OBJECTIVE:To explore the role and mechanism of Qufeng Jiejing(祛风解痉,QFJJ)formula in the asthma progression.METHODS:The Bagg Albino/c mice treated with Ovalbumin and AL(OH)3,and airway smooth muscle cells(ASMCs)trea...OBJECTIVE:To explore the role and mechanism of Qufeng Jiejing(祛风解痉,QFJJ)formula in the asthma progression.METHODS:The Bagg Albino/c mice treated with Ovalbumin and AL(OH)3,and airway smooth muscle cells(ASMCs)treated with platelet-derived growth factor(PDGF)-BB to establish a asthma model in vivo and in vitro.The cell morphology was observed with microscope and immunofluorescence staining.The cell viability was assessed with methyl thiazolyl tetrazolium assay.The tumor necrosis factor-αlpha(TNF-α),interleukin-1beta(IL-1β),laminin,fibronectin and collagen IV levels in the ASMCs were detected with corresponding enzyme linked immunosorbent assay kits.Transwell and wound healing assays were conducted to test the cell migration.The TGF-β1,Smad2 and Smad3 levels were measured with Western blot.RESULTS:We found that QFJJ formula treatment dramatically decreased the cell viability,TNF-α,IL-1β,laminin,fibronectin and collagen IV levels in the PDGFBB stimulated ASMCs.Additionally,the protein levels of TGF-β1,Smad2 and Smad3 in the PDGF-BB stimulated ASMCs were prominently depleted after QFJJ formula treatment.Besides,SRI treatment neutralized the role of QFJJ formula in the PDGF-BB stimulated ASMCs.CONCLUSION:QFJJ formula effectively relieved the asthma progression through ameliorate the ASMCs function,which was achieved through suppressing the TGF-β1/Smads signaling pathway.展开更多
Vein graft(VG)failure(VGF)is associated with VG intimal hyperplasia,which is characterized by abnormal accumulation of vascular smooth muscle cells(VSMCs).Most neointimal VSMCs are derived from pre-existing VSMCs via ...Vein graft(VG)failure(VGF)is associated with VG intimal hyperplasia,which is characterized by abnormal accumulation of vascular smooth muscle cells(VSMCs).Most neointimal VSMCs are derived from pre-existing VSMCs via a process of VSMC phenotypic transition,also known as dedifferentiation.There is increasing evidence to suggest that ginger or its bioactive ingredients may block VSMC dedifferentiation,exerting vasoprotective functions;however,the precise mechanisms have not been fully characterized.Therefore,we investigated the effect of ginger on VSMC phenotypic transition in VG remodeling after transplantation.Ginger significantly inhibited neointimal hyperplasia and promoted lumen(L)opening in a 3-month VG,which was primarily achieved by reducing ferroptotic stress.Ferroptotic stress is a pro-ferroptotic state.Contractile VSMCs did not die but instead gained a proliferative capacity and switched to the secretory type,forming neointima(NI)after vein transplantation.Ginger and its two main vasoprotective ingredients(6-gingerol and 6-shogaol)inhibit VSMC dedifferentiation by reducing ferroptotic stress.Network pharmacology analysis revealed that 6-gingerol inhibits ferroptotic stress by targeting P53,while 6-shogaol inhibits ferroptotic stress by targeting 5-lipoxygenase(Alox5),both promoting ferroptosis.Furthermore,both ingredients co-target peroxisome proliferator-activated receptor gamma(PPARγ),decreasing PPARγ-mediated nicotinamide adenine dinucleotide phosphate(NADPH)oxidase 1(Nox1)expression.Nox1 promotes intracellular reactive oxygen species(ROS)production and directly induces VSMC dedifferentiation.In addition,Nox1 is a ferroptosis-promoting gene that encourages ferroptotic stress production,indirectly leading to VSMC dedifferentiation.Ginger,a natural multi-targeted ferroptotic stress inhibitor,finely and effectively prevents VSMC phenotypic transition and protects against venous injury remodeling.展开更多
Background:Mechanical ventilation(MV)provides life support for patients with severe respiratory distress but can simultaneously cause ventilator-induced lung injury(VILI).However,due to a poor understanding of its mec...Background:Mechanical ventilation(MV)provides life support for patients with severe respiratory distress but can simultaneously cause ventilator-induced lung injury(VILI).However,due to a poor understanding of its mechanism,there is still a lack of effective remedies for the often-deadly VILI.Recent studies indicate that the stretch associated with MV can enhance the secretion of extracellular vesicles(EVs)and induce endoplasmic reticulum(ER)stress in airway smoothmuscle cells(ASMCs),both of which can contribute to VILI.But whetherMVassociated stretch enhances the secretion of EVs via ER stress in ASMCs as an underlying mechanism of VILI remains unknown.Methods:In this study,we exposed cultured human ASMCs to stretch(13%strain)and mouse models to MV at tidal volume(18 mL/kg).Subsequently,the amount of secreted EVs in the culture medium of ASMCs and the bronchoalveolar lavage fluid(BALF)of mousemodels was quantitatively evaluated by ultracentrifugation,transmission electron microscopy,Western blot,flow cytometry,and nanoparticle tracking analysis.The cultured ASMCs and the lung tissues of mouse models were assessed for expression of biomarkers of EVs(cluster of differentiation antigen 63,CD63),ER stress(heat shock protein family A member 5,HSPA5),and EVs regulating molecule Rab27a by immunofluorescence microscopy,immunohistochemistry(IHC)and enzyme-linked immunosorbent