were divided into 4 groups, including EPO pretreatment group （ EPO 10 U/ml 24 h before H/R）, EPO pretreatment ＋ PDTC group（EPO 10 U/ml and PDTC 5 μg/ml 24 h before H/R）, PDTC group （PDTC 5 μg /ml 24 h before H/R） and eomrolgroup. Before and after the H/R, assay of LDH concentration in the culture medium, the survival rate of the myocytes tested by MTT chromatometry and the apoptosis by flow cytometry were undertaken. Activation of NF-κB was determined by EMSA before and after H/R. Results：EPO pretreatment markedly reduced the LDH concentration in the medium, elevated the survival rate of myocytes and inhibited the apoptosis after H/R. Addition of PDTC during the pretreatment abol- ished the protective effects of EPO pretreatment. NF-κB was markedly activated during EPO pretreatment and PDTCinhibited the activation. However, after H/R, the activity of NF-κB in myocytes with EPO pretreatment was significantly inhibited compared to the other myocytes. Conclusion：NF-κB is significantly activated during EPO pretreatment, but is inhibited after H/R, which is correlated with the protective effects of EPO pretreatment on cardiac myocytes with H/R. This phenomenon can be explained as the negative feedback mechanism of the activation of NF-κB.展开更多
Objective: To investigate the mechanism of Cornus officinalis Total Glycosides (COTG) on myocardial protection, by studying effects of COTG on cardiomyocyte apoptosis induced by hypoxia/reoxygenation and calcium conce...Objective: To investigate the mechanism of Cornus officinalis Total Glycosides (COTG) on myocardial protection, by studying effects of COTG on cardiomyocyte apoptosis induced by hypoxia/reoxygenation and calcium concentration in rats. Methods: The myocardial cells of born 1-3d SD rats were isolated by enzyme digestion, cultured for 3 days. Cells were divided into five groups: Control group, H/R group, Cornus officinalis Total Glycosides low-dose group (LDG), Cornus officinalis Total Glycosides middle-dose group (MDG) and Cornus officinalis Total Glycosides high-dose group (HDG). Three drug groups were pretreated with different doses of Cornus officinalis Total Glycosides before hypoxia/reoxygenation treatment. The apoptotic rate was determined by flow cytometry assay, the intracellular free calcium concentration was examined by flow cytometry, and the ultrastructure of myocardial cells was observed under transmission electron microscope. Results: The results revealed that Cornus officinalis Total Glycosides pretreatment decreased apoptosis rate, but the effect of lower dosage is not significant. Furthermore, Cornus officinalis Total Glycosides can attenuate mitochondrial calcium overload, improve mitochondrial morphology and inhibit cardiomyocyte apoptosis caused by H/R. Conclusion: Cornus officinalis Total Glycosides pretreatment can inhibit cardiomyocyte apoptosis and calcium overload during H/R injury. However, the underlying mechanisms require us to further study.展开更多
Phytochemical investigations from the roots of Cynanchum stauntonii led to obtain four new C_（21） steroidal glycosides（1–4） and one known compound stauntoside F（5）. Their chemical structures were characterized ...Phytochemical investigations from the roots of Cynanchum stauntonii led to obtain four new C_（21） steroidal glycosides（1–4） and one known compound stauntoside F（5）. Their chemical structures were characterized by sophisticated analyses of IR, HRESI-TOF-MS, 1D, and 2D-NMR data, together with chemical methods, which showed interesting 13,14：14,15-disecopregnane-type skeleton or 14,15-secopregnane-type skeleton C_（21） steroidal glycosides. Among them, compound 1 was determined to be glaucogenin C 3-O-b-D-glucopyranosyl-（1 → 4）-b-D-cymaropyranosyl-（1 → 4）-b-D-digitoxopyranosyl-（1 → 4）-b-D-thevetopyranoside. Compound 2 was characterized to be hirundigenin 3-O-a-L-diginopyranosyl-（1 → 4）-b-D-cymaropyranosyl-（1 → 4）-b-D-digitoxopyranosyl-（1 → 4）-b-D-30-demethyl-thevetopyranoside. Compound 3 was identified to be（14S,16 S,20R）-14,16-14,20-15,20-triepoxy-14,15-secopregn-5-en-3-ol-3-O-a-L-cymaropyranosyl-（1 → 4）-b-D-digitoxopyranosyl-（1 → 4）-b-D-oleandropyranoside.Compound 4 was identified to be（14S,16 S,20R）-14,16-14,20-15,20-triepoxy-14,15-secopregn-5-en-3-ol-3-O-a-L-cymaropyranosyl-（1 → 4）-b-D-cymaropyranosyl-（1 → 4）-b-D-digitoxopyranosyl-（1 → 4）-b-Dthevetopyranoside. Among them, compound 2 was hirundigenin type C21 steroidal glycoside that existed in nature as epimers due to the presence of 14-hemiketal hydroxyl group in its structure. In addition, the anti-inflammatory and cardiomyocyte protective effects of compounds 1–4 were evaluated. We found that they exhibited significant protective effects on hypoxia/reoxygenation induced cardiomyocyte injury, but did not showed obvious anti-inflammatory function.展开更多
AIM: To investigate the protective effect and mechanisms of ghrelin postconditioning against hypoxia/reoxygenation (H/R)-induced injury in human gastric epithelial cells. METHODS: The model of H/R injury was establish...AIM: To investigate the protective effect and mechanisms of ghrelin postconditioning against hypoxia/reoxygenation (H/R)-induced injury in human gastric epithelial cells. METHODS: The model of H/R injury was established in gastric epithelial cell line (GES-1) human gastric epithelial cells. Cells were divided into seven groups: normal control group (N); H/R postconditioning group; DMSO postconditioning group (DM); ghrelin postconditioning group (GH); D-Lys3-GHRP-6 + ghrelin postconditioning group (D + GH); capsazepine + ghrelin postconditioning group (C + GH); and LY294002 + ghrelin postconditioning group (L + GH). 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to detect GES-1 cell viability. Hoechst 33258 fluorochrome staining and flow cytometry were conducted to determine apoptosis of GES-1cells. Spectrophotometry was performed to determine release of lactate dehydrogenate (LDH). Protein expression of Bcl-2, Bax, Akt, and glycogen synthase kinase (GSK)-3β was determined by western blotting. Expression of vanilloid receptor subtype 1 (VR1), Akt and GSK-3β was observed by immunocytochemistry. RESULTS: Compared with the H/R group, cell viability of the GH group was significantly increased in a dosedependent manner (55.9% ± 10.0% vs 69.6% ± 9.6%, 71.9% ± 17.4%, and 76.3% ± 13.3%). Compared with the H/R group, the percentage of apoptotic cells in the GH group significantly decreased (12.38% ± 1.51% vs 6.88% ± 0.87%). Compared with the GH group, the percentage of apoptotic cells in the D + GH group, C + GH group and L + GH groups significantly increased (11.70% ± 0.88%, 11.93% ± 0.96%, 10.20% ± 1.05% vs 6.88% ± 0.87%). There were no significant differences in the percentage of apoptotic cells between the H/R and DM groups (12.38% ± 1.51% vs 13.00% ± 1.13%). There was a significant decrease in LDH release following ghrelin postconditioning compared with the H/R group (561.58 ± 64.01 U/L vs 1062.45 ± 105.29 U/L). There was a significant increase in LDH release in the D + GH, C + GH and L + GH groups compared with the GH group (816.89 ± 94.87 U/L, 870.95 ± 64.06 U/L, 838.62 ± 118.45 U/L vs 561.58 ± 64.01 U/L). There were no significant differences in LDH release between the H/R and DM groups (1062.45 ± 105.29 U/L vs 1017.65 ± 68.90 U/L). Compared with the H/R group, expression of Bcl-2 and Akt increased in the GH group, whereas expression of Bax and GSK3β decreased. Compared with the GH group, expression of Bcl-2 decreased and Bax increased in the D + GH, C + GH and L + GH groups, and Akt decreased and GSK-3β increased in the L + GH group. The H/R group also upregulated expression of VR1 and GSK-3β and downregulated Akt. The number of VR1-positive and Akt-positive cells in the GH group significantly increased, whereas the number of GSK-3β-positive cells significantly decreased. These effects of ghrelin were reversed by capsazepine and LY294002.CONCLUSION: Ghrelin postconditioning protected against H/R-induced injury in human gastric epithelial cells, which indicated that this protection might be associated with GHS-R, VR1 and the PI3K/Akt signaling pathway.展开更多
BACKGROUND:There are currently no effective drugs to mitigate the ischemia/reperfusion injury caused by fluid resuscitation after hemorrhagic shock(HS).The aim of this study was to explore the potential of the histone...BACKGROUND:There are currently no effective drugs to mitigate the ischemia/reperfusion injury caused by fluid resuscitation after hemorrhagic shock(HS).The aim of this study was to explore the potential of the histone deacetylase 6(HDAC6)-specific inhibitor tubastatin A(TubA)to suppress nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)inflammasome activation in macrophages under hypoxia/reoxygenation(H/R)conditions.METHODS:The viability of RAW264.7 cells subjected to H/R after treatment with different concentrations of TubA was assessed using a cell-counting kit-8(CCK8)assay.Briefly,2.5μmol/L TubA was used with RAW264.7 cells under H/R condition.RAW264.7 cells were divided into three groups,namely the control,H/R,and TubA groups.The levels of reactive oxygen species(ROS)in the cells were detected using fluorescence microscopy.The protein expression of HDAC6,heat shock protein 90(Hsp90),inducible nitric oxide synthase(iNOS),NLRP3,gasdermin-D(GSDMD),Caspase-1,GSDMD-N,and Caspase-1 p20 was detected by western blotting.The levels of interleukin-1β(IL-1β)and IL-18 in the supernatants were detected using enzyme-linked immunosorbent assay(ELISA).RESULTS:HDAC6,Hsp90,and iNOS expression levels were significantly higher(P<0.01)in the H/R group than in the control group,but lower in the TubA group than in the H/R group(P<0.05).When comparing the H/R group to the control group,ROS levels were significantly higher(P<0.01),but significantly reduced in the TubA group(P<0.05).The H/R group had higher NLRP3,GSDMD,Caspase-1,GSDMD-N,and Caspase-1 p20 expression levels than the control group(P<0.05),however,the TubA group had significantly lower expression levels than the H/R group(P<0.05).IL-1βand IL-18 levels in the supernatants were significantly higher in the H/R group compared to the control group(P<0.01),but significantly lower in the TubA group compared to the H/R group(P<0.01).CONCLUSION:TubA inhibited the expression of HDAC6,Hsp90,and iNOS in macrophages subjected to H/R.This inhibition led to a decrease in the content of ROS in cells,which subsequently inhibited the activation of the NLRP3 inflammasome and the secretion of IL-1βand IL-18.展开更多
Background:Anisodine hydrobromide(AT3),an anti-cholinergic agent,could be delivered to the brain across the blood-brain barrier and has been used clinically for the treatment of cerebral ischemia/reperfusion injury.En...Background:Anisodine hydrobromide(AT3),an anti-cholinergic agent,could be delivered to the brain across the blood-brain barrier and has been used clinically for the treatment of cerebral ischemia/reperfusion injury.Endothelial dysfunction can be caused by hypoxia/reoxygenation(H/R)via oxidative stress and metabolic alterations.The present study investigated whether AT3 regulates the production of nitric oxide(NO)and reactive oxygen species(ROS),and the HIF-1αpathway via regulation of muscarinic acetylcholine receptors(mAChRs)in brain microvascular endothelial cells after H/R exposure.Methods:Under H/R conditions,hCMEC/D3 cerebral microvascular endothelial cells were treated with AT3.Specific inhibitors of M2-and M4-mAChRs were used to explore the mechanism by which AT3 influences oxidative stress in endothelial cells.Then,mAChRs expression was detected by western blotting and NO production was detected by Greiss reaction.The intracellular ROS level was measured using DCFH-DA probes.The expression of hypoxia-inducible transcription factor 1α(HIF-1α)was also detected.Results:While H/R induced the expression of M2-and M4-mAChRs,AT3 suppressed the H/R-upregulated M2-and M4-mAChRs.H/R also induced the production of NO,ROS,and apoptosis.AT3 and M4-mAChR inhibitors inhibited the H/R-induced production of NO and ROS and apoptosis.HIF-1αwas induced by H/R,but was suppressed by AT3.Conclusion:Thus,the in vitro evidence shows that AT3 protects against H/R injury in cerebral microvascular endothelial cells via inhibition of HIF-1α,NO and ROS,predominantly through the downregulation of M4-mAChR.The findings offer novel understandings regarding AT3-mediated attenuation of endothelial cell apoptosis and cerebral ischemia/reperfusion injury.展开更多
Objective:To investigate to the expression effect of hypoxia and hypoxia/reoxygenation on ROS,MAPKs and cell apoptosis in H9c2 cardiomyocytes.Methods:H9c2 cells were treated with cobalt chloride(CoCl2)to establish the...Objective:To investigate to the expression effect of hypoxia and hypoxia/reoxygenation on ROS,MAPKs and cell apoptosis in H9c2 cardiomyocytes.Methods:H9c2 cells were treated with cobalt chloride(CoCl2)to establish the chemical hypoxia and hypoxia/reoxygenation-induced cardiomyocyte injury model.CoCl2 was used to process cells at different concentrations from 150-2400μmol/L and different time from4-24 h;H9c2 cells viability was detected by MTT,and the intracellular ROS level was measured by 2’,7’-dichlorflμoresceindiacetate(DCFH-DA)and dihydroethidiμm(DHE)staining and photoflurography.The active expression level of mitogen-activated protein kinases(MAPKs)(including JNK,ERK and p38)and caspase-3.Results:At the concentration from 300 to 1200μmol/L,CoCl2 does/time-dependently inhibited the cell viability in H9c2 cells(P<0.01).Compared with control group,the ROS levels in hypoxia group were significantly increased(P<0.05).In hypoxia group,the active expression levels of p-JNK,p-p38 and caspase-3 was higher than those in control group(P<0.05).However,the expression of p-ERK wasn’t significant differernce.Furthermore,all the expression levels of ROS,p-JNK,p-ERK,p-p38 and caspase-3 in H/R group were significantly raised compared with hypoxia group(P<0.01).Conclusions:Reoxygenation further aggravate chemical hypoxia induced cardiomyocyte oxidative stress injury by activating ROS/MAPKs signals,suggesting the role of myocardial ischemia/reperfusion injury in the pathogenesis of ischemic heart disease.展开更多
Stroke is a leading cause of death worldwide. Up to one thousand potential drugs or interventions have been developed to treat stroke, out of which;60 have gone on to clinical trials. However, none of them has been su...Stroke is a leading cause of death worldwide. Up to one thousand potential drugs or interventions have been developed to treat stroke, out of which;60 have gone on to clinical trials. However, none of them has been successful. New insights into the molecular and cellular mechanisms of ischemia-induced injury are needed for discovering new therapeutic targets. Recently, Drosophila has been used to uncover new hypoxia-related genes. In this study, we describe an efficient and reliable assay with a sophisticated apparatus for studying the effects of oxygen deprivation on flies. Using this assay, wild-type flies were exposed to an anoxic environment for varying lengths of time, then the cumulative death rate and mobility recovery were systematically analyzed. We found that anoxia for over one hour caused lethality. The cumulative death rate on day 5 after anoxia was linearly and positively correlatedwith the duration of anoxia, and reached 50% when the duration was 2.5 h–3 h. We also found that the mobility recovery in normoxia was slow, as the climbing ability remained largely unchanged 4 h–6 h after 2.5-h of anoxia.We suggest that 2.5 h–3 h of anoxia and 4 h–6 h of recovery before mobility analysis are appropriate for future use of the anoxia assay.展开更多