assay(ELISA),respectively.MicroRNAs(miRNAs)in EVs from ASMCs were measured with miRNA whole genome sequencing(miRNA-Seq).Results:We found that stretch enhanced EV secretion from cultured ASMCs.In addition,the cultured ASMCs and the mouse models were either or not pretreated with ER stress inhibitor(tauroursodeoxycholic acid,TUDCA)/EV secretion inhibitor(GW4869)prior to stretch or MV.We found that MV-associated stretch enhanced the expression of CD63,HSPA5,and Rab27a in cultured ASMCs and BALF/lung tissues of mousemodels,which could all be attenuated with TUDCA/GW4869 pretreatment.miRNA-Seq data show that differentially expressed miRNAs in EVsmainlymodulate gene transcription.Furthermore,the EVs fromcultured ASMCs under stretch tended to enhance detachment and expression of inflammatory cytokines,i.e.,transforming growth factor-β1(TGF-β1),interleukin-10(IL-10)in cultured airway epithelial cells.The expression of TGF-β1 and IL-10 in BALF of the mouse models also increased in response to MV,which was attenuated together with partial improvement of lung injury by pretreatment with TUDCA,GW4869/Rab27a siRNAs.Conclusion:Taken together,our data indicate thatMV-associated stretch can enhance the secretion of EVs from ASMCs via ER stress signaling to mediate airway inflammation and VILI,which provides new insight for further exploring EVs for the diagnosis and treatment of VILI.展开更多
Angiotensin II (Ang II) is the main mediator of the Renin-Angiotensin-System acting on AT<sub>1</sub> and other AT receptors. It is regarded as a pleiotropic agent that induces many actions, including func...Angiotensin II (Ang II) is the main mediator of the Renin-Angiotensin-System acting on AT<sub>1</sub> and other AT receptors. It is regarded as a pleiotropic agent that induces many actions, including functioning as a growth factor, and as a contractile hormone, among others. The aim of this work was to examine the impact of Ang II on the expression and function of α<sub>1</sub>-adrenergic receptors (α<sub>1</sub>-ARs) in cultured rat aorta, and aorta-derived smooth muscle cells. Isolated Wistar rat aorta was incubated for 24 h in DMEM at 37˚C, then subjected to isometric tension and to the action of added norepinephrine, in concentration-response curves. Ang II was added (1 × 10<sup>−5</sup> M), and in some experiments, 5-Methylurapidil (α<sub>1A</sub>-AR antagonist), AH11110A (α<sub>1B</sub>-AR antagonist), or BMY-7378 (α<sub>1D</sub>-AR antagonist), were used to identify the α<sub>1</sub>-AR involved in the response. Desensitization of the contractile response to norepinephrine was observed due to incubation time, and by the Ang II action. α<sub>1D</sub>-AR was protected from desensitization by BMY-7378;while RS-100329 and prazosin partially mitigated desensitization. In another set of experiments, isolated aorta-derived smooth muscle cells were exposed to Ang II and α<sub>1</sub>-ARs proteins were evaluated. α<sub>1D</sub>-AR increased at 30 and 60 min post Ang II exposure, the α<sub>1A</sub>-AR diminished from 1 to 4 h, while α<sub>1B</sub>-AR remained unchanged over 24 h of Ang II exposure. Ang II induced an increase of α<sub>1D</sub>-AR at short times, and BMY-7378 protected α<sub>1D</sub>-AR from desensitization.展开更多
To investigate whether the expression of exogenous heme oxygenase-1 (HO-1) gene within vascular smooth muscle cells (VSMC) could protect the cells from free radical attack and inhibit cell proliferation, we establishe...To investigate whether the expression of exogenous heme oxygenase-1 (HO-1) gene within vascular smooth muscle cells (VSMC) could protect the cells from free radical attack and inhibit cell proliferation, we established an in vitro transfection of human HO-1 gene into rat VSMC mediated by a retroviral vector. The results showed that the profound expression of HO-1 protein as well as HO activity was 1.8- and 2.0-fold increased respectively in the transfected cells compared to the non-transfected ones. The treatment of VSMC with different concentrations of H2O2 led to the remarkable cell damage as indicated by survival rate and LDH leakage. However, the resistance of the HO-1 transfected VSMC against H2O2 was significantly raised. This protective effect was dramatically diminished when the transfected VSMC were pretreated with ZnPP-IX, a specific inhibitor of HO, for 24 h. In addition, we found that the growth potential of the transfected cells was significantly inhibited directly by increased activity of HO-1, and this effect might be related to decreased phosphorylation of MAPK. These results suggest that the overexpression of introduced hHO-1 is potentially able to reduce the risk factors of atherosclerosis, partially due to its cellular protection against oxidative injury and to its inhibitory effect on cellular proliferation.展开更多
Objective： To investigate the differences of primary culture, purification and biological characteristics between endothelial cells and smooth muscle cells from rat aorta. Methods： Endothelial cells were obtained us...Objective： To investigate the differences of primary culture, purification and biological characteristics between endothelial cells and smooth muscle cells from rat aorta. Methods： Endothelial cells were obtained using the vascular ring adherence, collagenase digestion method and an improved vascular ring adherence method, while smooth muscle cells were separated from tissue sections of rat aorta. Clones of endothelial cells were selected by limiting dilution assay. Both cell types were identified using specific cell immunofluorescent markers, and phase contrast microscopy was used to observe the morphological disparity between endothelial cells and smooth muscle cells at the single cell and colony level. Cell proliferation was determined by the cell counting kit-8. Differences between endothelial cells and smooth muscle cells were evaluated in trypsin digestion time, attachment time and recovery after cryopreservation. Results： Endothelial cells were obtained by all three methods. The improved vascular ring method provided the most reproducible results. Cells were in good condition, and of high purity. Smooth muscle cells were cultured successfully by the tissue fragment culture method. Clonal expansion of single endothelial cells was attained. The two cell types expressed their respective specific markers, and the rate of proliferation of smooth muscle cells exceeded that of endothelial cells. Endothelial cells were more sensitive to trypsin digestion than smooth muscle cells. In addition, they had a shorter adherence time and better recovery following cryopreservation than smooth muscle cells. Conclusion： The improved vascular ring method was optimal for yielding endothelial cells. Limiting dilution is a novel and valid method for purifying primary endothelial cells from rat aorta. Primary rat endothelial cell and vascular smooth muscle cell cultures exhibited different morphological characteristics, proliferation rate, adherence time, susceptibility to trypsin digestion and recovery after cryopreservation. Our research can be a good foundation for further application in the regeneration of blood vessel.展开更多
BACKGROUND Epstein-Barr virus associated smooth muscle tumor(EBV-SMT)is a rare oncological entity.However,there is an increasing incidence of EBV-SMTs,as the frequency of organ transplantation and immunosuppression gr...BACKGROUND Epstein-Barr virus associated smooth muscle tumor(EBV-SMT)is a rare oncological entity.However,there is an increasing incidence of EBV-SMTs,as the frequency of organ transplantation and immunosuppression grows.EBV-SMT diagnosis relies on histopathology and immunochemical staining to distinguish it from post-transplant lymphoproliferative disorder(PTLD).There is no clear consensus on the treatment of EBV-SMTs.However,surgical resection,chemotherapy,radiation therapy,and immunosuppression reduction have been explored with varying degrees of success.CASE SUMMARY Our case series includes six cases of EBV-SMTs across different age groups,with different treatment modalities,adding to the limited existing literature on this rare tumor.The median latency time between immunosuppression and disease diagnosis is four years.EBV-SMTs present with variable degrees of aggressiveness and seem to have worse clinical outcomes in patients with tumor multiplicity and worse immunocompetency.CONCLUSION It is imperative to continue building on this knowledge and keeping EBV-SMTs on the differential in immunocompromised individuals.展开更多
Objective： To determine the effect of different concentrations of Radix Saposhnikoviae （RS） on the contraction of smooth muscle strips and the Ca2＋ mobilization of cultured smooth muscle ceils of rat colon and its...Objective： To determine the effect of different concentrations of Radix Saposhnikoviae （RS） on the contraction of smooth muscle strips and the Ca2＋ mobilization of cultured smooth muscle ceils of rat colon and its possible mechanism of action. Methods： Strips of rat colon longitudinal muscle were prepared and smooth muscle cells from rat colon were isolated and cultured. In the experiments, in vitro muscle strips were suspended in an organ bath and the contraction of the strips was recorded. In the cell- experiments, intracellular Ca2＋ was assessed using fluorescent intensity （FI） of smooth muscle cells loaded with Fluo-4/AM, measured with a laser scanning confocal microscope and related software. Results： In the in vitro experiment, RS （0.02, 0.2, 2, 20 g/L） inhibited contraction of muscle strips in a concentration-dependent manner, and this inhibition was significant for the three higher RS concentrations （P 〈 0.01） for both Peak （the maximal contraction amplitude） and Area （the area under curves）. Similarly, RS inhibited Ach-induced contraction. In these experiments the inhibition of the Peak values in the RS 2 and 20 g/L groups was significant （P 〈 0.01）, as was the inhibition of the Area values in all RS groups （P 〈 0.05）. Naloxone and propranolol did not significantly affect the inhibitory effect of RS on smooth muscle contractility, while phentolamine significantly reduced the inhibitory effect （P 〈 0.01）. In experiments using primary smooth muscle cell cultures in Ca2＋ - containing buffer, the post-treatment fluorescence of cells in the RS 0.2, 2 and 20 g/L groups differed significantly from pre-treatment values （P 〈 0.05）, and the percent inhibition of fluorescence in the RS 2 g/L and 20 g/L groups was significant （P 〈 0.01）. However, in Ca2＋-free buffer, FS had no significant effect on cell fluorescence. Conclusion： RS inhibited both the spontaneous and Ach-stimulated contraction of rat colonic smooth muscle strips. This RS effect appeared to involve α -adrenoceptors, but not β -adrenoceptors or opioid receptors. In cultured primary smooth muscle cells, RS reduced the mobilization of Ca2＋ from extracellular sources, but may had no effect on the release of Ca2＋ from sarcoplasmic reticulum and endoplasmic reticulum.展开更多