OBJECTIVE:To evaluate the anti-apoptotic efficacy of Qingnao Yizhi formula(清脑益智方,QNYZ)in cultured cerebral cortical neuronal cells(CNCs)and the regulation of the NogoA-Nogo receptor(NgR)/Rho-Rho kinase(ROCK)signa...OBJECTIVE:To evaluate the anti-apoptotic efficacy of Qingnao Yizhi formula(清脑益智方,QNYZ)in cultured cerebral cortical neuronal cells(CNCs)and the regulation of the NogoA-Nogo receptor(NgR)/Rho-Rho kinase(ROCK)signaling pathway.METHODS:Primary cultured CNCs were randomly divided into the following groups:normal control group(N-C),hypoxia-reoxygenation group(H/R),high-dose QNYZ group(Q-H),low-dose QNYZ group(Q-L)butylphthalide(NBP)group,and Y-27632(a selective ROCK transduction pathway inhibiter)group.Except those in the N-C group,CNCs were placed in hypoxic conditions for 24 h and then in reoxygenation conditions for 24 h.Cell media was changed every 48 h,and various assays were performed on the 7 th day.Cell viability was evaluated by measuring mitochondrial dehydrogenase activity,using a CCK-8 assay,in triplicate.Synapsin(SYN)protein concentrations were evaluated by enzyme-linked immunosorbent assay.NogoA and RhoA protein expression were evaluated through Western blotting.The gene expression of NogoA,NgR,RhoA,and ROCK was evaluated by reverse transcription-polymerase chain reaction.Cell apoptosis was measured using a terminal deoxynucleotidyl transferase biotin-d UTP nick end labeling assay.RESULTS:Compared with the N-C group,the cell viability of the H/R group decreased significantly(P<0.05).The cell viability values for the Q-H and Q-L groups increased compared with that for the H/R group,and the difference was significant for the Q-H group(P<0.05).The NogoA and RhoA protein levels and the NogoA,NgR,RhoA,and ROCK m RNA expression levels increased in the H/R group,compared with the N-C group,and decreased significantly in the Q-H and Q-L groups(P<0.05)and in the Y-27632 group(P<0.05)compared with the H/R group.The SYN levels in the Q-H,Q-L,and NBP groups significantly increased compared with that in the H/R group(P<0.05).Compared with the H/R group,the numbers of apoptotic cells in the Q-H,Q-L,and NBP groups significantly decreased(P<0.05).CONCLUSION:The presented study demonstrated that QNYZ exerted anti-apoptotic effects on H/R-induced CNCs,possibly through the modulation of the NogoA-NgR/Rho-ROCK signaling pathway and the promotion of synaptic plasticity in H/R CNCs.展开更多
OBJECTIVE:To investigate the protective effect of resveratrol on cardiomyocytes after hypoxia/reoxygenation intervention based on PTEN-induced putative kinase protein 1/Parkinson disease protein 2(PINK1/PARKIN)signali...OBJECTIVE:To investigate the protective effect of resveratrol on cardiomyocytes after hypoxia/reoxygenation intervention based on PTEN-induced putative kinase protein 1/Parkinson disease protein 2(PINK1/PARKIN)signaling pathway.METHODS:3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was used to detect the effect of resveratrol on the viability of H9C2 cells;the hypoxia/reoxygenation(H/R)model was established in tri-gas incubator;2’,7’-Dichlorofluorescin diacetate staining was used to measure the content of reactive oxygen species(ROS);the changes of mitochondrial membrane potential was determined by 5,5’,6,6’-Tetrachloro-1,1’,3,3’-tetraethyl-imidacarbocyanine iodide staining;the changes of mitochondrial respiratory chain complex activity was evaluated by enzyme activity kits;flow cytometry was used to detect the ratio of apoptotic cells;transmission electron microscope was used to observe the ultrastructure of H9C2 cells;Western blot was used to detect the protein changes of mitochondrial 20 k Da outer membrane protein(TOM20),translocase of inner mitochondrial membrane 23(TIM23),presenilins associated rhomboid-like protein(PARL),PINK1,PARKIN and mitofusin 1(Mfn1),mitofusin 2(Mfn2),phosphotyrosine independent ligand for the Lck SH2 domain of 62 k Da(P62),microtubule-associated protein 1 light chain 3 beta(LC3B);the m RNA levels of PINK1 and PARKIN was detected by quantitative polymerase chain reaction;immunoprecipitation assay was used to detect the interaction between PARKIN and Ubiquitin.RESULTS:Resveratrol could inhibit the proliferation of H9C2 cells in a time-and concentration-dependent manner;however,pretreatment with low cytotoxic resveratrol could reduce the H/R-induced increase in cellular ROS levels,alleviate the loss of mitochondrial membrane potential induced by H/R,inhibit H/R-induced apoptosis of H9C2 cells,and protect the mitochondrial structure and respiratory chain of H9C2 cells from H/R damage.Resveratrol could further increase the levels of p62,PINK1,PARKIN protein,the expression of PINK1,PARKIN m RNA and the ratio of LC3BⅡ/LC3BⅠin H/Rinduced H9C2 cells,inhibit the interaction between PARKIN and Ubiquitin in H/R-induced H9C2 cells,and further reduce the expression of TOM20,TIM23,PARL,Mfn1 and Mfn2 protein in H/R-induced H9C2 cells.The effect of resveratrol is consistent with that of autophagy activator on H/R-induced H9C2 cells.CONCLUSIONS:Resveratrol can protect H9C2 cells from H/R injury,which may be related to resveratrol promoting mitochondrial autophagy by activating PINK1/PARKIN signaling pathway.展开更多
BACKGROUND: Retinoid X receptor(RXR) plays a central role in the regulation of intracellular receptor signaling pathways. The activation of RXR has protective effect on H2O2-induced apoptosis of H9c2 ventricular cells...BACKGROUND: Retinoid X receptor(RXR) plays a central role in the regulation of intracellular receptor signaling pathways. The activation of RXR has protective effect on H2O2-induced apoptosis of H9c2 ventricular cells in rats. But the protective effect and mechanism of activating RXR in cardiomyocytes against hypoxia/reoxygenation(H/R)-induced oxidative iniury are still unclear.METHODS: The model of H/R injury was established through hypoxia for 2 hours and reoxygenation for 4 hours in H9c2 cardiomyocytes of rats. 9-cis-retinoic acid(9-cis RA) was obtained as an RXR agonist, and HX531 as an RXR antagonist. Cultured cardiomyocytes were randomly divided into four groups: sham group, H/R group, H/R+9-cis RA-pretreated group(100 nmol/L 9-cis RA), and H/R+9-cis RA+HX531-pretreated group(2.5 μmol/L HX531). The cell viability was measured by MTT, apoptosis rate of cardiomyocytes by flow cytometry analysis, and mitochondrial membrane potential(ΔΨm) by JC-1 fluorescent probe, and protein expressions of Bcl-2, Bax and cleaved caspase-9 with Western blotting. All measurement data were expressed as mean±standard deviation, and analyzed using one-way ANOVA and the Dunnett test. Differences were considered signif icant when P was <0.05.RESULTS: Pretreatment with RXR agonist enhanced cell viability, reduced apoptosis ratio, and stabled ΔΨm. Dot blotting experiments showed that under H/R stress conditions, Bcl-2 protein level decreased, while Bax and cleaved caspase-9 were increased. 9-cis RA administration before H/R stress prevented these effects, but the protective effects of activating RXR on cardiomyocytes against H/R induced oxidative injury were abolished when pretreated with RXR pan-antagonist HX531.CONCLUSION: The activation of RXR has protective effects against H/R injury in H9c2 cardiomyocytes of rats through attenuating signaling pathway of mitochondria apoptosis.展开更多
基金supported by the National Natural Science Foundation of China grant (NSFC81970247)。
文摘Background:Administration of propofol,an intravenous anesthetic with antioxidant property,immediately at the onset of post-ischemic reperfusion(propofol postconditioning,P-PostC) has been shown to confer cardioprotection against ischemia–reperfusion(I/R) injury,while the underlying mechanism remains incompletely understood.The forkhead box O(FoxO) transcription factors are reported to play critical roles in activating cardiomyocyte survival signaling throughout the process of cellular injuries induced by oxidative stress and are also involved in hypoxic postconditioning mediated neuroprotection,however,the role of FoxO in postconditioning mediated protection in the heart and in particular in high glucose condition is unknown.Methods:Rat heart-derived H9c2 cells were exposed to high glucose(HG) for 48 h,then subjected to hypoxia/reoxygenation(H/R,composed of 8 h of hypoxia followed by 12 h of reoxygenation) in the absence or presence of postconditioning with various concentrations of propofol(P-PostC) at the onset of reoxygenation.After having identified the optical concentration of propofol,H9c2 cells were subjected to H/R and P-PostC in the absence or presence of FoxO1 or FoxO3a gene silencing to explore their roles in P-PostC mediated protection against apoptotic and autophagic cell deaths under hyperglycemia.Results:The results showed that HG with or without H/R decreased cell viability,increased lactate dehydrogenase(LDH) leakage and the production of reactive oxygen species(ROS) in H9c2 cells,all of which were significantly reversed by propofol(P-PostC),especially at the concentration of 25 μmol/L(P25)(P<0.05,NC vs.HG;HG vs.HG+HR;HG+HR+P12.5 or HG+HR+P25 or HG+HR+P50 vs.HG+HR).Moreover,we found that propofol(P25) decreased H9c2 cells apoptosis and autophagy that were concomitant with increased FoxO1 and FoxO3a expression(P<0.05,HG+HR+P25 vs.HG+HR).The protective effects of propofol(P25) against H/R injury were reversed by silencing FoxO1 or FoxO3a(P<0.05,HG+HR+P25 vs.HG+HR+P25+siRNA-1 or HG+HR+P25+siRNA-5).Conclusions:It is concluded that propofol postconditioning attenuated H9c2 cardiac cells apoptosis and autophagy induced by H/R injury through upregulating FoxO1 and FoxO3a under hyperglycemia.