AIM:To investigate the pathway(s)mediating rat antral circular smooth muscle contractile responses to the cholinomimetic agent,bethanechol and the subtypes of muscarinic receptors mediating the cholinergic contraction...AIM:To investigate the pathway(s)mediating rat antral circular smooth muscle contractile responses to the cholinomimetic agent,bethanechol and the subtypes of muscarinic receptors mediating the cholinergic contraction. METHODS:Circular smooth muscle strips from the antrum of Sprague-Dawley rats were mounted in muscle baths in Krebs buffer.Isometric tension was recorded.Cumulative concentration-response curves were obtained for(+)-cis- dioxolane(cD),a nonspecific muscarinic agonist,at 10^(-8)- 10^(-4)mol/L,in the presence of tetrodotoxin(TTX,10^(-7)mol/L). Results were normalized to cross sectional area.A repeat concentration-response curve was obtained after incubation of the muscle for 90 min with antagonists for M1(pirenzepine), M2(methoctramine)and M3(darifenadn)muscarinic receptor subtypes.The sensitivity to PTX was tested by the ip injection of 100 mg/kg of PTX 5 d before the experiment.The antral circular smooth muscles were removed from PTX-treated and non-treated rats as strips and dispersed smooth muscle cells to identify whether PTX-linked pathway mediated the contractility to bethanechol. RESULTS:A dose-dependent contractile response observed with bethanechol,was not affected by TTx.The pretreatment of rats with pertussis toxin decreased the contraction induced by bethanechol.Lack of calcium as well as the presence of the L-type calcium channel blocker,nifedipine,also inhibited the cholinergic contraction,with a reduction in response from 2.5±0.4 g/mm^2 to 1.2±0.4 g/mm^2(P<0.05).The dose- response curves were shifted to the right by muscarinic antagonists in the following order of affinity:darifenacin (M_3)>methocramine(M_2)>pirenzepine(M_1). CONCLUSION:The muscarinic receptors-dependent contraction of rat antral circular smooth muscles was linked to the signal transduction pathway(s)involving pertussis-toxin sensitive GTP-binding proteins and to extracellular calcium via L-type voltage gated calcium channels.The presence of the residual contractile response after the treatment with nifedipine,suggests that an additional pathway could mediate the cholinergic contraction.The involvement of more than one muscarinic receptor(functionally predominant type 3 over type 2)also suggests more than one pathway mediating the cholinergic contraction in rat antrum.展开更多
The purpose of this study was to examine the relaxation effect of CY on the vascular smooth muscle （VSM） from rabbits. Experiments were carried out on isolated thoracic aorta of rabbits. CY （3 x 103 mM- 3 mM） coul...The purpose of this study was to examine the relaxation effect of CY on the vascular smooth muscle （VSM） from rabbits. Experiments were carried out on isolated thoracic aorta of rabbits. CY （3 x 103 mM- 3 mM） could relax the VSM preparations pre-contracted by adrenaline （AD）, noradrenaline （NE）, high-K^＋ solution or BaCl2 with respective EC50 values of （0.3 1±0.11） mM, 0.19±0.03 mM, 0.20±0.04 mM and 0.25±0.04 mM. Moreover, CY （10-2 mM, 0.1 mM and 1 mM） inhibited norepinephrine （NE）, CaCl2 and KCl-induced vasoconstriction in a concentration dependent manner. The phasic contraction produced by NE was concentration dependently attenuated with CY （10^-2 mM, 0.1 mM and 1 mM） in calcium-free medium, similar to that caused by verapamil. The present findings suggest that CY relaxed thoracic aortic rings by blocking voltage-dependent Ca^2＋ channels. The inhibition of intracellular Ca^2＋ release may be one of the main vasorelaxant mechanisms of CY.展开更多
OBJECTIVE: To investigate the influence of serum from asthmatic rat treated with acupuncture(acupuncture serum) on the contractile responses of airway smooth muscle cells(ASMCs).METHODS: OVA-induced asthmatic rats wer...OBJECTIVE: To investigate the influence of serum from asthmatic rat treated with acupuncture(acupuncture serum) on the contractile responses of airway smooth muscle cells(ASMCs).METHODS: OVA-induced asthmatic rats were subject to acupuncture treatment at the points of Dazhui(GV 14), Fengmen(BL 12), and Feishu(BL 13).The resulting serum was collected, as well as serum from normal animals. Contraction of ASMCs was induced with 10 μM acetylcholine(Ach), and acupuncture serum or normal serum was supplemented 15 min later. The effects of the sera on the changes in cell length, width, and surface areawere monitored in real time via a live cell imaging system.RESULTS: The contraction rates of ASMCs 15 min and 30 min after Ach stimulation reached 38.2% ±3.8% and 49.7% ± 4.6%, both of which were significantly higher than unstimulated control cells(P <0.01). Acupuncture serum at the 1∶100 dosage reduced the contraction rate of ASMCs from 40.1% ±5.1% to 17.6% ± 6.5%(P < 0.01). Thus, the treatment significantly inhibited the Ach-induced contractile responses of ASMCs and yielded a relaxation efficiency of 58%, which was greater than the effects obtained with a 1∶250 dosage. Treatment with acupuncture serum at 1∶500 dosage or normal serum displayed no clear effect of suppressing Ach-induced contraction of ASMCs.CONCLUSION: Our data suggest that acupuncture serum has the effect of inhibiting Ach-induced contraction of ASMCs, thereby promoting relaxation of the cells.展开更多
The aim of this study was to establish a method of isolating and culturing smooth muscle cells from the ductus deferens of rats. Smooth muscle cells were prepared from ductus deferens by explanting technique after dis...The aim of this study was to establish a method of isolating and culturing smooth muscle cells from the ductus deferens of rats. Smooth muscle cells were prepared from ductus deferens by explanting technique after dissection of adventitia and intimae, and cultured in vitro. The identification of the smooth muscle cells were verified by using anti u-smooth muscle actin （a-SMA） immunohistochemistry studies. The result suggested that the cells are multi-morphous, showing long fusiform or star shapes. The apophysis of cells contacted and coalesced to each other, in some regions the cells overlapped in multilayer, while in the other regions they formed monolayer that fluctuated and showed a ＂peak-valley＂ shape. They presented a positive reaction through immunohistochemistry studies. The purity of the cells was more than 99% through this method. The culturing of smooth muscle cells by explanting technique is simple and stable.展开更多
Objective: To investigate the effect of intravascular in radiation on thearterial wall smooth muscle cells (SMCs) proliferation and apoptosis after iliac artery bollominjury in figs. Methods: Twenty-seven miniature fi...Objective: To investigate the effect of intravascular in radiation on thearterial wall smooth muscle cells (SMCs) proliferation and apoptosis after iliac artery bollominjury in figs. Methods: Twenty-seven miniature figs were divided into three groups. All pigsunderwent iliac artery balloon over-stretch. An^(192) Ir source through afterloader was positionedat the injuried segments to give 10 Gy in 9 pigs and 20 Gy in the other 9 pigs, and the rest 9 pigswere, used as control group. The pigs were killed on the 3rd, 10th and 28th days respectively forobservation. The injured segments were processed to examine SMCs proliferation by proliferation cellnuclear antigen (PCNA) and apopto-sis by terminal deoxynucleotidyl transferase-mediated dUTPnick-end labeling (TUNEL). Results: PC-NA index analysis has some that SMCs proliferation inneointima was significantly inhibited in irradiation group on the 10th and 28th days. The value forintimal SMCs apoptosis in control vs 10 Gy and 20 Gy irradiation groups were: (1. 185+-0. 49)% vs(2. 27+-0. 49)%(P>0. 05) and (1. 85+-0. 49)% vs (2. 53+-0. 45)%(P<0. 05), at the 10th day; (1.61+-0. 35)% vs (3. 11+-0. 51)%(P<0. 05), and (1.61+-0. 35)% vs (7. 05+-1. 82)% (P<0. 05), on the28th day. In irradiated arteries, the maximal incidence of intimal SMCs apoptosis was (7. 05+--1.82)% in 20 Gy group vs (3. 11+-0. 51)% in 10 Gy group (P<0. 05), on the 28th day. In the same doseirradiation group, the incidence of intimal SMCs apoptosis was higher on the 28th day than that onthe 10th day. Conclusion: Intra-arterial gamma irradiation can inhibit intimal SMCs proliferationand stimulate SMCs apoptosis in balloon-in jured arteries. These may be contributive to preventionof restenosis of arteries after balloon injury.展开更多
The effects of sodium ferulate(SF), a water-soluble element of Chinese medicine Angelica sinensis diels, on cell-mediated oxidative modification of human low density lipoprotein(LDL) and proliferation of rabbit aortic...The effects of sodium ferulate(SF), a water-soluble element of Chinese medicine Angelica sinensis diels, on cell-mediated oxidative modification of human low density lipoprotein(LDL) and proliferation of rabbit aortic smooth muscle cells(SMCs) were investigated. Using experimental models of proliferation of cultured rabbit aortic SMCs induced by oxidized LDL(ox-LDL), the extent of oxidation was determined by thiobarbituric acid reactive substances(TBARS) method, MTT colorimetry and 3H-thymidine(3H-TdR) incorporation were used to observe proliferation of SMCs. It showed that SF effectively inhibited cell-mediated oxidation induced by Cu2+ in a concentration-dependent manner. At the final concentration of 40, 80, 120 gmL-1, SF could significantly inhibit 3H-TdR incorporation and cell Proliferation in a dose-dependent manner. The results indicated that SF could, in vitro protect LDL against oxidative modification and inhibit the proliferation of SMC, which might be due to its free radical scavenging capacity.展开更多
Aim This study was to evaluate the effect of arsenic trioxide （As2O3） on the transgenic TNF-α promoter activity in cultured vascular smooth muscle cells （VSMCs） and THP-1 monocytes. Methods Human TNF-α promoter ...Aim This study was to evaluate the effect of arsenic trioxide （As2O3） on the transgenic TNF-α promoter activity in cultured vascular smooth muscle cells （VSMCs） and THP-1 monocytes. Methods Human TNF-α promoter was constructed by reporter gene system and was transiently transfected into VSMCs and THP-1 in vitro. The promoter activity was tested by luciferase activity with or without LPS and Ang Ⅱ stimulation, before and after different dosage of As2O3 treatment. Results 1. TNF-α promoter effectively expressed in VSMCs and THP-1 compared with CMV promoter （58.3% and 80.9%, respectively）. Both LPS and Ang Ⅱ significantly up-regulated TNF-α promoter activity （P〈0.05）. 2. As2O3 significantly inhibited, both intact and LPS/Ang Ⅱ stimulated promoter activity, in a dose dependent manner （P〈0.05）, and in both cell type. Conclusion These results manifested that, the inhibition effect of As2O3 on the activity of human TNF-α promoter indicated its potential inhibition on pro-inflammatory cytokine genes expression at transcriptional level and its potential anti-inflammatory property in the cardiovascular system.展开更多