基金supported by the Natural Science Foundation of Jiangsu Province of China,No.BK20171180(to XRW)
文摘Rosmarinic acid,a common ester extracted from Rosemary,Perilla frutescens,and Salvia miltiorrhiza Bunge,has been shown to have protective effects against various diseases.This is an investigation into whether rosmarinic acid can also affect the changes of white matter fibers and cognitive deficits caused by hypoxic injury.The right common carotid artery of 3-day-old rats was ligated for 2 hours.The rats were then prewarmed in a plastic container with holes in the lid,which was placed in 37°C water bath for 30 minutes.Afterwards,the rats were exposed to an atmosphere with 8% O2 and 92% N2 for 30 minutes to establish the perinatal hypoxia/ischemia injury models.The rat models were intraperitoneally injected with rosmarinic acid 20 mg/kg for 5 consecutive days.At 22 days after birth,rosmarinic acid was found to improve motor,anxiety,learning and spatial memory impairments induced by hypoxia/ischemia injury.Furthermore,rosmarinic acid promoted the proliferation of oligodendrocyte progenitor cells in the subventricular zone.After hypoxia/ischemia injury,rosmarinic acid reversed to some extent the downregulation of myelin basic protein and the loss of myelin sheath in the corpus callosum of white matter structure.Rosmarinic acid partially slowed down the expression of oligodendrocyte marker Olig2 and myelin basic protein and the increase of oligodendrocyte apoptosis marker inhibitors of DNA binding 2.These data indicate that rosmarinic acid ameliorated the cognitive dysfunction after perinatal hypoxia/ischemia injury by improving remyelination in corpus callosum.This study was approved by the Animal Experimental Ethics Committee of Xuzhou Medical University,China (approval No.20161636721) on September 16,2017.
文摘Objective: To establish the rat model with myocardial hypoxia/reoxygenation (H/R) injury, and investigate the protective effect of EPO pretreatment on the myocardium. Methods: Sixty male adult Wistar rats were randomly divided into 3 groups: control group, H/R group, and EPO group, 20 in each group. The rats in EPO group accepted injection of 5 000 U/kg recombinant human erythropoietin (RHuEPO) through vein, and the other rats accepted the injection of the same volume of saline. Twenty-four hours after the injection, rats in the EPO and H/R groups were put into the hypoxia environment for 12 h and then returned to the normoxic environment for 2 h, and then the samples of blood and myocardium were collected. Serum myocardial enzyme activity, apoptosis, ultrastructure, myocardial MDA contents, EPO receptor (EPOR) expression in cardiac myocytes and cardiac functions were tested. Results: EPOR expression was positive in cardiac myocytes of adult rat according to the result of immunonistochemitry assaying. Compared to those in H/R group, rats in EPO group presented lighter injury of myocardial ultrastructure, the reduction of serum myocardial enzyme activity, inhibition of apoptosis, the better recovery of cardiac functions, and the less production of oxygen-derived free radicals. Conclusion: Adult rat cardiac myocytes could express EPOR, and EPO pretreatment produced protective effects on myocardium with H/R injury.
基金the National Key R&D Program of China(2017YFC0908700,2017YFC0908703)National Natural Science Foundation of China(81772036,81671952,81873950,81873953,81570401,81571934)+4 种基金National S&T Fundamental Resources Investigation Project(2018FY100600,2018FY100602)Taishan Pandeng Scholar Program of Shandong Province(tspd20181220)Taishan Young Scholar Program of Shandong Province(tsqn20161065,tsqn201812129)Key R&D Program of Shandong Province(2018GSF118003)the Fundamental Research Funds of Shandong University(2018JC011).
文摘BACKGROUND:Disturbance of mitochondrial fi ssion and fusion(termed mitochondrial dynamics)is one of the leading causes of ischemia/reperfusion(I/R)-induced myocardial injury.Previous studies showed that mitochondrial aldehyde dehydrogenase 2(ALDH2)conferred cardioprotective effect against myocardial I/R injury and suppressed I/R-induced excessive mitophagy in cardiomyocytes.However,whether ALDH2 participates in the regulation of mitochondrial dynamics during myocardial I/R injury remains unknown.METHODS:In the present study,we investigated the effect of ALDH2 on mitochondrial dynamics and the underlying mechanisms using the H9c2 cells exposed to hypoxia/reoxygenation(H/R)as an in vitro model of myocardial I/R injury.RESULTS:Cardiomyocyte apoptosis was significantly increased after oxygen-glucose deprivation and reoxygenation(OGD/R),and ALDH2 activation largely decreased the cardiomyocyte apoptosis.Additionally,we found that both ALDH2 activation and overexpression significantly inhibited the increased mitochondrial fission after OGD/R.Furthermore,we found that ALDH2 dominantly suppressed dynamin-related protein 1(Drp1)phosphorylation(Ser616)and adenosine monophosphate-activated protein kinase(AMPK)phosphorylation(Thr172)but not interfered with the expression levels of mitochondrial shaping proteins.CONCLUSIONS:We demonstrate the protective effect of ALDH2 against cardiomyocyte H/R injury with a novel mechanism on mitochondrial fission/fusion.
基金supported by the National Natural Science Foundation of China(No.81470418 and No.81770230)。
文摘Morphological and functional abnormalities of vascular endothelial cells(VECs) are risk factors of ischemiareperfusion in skin flaps.Signaling pathway mediated by interleukin-1 receptor(IL-1 R) is essential to hypoxia/reoxygenation(H/R) injury of VECs.While the TIR/BB-loop mimetic(AS-1) disrupts the interaction between IL-1 R and myeloid differentiation primary-response protein 88(MyD88),its role in the VECs dysfunction under H/R is unclear.In this study,we first showed that there was an infiltration of inflammatory cells and the apoptosis of VECs by using a skin flap section from patients who received flap transplantation.We then showed that the H/R treatment induced apoptosis and loss of cell migration of endothelial cell line H926 were attenuated by AS-1.Furthermore,our data suggested that AS-1 inhibits the interaction between IL-1 R and MyD88,and subsequent phosphorylation of IκB and p38 pathway,as well as the nuclear localization of NF-κB subunit p65/p50.Thus,this study indicated that the protective role of AS-1 in H/R induced cellular injury may be due to the AS-1 mediated down-regulation of IL-1 R signaling pathway.
基金the National Natural Science Foundation of China, No.81070957the Natural Science Foundation of Shanxi Province, No.2008011082-1
文摘Astrocytes in an in vitro murine astrocyte model of oxygen and glucose deprivation/hypoxia and reoxygenation were treated with different concentrations of triptolide （250, 500, 1 000 ng/mL） in a broader attempt to elucidate the protection and mechanism underlying triptolide treatment on astrocytes exposed to hypoxia/reoxygenation injury. The results showed that the matrix metalloproteinase-9, interleukin-1β, tumor necrosis factor α and interleukin-6 expressions were significantly decreased after triptolide treatment in the astrocytes exposed to hypoxia/ reoxygenation injury, while interleukin-10 expression was upregulated. In addition, the vitality of the injured astrocytes was enhanced, the triptolide＇s effect was apparent at 500 ng/mL. These experimental findings indicate that triptolide treatment could protect astrocytes against hypoxia/ reoxygenation injury through the inhibition of inflammatory response and the reduction of matrix metalloproteinase-9 expression.
基金the Natural Science Foundation of Shandong Province, No. Y2004C04
文摘BACKGROUND： Phycocyanin can relieve decrease of mitochondrial membrane potential through reducing production of active oxygen so as to protect neurons after hypoxia/reoxygenation. OBJECTIVE： To observe the effect of phycocyanin on activity of PC12 cells and mitochondrial membrane potential after hypoxia/reoxygenation. DESIGN： Randomized controlled study SETTING ： Cerebrovascular Disease Institute of Affiliated Hospital, Medical College of Qingdao University MATERIALS： The experiment was carried out at the Key Laboratory of Prevention and Cure for cerebropathia in Shandong Province from October to December 2005. PC12 cells, rat chromaffin tumor cells, were provided by Storage Center of Wuhan University; phycocyanin was provided by Ocean Institute of Academia Sinica; Thiazoyl blue tetrazolium bromide （MTT） and rhodamine 123 were purchased from Sigma Company, USA; RPMI-1640 medium, fetal bovine serum and equine serum were purchased from Gibco Company, USA. METHODS： ① Culture of PC12 cells： PC12 cells were put into RPMI-1640 medium which contained 100 g/L heat inactivation equine serum and 0.05 volume fraction of fetal bovine serum and incubated in CO2 incubator at 37℃. Number of cells was regulated to 4 × 10^5 L 1, and cells were inoculated at 96-well culture plate. The final volume was 100μL. ② Model establishing and grouping： Cultured PC12 cells were randomly divided into three groups： phycocyanin group, model control group and non-hypoxia group. At 24 hours before hypoxia, culture solution in phycocyanin group was added with phycocyanin so as to make sure the final concentration of 3 g/L , but cells in model control group did not add with phycocyanin. Cells in non-hypoxia group were also randomly divided into adding phycocyanin group （the final concentration of 3 g/L） and non-adding phycocyanin group. Cells in model control group and phycocyanin group were cultured with hypoxia for 1 hour and reoxygenation for 1, 2 and 3 hours; meanwhile, cells in non-hypoxia group were cultured with oxygen and were measured at 1 hour after hypoxia/reoxygenation. ③ Detecting items： At 1, 2 and 3 hours after reoxygenation, absorbance （A value） of PC12 cells was measured with MTT technique so as to observe activity and quantity of cells. Fluorescence intensity of PC12 cells marked by rhodamine 123 was measured with confocal microscope in order to observe changes of mitochondrial membrane potential. MAEN OUTCOME MEASURES： Comparisons between quantity and activity of PC12 cells and mitochondria membrane potential at 1, 2 and 3 hours after reoxygenation. RESULTS： ① Effect of phycocyanin on quantity and activity of PC12 cells： A value was 0.924±0.027 in adding phycocyanin group and 0.924±0.033 in non-adding phycocyanin group. A value was lower in model control group and phycocyanin group than that in non-hypoxia group at 1, 2 and 3 hours after reoxygenation （0.817±0.053, 0.838±0.037, 0.875±0.029; 0.842±0.029, 0.872±0.025, 0.906±0.023, P 〈 0.05）. A value was higher in phycocyanin group than that in model control group at 1, 2 and 3 after culture （P 〈 0.05）. With culture time being longer, A value was increased gradually in phycocyanin group and model control group after reoxygenation （P 〈 0.05）. ~ Effect of phycocyanin on mitochondrial membrane potential of PC12 cells： Fluorescence intensity was 2.967±0.253 in adding phycocyanin group and 2.962±0.294 in non-adding phycocyanin group. Fluorescence intensity was lower in model control group and phycocyanin group than that in non-hypoxia group at 1, 2 and 3 hours after hypoxia/reoxygenation （1.899±0.397, 2.119±0.414, 2.287±0.402; 2.191±0.377, 2.264±0.359, 2.436±0.471, P 〈 0.05）; but it was higher in phycocyanin group than that in model control group at 1, 2 and 3 after reoxygenation （P 〈 0.05）. With culture time being longer, fluorescence intensity was increased gradually in phycocyanin group and model control group after reoxygenation （P 〈 0.05）. CONCLUSION： Phycocyanin and reoxygenation can protect PC12 cells after hypoxia injury through increasing mitochondrial membrane potential and cellular activity, and the effect is improved gradually with prolonging time of reoxygenation.
基金supported by Jiangsu Provincial Science Foundation of China(BK2006229)
文摘Objective：Carbamylated EPO（CEPO） is a derivative of erythropoietin（EPO） by subjecting it to carbamylation. It does not stimulate erythropoiesis, but effectively protects tissue from injury. The present study was to investigate the effect of CEPO treatment using in vitro models of hypoxia/reoxygenation（H/R）. Methods：Cardiomyocytes were exposed to hypoxia（95% N2 and 5% CO2） for 1 hour followed by 4 hours of reoxygenation（95% O2 and 5% CO2）. CEPO was administered after hypoxia, just before reoxygenation. The apoptotic cardiomyocytes were determined by flow cytometry. The level of protein was assessed by western blot analysis. Results： CEPO treatment significantly decreased the apoptotic cardiomyocytes by 54.20% compared with H/R group. Western blot analysis showed that CEPO administration increased the level of Bcl-2（an antiapoptotic protein） by 62.22% compared with H/R group. Conclusion： Acute administration of CEPO protected cardiomyocytes from H/R-induced apoptosis. CEPO protected cardiomyocytes with a concomitant upregulation of Bcl-2 after H/R injury.
文摘[Objectives]To explore the protection mechanism of crocin against ischemia-reperfusion injury of myocardial cells.[Methods]Newborn male SD rats were selected,left ventricular cardiomyocytes(CMs)were isolated,and a hypoxia/reoxygenation model of CMs was established to simulate the process of ischemia/reperfusion injury.The cells were randomly divided into four groups:normal cell group(control group),crocin group),hypoxia/reoxygenation group(H/R group),hypoxia/reoxygenation+crocin group(H/R+crocin group).H/R+crocin group selected the concentration of crocin 1,10,and 100μmol/L,and determined the optimal concentration of crocin by detecting the cell proliferation ability.After the cells were pretreated using the optimal concentration of crocin,the levels of superoxide anion,cell proliferation,apoptosis and Nox2 levels in each group of cells were detected.[Results]Compared with the control group,the proliferation ability of CMs after hypoxia-reoxygenation injury was reduced(P<0.05),while cell apoptosis and intracellular superoxide anion levels were significantly increased(P<0.01);the CMs pretreated with crocin can reduce the level of Nox2(P<0.01),increase the cell proliferation ability of CMs,reduce cell apoptosis,and accordingly reduce the level of superoxide anion in the cell(P<0.05).[Conclusions]Crocin protects CMs from hypoxia/reoxygenation injury through down-regulating the level of Nox2 and reducing oxidative stress injury.