Objective: This study aims to investigate the effects of urocortin (Ucn) on the viability of endothelial cells (ECV304) and rat vascular muscle cells (VSMC). Methods: Rat aortic VSMC were isolated from the rats' t...Objective: This study aims to investigate the effects of urocortin (Ucn) on the viability of endothelial cells (ECV304) and rat vascular muscle cells (VSMC). Methods: Rat aortic VSMC were isolated from the rats' thoracic aorta. We studied the effect of Ucn on the viability of ECV304 cells and VSMC by using a tetrazolium (MTT) assay.Results: Ucn (10 -7 mol/L) inhibited the viability of ECV304 cells and VSMC. Inhibition rates are 13% and 15%, respectively(P<0.05, compared with Control). This inhibition was not dependent on the affecting time and was not affected by the addition of ATP-sensitive potassium channel (KATP channel) blocker, glybenclamide (Gly, 10 mol/L). Conclusion: Ucn inhibits the viability of ECV304 and VSMC. Our results suggest that Ucn may be a new vasoactive agent and may have a beneficial effect in the process of vascular remodeling (VR).展开更多
\ The effects of tetrandrine (Tet) on cytosolic free calcium ([Ca2+]i) in subcultured bovine aortic smooth muscle cells (SMC) were studied by Fura2 and ARCMMIC cation measurement system. Tet (1～100 μmol·L-1) ...\ The effects of tetrandrine (Tet) on cytosolic free calcium ([Ca2+]i) in subcultured bovine aortic smooth muscle cells (SMC) were studied by Fura2 and ARCMMIC cation measurement system. Tet (1～100 μmol·L-1) had no effect on the resting [Ca2+]i, but had inhibitory effects on [Ca2+]i elevation induced by high K+, 5HT, ATP, Ang II and NE in the presence of extracellular Ca2+. High concentration of Tet also inhibited Pheinduced [Ca2+]i elevation in absence of extracellular Ca2+. Tet (1～100 μmol·L-1) inhibited KCl (60 mmol·L-1) induced [Ca2+]i elevation in dosedependent manner, the IC50 value was 9.2 (95% confidence limits: 5.7～14.9) mmol·L-1. The results suggested that Tet had blocking effects on both VOC and ROC in bovine aortic SMC. It appears that the mechanisms of blocking effect of Tet on ROC might be primarily due to its Ca2+ entry blocking effects.展开更多
Vascular smooth muscle cell （VSMC） differentiation and proliferation are two important physiological proc- esses during vascular development. The phenotypic alteration from differentiated to proliferative VSMC contr...Vascular smooth muscle cell （VSMC） differentiation and proliferation are two important physiological proc- esses during vascular development. The phenotypic alteration from differentiated to proliferative VSMC contrib- utes to the development of several major cardiovascular diseases including atherosclerosis, hypertension, resteno- sis after angioplasty or bypass, diabetic vascular complications, and transplantation arteriopathy. Since the VSMC phenotype in these pathological conditions resembles that of developing VSMC during embryonic development, understanding of the molecular mechanisms that control VSMC differentiation will provide fundamental insights into the pathological processes of these cardiovascular diseases. Although VSMC differentiation is usually ac- companied by an irreversible cell cycle exit, VSMC proliferation and differentiation occur concurrently during embryonic development. The molecular mechanisms simultaneously regulating these two processes, however, remain largely unknown. Our recent study demonstrates that cell division cycle 7, a key regulator of cell cycle, promotes both VSMC differentiation and proliferation through different mechanisms during the initial phase of VSMC differentiation. Conversely, Kriappel-like factor 4 appears to be a repressor for both VSMC differentia- tion and proliferation. This review attempts to highlight the novel role of cell division cycle 7 in TGF-β-induced VSMC differentiation and proliferation. The role of K141ppel-like factor 4 in suppressing these two processes will also be discussed.展开更多
This study compared tankyrase 1 expression and autophagy quantity between erectile dysfunction （ED） and non-ED rats＇ corpus cavernosum smooth muscle cells （CSMCs）. This study aslo explored the effect and possible...This study compared tankyrase 1 expression and autophagy quantity between erectile dysfunction （ED） and non-ED rats＇ corpus cavernosum smooth muscle cells （CSMCs）. This study aslo explored the effect and possible mechanism of tankyrase 1 on autophagy and cell proliferation in ageing ED rats＇ CSMCs. The intracavernous pres- sure and mean systemic arterial pressure were measured to investigate erectile function so that eight 24-month-old ED and eight 8-month-old male Wistar rats were choosed respectively. The rat CSMCs were isolated and cultured by enzyme digestion, in which tankyrase 1 expression and autophagy quantity were compared. Tankyrase 1 over-expression was induced with plasmid transfection by Lipofectamine^TM. The effect of tankyrase 1 overexpression on proliferation, autophagy and mTOR pathway in 24-month-old ED rats＇ CSMCs was measured by the cell growth curve in MTT assay, cell cycle analysis in flow cytometry （FCM）, key protein expression in Western blot, autophagy quantity in transmission electron microscopy, monodansylcadaverine staining and GFP-LC3 fluorescence. The primary CSMCs were confirmed by immunofluorescence, and the purity was 99.1% in FCM. Compared with that of 8-month-old rats, tankyrase 1 expression and autophagy quantity significantly decreased in 24-month-old ED rats＇ primary CSMCs （P 〈 0.01）. Tankyrase 1 overexpression significantly increased the growth rate （P 〈 0.05） and increased the S phase of cell cycle （P 〈 0.01）. The autophagosome quantity was remarkably increased （P 〈 0.01）, LC3-Ⅰ/Ⅱ and Beclin 1 were upregulated （P 〈 0.01 and P 〈 0.05）, and p-p70S6K （Thr389） was downregulated in 24-month-old ED rat CSMCs （P 〈 0.05）. In conclusion, Tankyrase 1 and autophagy decrease in the CSMCs from aging rats with ED, and tankyrase 1 may have a positive effect on proliferation by enhancing autophagy and regulating the mTOR signalling pathway.展开更多
The increased proliferation and migration of vascular smooth muscle cells （VSMCs） are key events in the development of atherosclerotic lesions. Baicalin, an herb-derived flavonoid compound, has been previously shown...The increased proliferation and migration of vascular smooth muscle cells （VSMCs） are key events in the development of atherosclerotic lesions. Baicalin, an herb-derived flavonoid compound, has been previously shown to induce apoptosis and growth inhibition in cancer cells through multiple pathways. However, the potential role of baicalin in regulation of VSMC proliferation and prevention of cardiovascular diseases remains unexplored. In this study, we show that pretreatment with baicalin has a dose-dependent inhibitory effect on PDGF-BB-stimulated VSMC pro- liferation, accompanied with the reduction of proliferating cell nuclear antigen （PCNA） expression. We also show that baicalin-induced growth inhibition is associated with a decrease in cyclin E-CDK2 activation and increase in p27 level in PDGF-stimulated VSMCs, which appears to be at least partly mediated by blockade of PDGF recep- tor [~ （PDGFR~）-extracellular signal-regulated kinase 1/2 （ERK1/2） signaling. In addition, baicalin was also found to inhibit adhesion molecule expression and cell migration induced by PDGF-BB in VSMCs. Furthermore, using an animal carotid arterial balloon-injury model, we found that baicalin significantly inhibited neointimal hyperplasia. Taken together, our results reveal a novel function of baicalin in inducing growth arrest of PDGF-stimulated VSMCs and suppressing neointimal hyperplasia after balloon injury, and suggest that the underlying mechanism involves the inhibition of cyclin E-CDK2 activation and the increase in p27 accumulation via blockade of the PDGFR^-ERK1/2 signaling cascade.展开更多
基金Supported by Science and Technology Innovation Project of China Academy of Chinese Medical Sciences of Study on the Mechanism of Qufeng Jiejing Formula in Regulating Mitogen-activated Protein Kinase Signaling Pathway to Inhibit Phenotypic Transformation of Airway Smooth Muscle Cells in Asthma(No.CI2021A01108)Cultivation Project of The National Natural Science Foundation of China of Xiyuan Hospital,China Academy of Chinese Medical Sciences of Research on the Role of Traditional Chinese Medicines-Qufeng Jiejing in the Proliferation,Migration and Phenotypic Transformation of Airway Smooth Muscle Cells in Asthma(No.XY20-17)。
文摘OBJECTIVE:To explore the role and mechanism of Qufeng Jiejing(祛风解痉,QFJJ)formula in the asthma progression.METHODS:The Bagg Albino/c mice treated with Ovalbumin and AL(OH)3,and airway smooth muscle cells(ASMCs)treated with platelet-derived growth factor(PDGF)-BB to establish a asthma model in vivo and in vitro.The cell morphology was observed with microscope and immunofluorescence staining.The cell viability was assessed with methyl thiazolyl tetrazolium assay.The tumor necrosis factor-αlpha(TNF-α),interleukin-1beta(IL-1β),laminin,fibronectin and collagen IV levels in the ASMCs were detected with corresponding enzyme linked immunosorbent assay kits.Transwell and wound healing assays were conducted to test the cell migration.The TGF-β1,Smad2 and Smad3 levels were measured with Western blot.RESULTS:We found that QFJJ formula treatment dramatically decreased the cell viability,TNF-α,IL-1β,laminin,fibronectin and collagen IV levels in the PDGFBB stimulated ASMCs.Additionally,the protein levels of TGF-β1,Smad2 and Smad3 in the PDGF-BB stimulated ASMCs were prominently depleted after QFJJ formula treatment.Besides,SRI treatment neutralized the role of QFJJ formula in the PDGF-BB stimulated ASMCs.CONCLUSION:QFJJ formula effectively relieved the asthma progression through ameliorate the ASMCs function,which was achieved through suppressing the TGF-β1/Smads signaling pathway.
基金supported by grants from the Natural Science Foundation of Shandong Province,China(Grant Nos.:ZR2019ZD28 and ZR2022QH008)the National Natural Science Foundation of China(Grant Nos.:82270301 and 82200465)+1 种基金China Postdoctoral Science Foundation(Grant No.:2023M731842)Shandong Postdoctoral Science Foundation,China(Grant No.:SDCX-ZG-202203013).