文摘Objective：To observe the protective effects of erythropoietin （EPO） pretreatment on cardiac myocyte with hypoxia/reoxygenation （H/R） injury and the role of NF-κBin this effects. Methods：After the H/R model of cardiac myocytes of neonatal rats was established, the cultured cardiac myocytes were divided into 4 groups, including EPO pretreatment group （ EPO 10 U/ml 24 h before H/R）, EPO pretreatment ＋ PDTC group（EPO 10 U/ml and PDTC 5 μg/ml 24 h before H/R）, PDTC group （PDTC 5 μg /ml 24 h before H/R） and eomrolgroup. Before and after the H/R, assay of LDH concentration in the culture medium, the survival rate of the myocytes tested by MTT chromatometry and the apoptosis by flow cytometry were undertaken. Activation of NF-κB was determined by EMSA before and after H/R. Results：EPO pretreatment markedly reduced the LDH concentration in the medium, elevated the survival rate of myocytes and inhibited the apoptosis after H/R. Addition of PDTC during the pretreatment abol- ished the protective effects of EPO pretreatment. NF-κB was markedly activated during EPO pretreatment and PDTCinhibited the activation. However, after H/R, the activity of NF-κB in myocytes with EPO pretreatment was significantly inhibited compared to the other myocytes. Conclusion：NF-κB is significantly activated during EPO pretreatment, but is inhibited after H/R, which is correlated with the protective effects of EPO pretreatment on cardiac myocytes with H/R. This phenomenon can be explained as the negative feedback mechanism of the activation of NF-κB.
文摘Objective: To investigate the mechanism of Cornus officinalis Total Glycosides (COTG) on myocardial protection, by studying effects of COTG on cardiomyocyte apoptosis induced by hypoxia/reoxygenation and calcium concentration in rats. Methods: The myocardial cells of born 1-3d SD rats were isolated by enzyme digestion, cultured for 3 days. Cells were divided into five groups: Control group, H/R group, Cornus officinalis Total Glycosides low-dose group (LDG), Cornus officinalis Total Glycosides middle-dose group (MDG) and Cornus officinalis Total Glycosides high-dose group (HDG). Three drug groups were pretreated with different doses of Cornus officinalis Total Glycosides before hypoxia/reoxygenation treatment. The apoptotic rate was determined by flow cytometry assay, the intracellular free calcium concentration was examined by flow cytometry, and the ultrastructure of myocardial cells was observed under transmission electron microscope. Results: The results revealed that Cornus officinalis Total Glycosides pretreatment decreased apoptosis rate, but the effect of lower dosage is not significant. Furthermore, Cornus officinalis Total Glycosides can attenuate mitochondrial calcium overload, improve mitochondrial morphology and inhibit cardiomyocyte apoptosis caused by H/R. Conclusion: Cornus officinalis Total Glycosides pretreatment can inhibit cardiomyocyte apoptosis and calcium overload during H/R injury. However, the underlying mechanisms require us to further study.
基金supported by grants from Science and Technology Planning Project of Guangdong Province,China(No.2015B030301005)
文摘Phytochemical investigations from the roots of Cynanchum stauntonii led to obtain four new C_（21） steroidal glycosides（1–4） and one known compound stauntoside F（5）. Their chemical structures were characterized by sophisticated analyses of IR, HRESI-TOF-MS, 1D, and 2D-NMR data, together with chemical methods, which showed interesting 13,14：14,15-disecopregnane-type skeleton or 14,15-secopregnane-type skeleton C_（21） steroidal glycosides. Among them, compound 1 was determined to be glaucogenin C 3-O-b-D-glucopyranosyl-（1 → 4）-b-D-cymaropyranosyl-（1 → 4）-b-D-digitoxopyranosyl-（1 → 4）-b-D-thevetopyranoside. Compound 2 was characterized to be hirundigenin 3-O-a-L-diginopyranosyl-（1 → 4）-b-D-cymaropyranosyl-（1 → 4）-b-D-digitoxopyranosyl-（1 → 4）-b-D-30-demethyl-thevetopyranoside. Compound 3 was identified to be（14S,16 S,20R）-14,16-14,20-15,20-triepoxy-14,15-secopregn-5-en-3-ol-3-O-a-L-cymaropyranosyl-（1 → 4）-b-D-digitoxopyranosyl-（1 → 4）-b-D-oleandropyranoside.Compound 4 was identified to be（14S,16 S,20R）-14,16-14,20-15,20-triepoxy-14,15-secopregn-5-en-3-ol-3-O-a-L-cymaropyranosyl-（1 → 4）-b-D-cymaropyranosyl-（1 → 4）-b-D-digitoxopyranosyl-（1 → 4）-b-Dthevetopyranoside. Among them, compound 2 was hirundigenin type C21 steroidal glycoside that existed in nature as epimers due to the presence of 14-hemiketal hydroxyl group in its structure. In addition, the anti-inflammatory and cardiomyocyte protective effects of compounds 1–4 were evaluated. We found that they exhibited significant protective effects on hypoxia/reoxygenation induced cardiomyocyte injury, but did not showed obvious anti-inflammatory function.
基金Supported by National Natural Science Foundation of China, No.30570671the Educational Department Science Research Foundation of Jiangsu Province, No. 99KJB310005 and 05KJB310134
文摘AIM: To investigate the protective effect and mechanisms of ghrelin postconditioning against hypoxia/reoxygenation (H/R)-induced injury in human gastric epithelial cells. METHODS: The model of H/R injury was established in gastric epithelial cell line (GES-1) human gastric epithelial cells. Cells were divided into seven groups: normal control group (N); H/R postconditioning group; DMSO postconditioning group (DM); ghrelin postconditioning group (GH); D-Lys3-GHRP-6 + ghrelin postconditioning group (D + GH); capsazepine + ghrelin postconditioning group (C + GH); and LY294002 + ghrelin postconditioning group (L + GH). 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to detect GES-1 cell viability. Hoechst 33258 fluorochrome staining and flow cytometry were conducted to determine apoptosis of GES-1cells. Spectrophotometry was performed to determine release of lactate dehydrogenate (LDH). Protein expression of Bcl-2, Bax, Akt, and glycogen synthase kinase (GSK)-3β was determined by western blotting. Expression of vanilloid receptor subtype 1 (VR1), Akt and GSK-3β was observed by immunocytochemistry. RESULTS: Compared with the H/R group, cell viability of the GH group was significantly increased in a dosedependent manner (55.9% ± 10.0% vs 69.6% ± 9.6%, 71.9% ± 17.4%, and 76.3% ± 13.3%). Compared with the H/R group, the percentage of apoptotic cells in the GH group significantly decreased (12.38% ± 1.51% vs 6.88% ± 0.87%). Compared with the GH group, the percentage of apoptotic cells in the D + GH group, C + GH group and L + GH groups significantly increased (11.70% ± 0.88%, 11.93% ± 0.96%, 10.20% ± 1.05% vs 6.88% ± 0.87%). There were no significant differences in the percentage of apoptotic cells between the H/R and DM groups (12.38% ± 1.51% vs 13.00% ± 1.13%). There was a significant decrease in LDH release following ghrelin postconditioning compared with the H/R group (561.58 ± 64.01 U/L vs 1062.45 ± 105.29 U/L). There was a significant increase in LDH release in the D + GH, C + GH and L + GH groups compared with the GH group (816.89 ± 94.87 U/L, 870.95 ± 64.06 U/L, 838.62 ± 118.45 U/L vs 561.58 ± 64.01 U/L). There were no significant differences in LDH release between the H/R and DM groups (1062.45 ± 105.29 U/L vs 1017.65 ± 68.90 U/L). Compared with the H/R group, expression of Bcl-2 and Akt increased in the GH group, whereas expression of Bax and GSK3β decreased. Compared with the GH group, expression of Bcl-2 decreased and Bax increased in the D + GH, C + GH and L + GH groups, and Akt decreased and GSK-3β increased in the L + GH group. The H/R group also upregulated expression of VR1 and GSK-3β and downregulated Akt. The number of VR1-positive and Akt-positive cells in the GH group significantly increased, whereas the number of GSK-3β-positive cells significantly decreased. These effects of ghrelin were reversed by capsazepine and LY294002.CONCLUSION: Ghrelin postconditioning protected against H/R-induced injury in human gastric epithelial cells, which indicated that this protection might be associated with GHS-R, VR1 and the PI3K/Akt signaling pathway.
基金supported by National Natural Science Foundation of China(82102315).
文摘BACKGROUND:There are currently no effective drugs to mitigate the ischemia/reperfusion injury caused by fluid resuscitation after hemorrhagic shock(HS).The aim of this study was to explore the potential of the histone deacetylase 6(HDAC6)-specific inhibitor tubastatin A(TubA)to suppress nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)inflammasome activation in macrophages under hypoxia/reoxygenation(H/R)conditions.METHODS:The viability of RAW264.7 cells subjected to H/R after treatment with different concentrations of TubA was assessed using a cell-counting kit-8(CCK8)assay.Briefly,2.5μmol/L TubA was used with RAW264.7 cells under H/R condition.RAW264.7 cells were divided into three groups,namely the control,H/R,and TubA groups.The levels of reactive oxygen species(ROS)in the cells were detected using fluorescence microscopy.The protein expression of HDAC6,heat shock protein 90(Hsp90),inducible nitric oxide synthase(iNOS),NLRP3,gasdermin-D(GSDMD),Caspase-1,GSDMD-N,and Caspase-1 p20 was detected by western blotting.The levels of interleukin-1β(IL-1β)and IL-18 in the supernatants were detected using enzyme-linked immunosorbent assay(ELISA).RESULTS:HDAC6,Hsp90,and iNOS expression levels were significantly higher(P<0.01)in the H/R group than in the control group,but lower in the TubA group than in the H/R group(P<0.05).When comparing the H/R group to the control group,ROS levels were significantly higher(P<0.01),but significantly reduced in the TubA group(P<0.05).The H/R group had higher NLRP3,GSDMD,Caspase-1,GSDMD-N,and Caspase-1 p20 expression levels than the control group(P<0.05),however,the TubA group had significantly lower expression levels than the H/R group(P<0.05).IL-1βand IL-18 levels in the supernatants were significantly higher in the H/R group compared to the control group(P<0.01),but significantly lower in the TubA group compared to the H/R group(P<0.01).CONCLUSION:TubA inhibited the expression of HDAC6,Hsp90,and iNOS in macrophages subjected to H/R.This inhibition led to a decrease in the content of ROS in cells,which subsequently inhibited the activation of the NLRP3 inflammasome and the secretion of IL-1βand IL-18.
基金funding from the National Natural Science Foundation of China(12272246)the Key Research and Development Projects of Sichuan Province(2023YFS0075).
文摘Background:Anisodine hydrobromide(AT3),an anti-cholinergic agent,could be delivered to the brain across the blood-brain barrier and has been used clinically for the treatment of cerebral ischemia/reperfusion injury.Endothelial dysfunction can be caused by hypoxia/reoxygenation(H/R)via oxidative stress and metabolic alterations.The present study investigated whether AT3 regulates the production of nitric oxide(NO)and reactive oxygen species(ROS),and the HIF-1αpathway via regulation of muscarinic acetylcholine receptors(mAChRs)in brain microvascular endothelial cells after H/R exposure.Methods:Under H/R conditions,hCMEC/D3 cerebral microvascular endothelial cells were treated with AT3.Specific inhibitors of M2-and M4-mAChRs were used to explore the mechanism by which AT3 influences oxidative stress in endothelial cells.Then,mAChRs expression was detected by western blotting and NO production was detected by Greiss reaction.The intracellular ROS level was measured using DCFH-DA probes.The expression of hypoxia-inducible transcription factor 1α(HIF-1α)was also detected.Results:While H/R induced the expression of M2-and M4-mAChRs,AT3 suppressed the H/R-upregulated M2-and M4-mAChRs.H/R also induced the production of NO,ROS,and apoptosis.AT3 and M4-mAChR inhibitors inhibited the H/R-induced production of NO and ROS and apoptosis.HIF-1αwas induced by H/R,but was suppressed by AT3.Conclusion:Thus,the in vitro evidence shows that AT3 protects against H/R injury in cerebral microvascular endothelial cells via inhibition of HIF-1α,NO and ROS,predominantly through the downregulation of M4-mAChR.The findings offer novel understandings regarding AT3-mediated attenuation of endothelial cell apoptosis and cerebral ischemia/reperfusion injury.
基金The foundation project of national natural science [No.(2018)40]。
文摘Objective:To investigate to the expression effect of hypoxia and hypoxia/reoxygenation on ROS,MAPKs and cell apoptosis in H9c2 cardiomyocytes.Methods:H9c2 cells were treated with cobalt chloride(CoCl2)to establish the chemical hypoxia and hypoxia/reoxygenation-induced cardiomyocyte injury model.CoCl2 was used to process cells at different concentrations from 150-2400μmol/L and different time from4-24 h;H9c2 cells viability was detected by MTT,and the intracellular ROS level was measured by 2’,7’-dichlorflμoresceindiacetate(DCFH-DA)and dihydroethidiμm(DHE)staining and photoflurography.The active expression level of mitogen-activated protein kinases(MAPKs)(including JNK,ERK and p38)and caspase-3.Results:At the concentration from 300 to 1200μmol/L,CoCl2 does/time-dependently inhibited the cell viability in H9c2 cells(P<0.01).Compared with control group,the ROS levels in hypoxia group were significantly increased(P<0.05).In hypoxia group,the active expression levels of p-JNK,p-p38 and caspase-3 was higher than those in control group(P<0.05).However,the expression of p-ERK wasn’t significant differernce.Furthermore,all the expression levels of ROS,p-JNK,p-ERK,p-p38 and caspase-3 in H/R group were significantly raised compared with hypoxia group(P<0.01).Conclusions:Reoxygenation further aggravate chemical hypoxia induced cardiomyocyte oxidative stress injury by activating ROS/MAPKs signals,suggesting the role of myocardial ischemia/reperfusion injury in the pathogenesis of ischemic heart disease.
基金supported by the National Key Basic Research Program of China(2013CB530900)the National Natural Science Foundation of China(81371400 and 81771416)Shanghai Municipal Commission of Health and Family Planning(201740153)
文摘Stroke is a leading cause of death worldwide. Up to one thousand potential drugs or interventions have been developed to treat stroke, out of which;60 have gone on to clinical trials. However, none of them has been successful. New insights into the molecular and cellular mechanisms of ischemia-induced injury are needed for discovering new therapeutic targets. Recently, Drosophila has been used to uncover new hypoxia-related genes. In this study, we describe an efficient and reliable assay with a sophisticated apparatus for studying the effects of oxygen deprivation on flies. Using this assay, wild-type flies were exposed to an anoxic environment for varying lengths of time, then the cumulative death rate and mobility recovery were systematically analyzed. We found that anoxia for over one hour caused lethality. The cumulative death rate on day 5 after anoxia was linearly and positively correlatedwith the duration of anoxia, and reached 50% when the duration was 2.5 h–3 h. We also found that the mobility recovery in normoxia was slow, as the climbing ability remained largely unchanged 4 h–6 h after 2.5-h of anoxia.We suggest that 2.5 h–3 h of anoxia and 4 h–6 h of recovery before mobility analysis are appropriate for future use of the anoxia assay.
基金Supported by the National Natural Science Foundation of China(Study on the Mechanism of NogoA-NgR/Rho-ROCK in Regulating the Synaptic Remodeling of VD,No.81202653)the China Postdoctoral Fund of Sciences(Study on the Mechanism of Qingnao Yizhi Formula Based on the PI3K-Akt-mTOR Signal Transduction Pathway in the Treatment of Vascular Dementia,No.20110490080)+1 种基金Science and Innovation Commission of Shenzhen(Mechanism Study of Hydroxysafflor Yellow A Regulating Mitochondrial Autophagy through ROS Mediated PINK1/parkin Pathway in the Treatment of Acute Cerebral Infarction,JCYJ20180302173504891)Science and Innovation Commission of Shenzhen(lncRNA Malat1 Mediates SDF1/CXCR4 Axis in Cerebral Angiogenesis after Acute Cerebral Infarction and the Intervention Mechanism of Hydroxysafflor Yellow A,JCYJ 20190812161807600)。
文摘OBJECTIVE:To evaluate the anti-apoptotic efficacy of Qingnao Yizhi formula(清脑益智方,QNYZ)in cultured cerebral cortical neuronal cells(CNCs)and the regulation of the NogoA-Nogo receptor(NgR)/Rho-Rho kinase(ROCK)signaling pathway.METHODS:Primary cultured CNCs were randomly divided into the following groups:normal control group(N-C),hypoxia-reoxygenation group(H/R),high-dose QNYZ group(Q-H),low-dose QNYZ group(Q-L)butylphthalide(NBP)group,and Y-27632(a selective ROCK transduction pathway inhibiter)group.Except those in the N-C group,CNCs were placed in hypoxic conditions for 24 h and then in reoxygenation conditions for 24 h.Cell media was changed every 48 h,and various assays were performed on the 7 th day.Cell viability was evaluated by measuring mitochondrial dehydrogenase activity,using a CCK-8 assay,in triplicate.Synapsin(SYN)protein concentrations were evaluated by enzyme-linked immunosorbent assay.NogoA and RhoA protein expression were evaluated through Western blotting.The gene expression of NogoA,NgR,RhoA,and ROCK was evaluated by reverse transcription-polymerase chain reaction.Cell apoptosis was measured using a terminal deoxynucleotidyl transferase biotin-d UTP nick end labeling assay.RESULTS:Compared with the N-C group,the cell viability of the H/R group decreased significantly(P<0.05).The cell viability values for the Q-H and Q-L groups increased compared with that for the H/R group,and the difference was significant for the Q-H group(P<0.05).The NogoA and RhoA protein levels and the NogoA,NgR,RhoA,and ROCK m RNA expression levels increased in the H/R group,compared with the N-C group,and decreased significantly in the Q-H and Q-L groups(P<0.05)and in the Y-27632 group(P<0.05)compared with the H/R group.The SYN levels in the Q-H,Q-L,and NBP groups significantly increased compared with that in the H/R group(P<0.05).Compared with the H/R group,the numbers of apoptotic cells in the Q-H,Q-L,and NBP groups significantly decreased(P<0.05).CONCLUSION:The presented study demonstrated that QNYZ exerted anti-apoptotic effects on H/R-induced CNCs,possibly through the modulation of the NogoA-NgR/Rho-ROCK signaling pathway and the promotion of synaptic plasticity in H/R CNCs.
基金Supported by the Open Fund of Key Laboratory of Dunhuang Medicine,Ministry of Education(DHYX20-09)the Youth Research Foundation of the Gansu University of Chinese Medicine(No.ZQ2017-14)。
文摘OBJECTIVE:To investigate the protective effect of resveratrol on cardiomyocytes after hypoxia/reoxygenation intervention based on PTEN-induced putative kinase protein 1/Parkinson disease protein 2(PINK1/PARKIN)signaling pathway.METHODS:3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was used to detect the effect of resveratrol on the viability of H9C2 cells;the hypoxia/reoxygenation(H/R)model was established in tri-gas incubator;2’,7’-Dichlorofluorescin diacetate staining was used to measure the content of reactive oxygen species(ROS);the changes of mitochondrial membrane potential was determined by 5,5’,6,6’-Tetrachloro-1,1’,3,3’-tetraethyl-imidacarbocyanine iodide staining;the changes of mitochondrial respiratory chain complex activity was evaluated by enzyme activity kits;flow cytometry was used to detect the ratio of apoptotic cells;transmission electron microscope was used to observe the ultrastructure of H9C2 cells;Western blot was used to detect the protein changes of mitochondrial 20 k Da outer membrane protein(TOM20),translocase of inner mitochondrial membrane 23(TIM23),presenilins associated rhomboid-like protein(PARL),PINK1,PARKIN and mitofusin 1(Mfn1),mitofusin 2(Mfn2),phosphotyrosine independent ligand for the Lck SH2 domain of 62 k Da(P62),microtubule-associated protein 1 light chain 3 beta(LC3B);the m RNA levels of PINK1 and PARKIN was detected by quantitative polymerase chain reaction;immunoprecipitation assay was used to detect the interaction between PARKIN and Ubiquitin.RESULTS:Resveratrol could inhibit the proliferation of H9C2 cells in a time-and concentration-dependent manner;however,pretreatment with low cytotoxic resveratrol could reduce the H/R-induced increase in cellular ROS levels,alleviate the loss of mitochondrial membrane potential induced by H/R,inhibit H/R-induced apoptosis of H9C2 cells,and protect the mitochondrial structure and respiratory chain of H9C2 cells from H/R damage.Resveratrol could further increase the levels of p62,PINK1,PARKIN protein,the expression of PINK1,PARKIN m RNA and the ratio of LC3BⅡ/LC3BⅠin H/Rinduced H9C2 cells,inhibit the interaction between PARKIN and Ubiquitin in H/R-induced H9C2 cells,and further reduce the expression of TOM20,TIM23,PARL,Mfn1 and Mfn2 protein in H/R-induced H9C2 cells.The effect of resveratrol is consistent with that of autophagy activator on H/R-induced H9C2 cells.CONCLUSIONS:Resveratrol can protect H9C2 cells from H/R injury,which may be related to resveratrol promoting mitochondrial autophagy by activating PINK1/PARKIN signaling pathway.
基金supported by grants from the National Natural Science Foundation of China(81270282,81070176,30600242,81170192,81200163)Wenzhou Science Technology Bureau Foundation(Y20100010)Education Foundation of Zhejiang Province(Y200906376)
文摘BACKGROUND: Retinoid X receptor(RXR) plays a central role in the regulation of intracellular receptor signaling pathways. The activation of RXR has protective effect on H2O2-induced apoptosis of H9c2 ventricular cells in rats. But the protective effect and mechanism of activating RXR in cardiomyocytes against hypoxia/reoxygenation(H/R)-induced oxidative iniury are still unclear.METHODS: The model of H/R injury was established through hypoxia for 2 hours and reoxygenation for 4 hours in H9c2 cardiomyocytes of rats. 9-cis-retinoic acid(9-cis RA) was obtained as an RXR agonist, and HX531 as an RXR antagonist. Cultured cardiomyocytes were randomly divided into four groups: sham group, H/R group, H/R+9-cis RA-pretreated group(100 nmol/L 9-cis RA), and H/R+9-cis RA+HX531-pretreated group(2.5 μmol/L HX531). The cell viability was measured by MTT, apoptosis rate of cardiomyocytes by flow cytometry analysis, and mitochondrial membrane potential(ΔΨm) by JC-1 fluorescent probe, and protein expressions of Bcl-2, Bax and cleaved caspase-9 with Western blotting. All measurement data were expressed as mean±standard deviation, and analyzed using one-way ANOVA and the Dunnett test. Differences were considered signif icant when P was <0.05.RESULTS: Pretreatment with RXR agonist enhanced cell viability, reduced apoptosis ratio, and stabled ΔΨm. Dot blotting experiments showed that under H/R stress conditions, Bcl-2 protein level decreased, while Bax and cleaved caspase-9 were increased. 9-cis RA administration before H/R stress prevented these effects, but the protective effects of activating RXR on cardiomyocytes against H/R induced oxidative injury were abolished when pretreated with RXR pan-antagonist HX531.CONCLUSION: The activation of RXR has protective effects against H/R injury in H9c2 cardiomyocytes of rats through attenuating signaling pathway of mitochondria apoptosis.