文摘Vein graft(VG)failure(VGF)is associated with VG intimal hyperplasia,which is characterized by abnormal accumulation of vascular smooth muscle cells(VSMCs).Most neointimal VSMCs are derived from pre-existing VSMCs via a process of VSMC phenotypic transition,also known as dedifferentiation.There is increasing evidence to suggest that ginger or its bioactive ingredients may block VSMC dedifferentiation,exerting vasoprotective functions;however,the precise mechanisms have not been fully characterized.Therefore,we investigated the effect of ginger on VSMC phenotypic transition in VG remodeling after transplantation.Ginger significantly inhibited neointimal hyperplasia and promoted lumen(L)opening in a 3-month VG,which was primarily achieved by reducing ferroptotic stress.Ferroptotic stress is a pro-ferroptotic state.Contractile VSMCs did not die but instead gained a proliferative capacity and switched to the secretory type,forming neointima(NI)after vein transplantation.Ginger and its two main vasoprotective ingredients(6-gingerol and 6-shogaol)inhibit VSMC dedifferentiation by reducing ferroptotic stress.Network pharmacology analysis revealed that 6-gingerol inhibits ferroptotic stress by targeting P53,while 6-shogaol inhibits ferroptotic stress by targeting 5-lipoxygenase(Alox5),both promoting ferroptosis.Furthermore,both ingredients co-target peroxisome proliferator-activated receptor gamma(PPARγ),decreasing PPARγ-mediated nicotinamide adenine dinucleotide phosphate(NADPH)oxidase 1(Nox1)expression.Nox1 promotes intracellular reactive oxygen species(ROS)production and directly induces VSMC dedifferentiation.In addition,Nox1 is a ferroptosis-promoting gene that encourages ferroptotic stress production,indirectly leading to VSMC dedifferentiation.Ginger,a natural multi-targeted ferroptotic stress inhibitor,finely and effectively prevents VSMC phenotypic transition and protects against venous injury remodeling.
基金funded by the Natural Science Foundation of China(NSFC),Grants No.12072048 to M.L.,12272063,and 11532003 to L.D.partially supported by the Science and Technology Innovation Leading Plan of High-Tech Industry in Hunan Province,China,Grant No.2020SK2018 to L.D.
文摘Background:Mechanical ventilation(MV)provides life support for patients with severe respiratory distress but can simultaneously cause ventilator-induced lung injury(VILI).However,due to a poor understanding of its mechanism,there is still a lack of effective remedies for the often-deadly VILI.Recent studies indicate that the stretch associated with MV can enhance the secretion of extracellular vesicles(EVs)and induce endoplasmic reticulum(ER)stress in airway smoothmuscle cells(ASMCs),both of which can contribute to VILI.But whetherMVassociated stretch enhances the secretion of EVs via ER stress in ASMCs as an underlying mechanism of VILI remains unknown.Methods:In this study,we exposed cultured human ASMCs to stretch(13%strain)and mouse models to MV at tidal volume(18 mL/kg).Subsequently,the amount of secreted EVs in the culture medium of ASMCs and the bronchoalveolar lavage fluid(BALF)of mousemodels was quantitatively evaluated by ultracentrifugation,transmission electron microscopy,Western blot,flow cytometry,and nanoparticle tracking analysis.The cultured ASMCs and the lung tissues of mouse models were assessed for expression of biomarkers of EVs(cluster of differentiation antigen 63,CD63),ER stress(heat shock protein family A member 5,HSPA5),and EVs regulating molecule Rab27a by immunofluorescence microscopy,immunohistochemistry(IHC)and enzyme-linked immunosorbent assay(ELISA),respectively.MicroRNAs(miRNAs)in EVs from ASMCs were measured with miRNA whole genome sequencing(miRNA-Seq).Results:We found that stretch enhanced EV secretion from cultured ASMCs.In addition,the cultured ASMCs and the mouse models were either or not pretreated with ER stress inhibitor(tauroursodeoxycholic acid,TUDCA)/EV secretion inhibitor(GW4869)prior to stretch or MV.We found that MV-associated stretch enhanced the expression of CD63,HSPA5,and Rab27a in cultured ASMCs and BALF/lung tissues of mousemodels,which could all be attenuated with TUDCA/GW4869 pretreatment.miRNA-Seq data show that differentially expressed miRNAs in EVsmainlymodulate gene transcription.Furthermore,the EVs fromcultured ASMCs under stretch tended to enhance detachment and expression of inflammatory cytokines,i.e.,transforming growth factor-β1(TGF-β1),interleukin-10(IL-10)in cultured airway epithelial cells.The expression of TGF-β1 and IL-10 in BALF of the mouse models also increased in response to MV,which was attenuated together with partial improvement of lung injury by pretreatment with TUDCA,GW4869/Rab27a siRNAs.Conclusion:Taken together,our data indicate thatMV-associated stretch can enhance the secretion of EVs from ASMCs via ER stress signaling to mediate airway inflammation and VILI,which provides new insight for further exploring EVs for the diagnosis and treatment of VILI.
文摘Angiotensin II (Ang II) is the main mediator of the Renin-Angiotensin-System acting on AT<sub>1</sub> and other AT receptors. It is regarded as a pleiotropic agent that induces many actions, including functioning as a growth factor, and as a contractile hormone, among others. The aim of this work was to examine the impact of Ang II on the expression and function of α<sub>1</sub>-adrenergic receptors (α<sub>1</sub>-ARs) in cultured rat aorta, and aorta-derived smooth muscle cells. Isolated Wistar rat aorta was incubated for 24 h in DMEM at 37˚C, then subjected to isometric tension and to the action of added norepinephrine, in concentration-response curves. Ang II was added (1 × 10<sup>−5</sup> M), and in some experiments, 5-Methylurapidil (α<sub>1A</sub>-AR antagonist), AH11110A (α<sub>1B</sub>-AR antagonist), or BMY-7378 (α<sub>1D</sub>-AR antagonist), were used to identify the α<sub>1</sub>-AR involved in the response. Desensitization of the contractile response to norepinephrine was observed due to incubation time, and by the Ang II action. α<sub>1D</sub>-AR was protected from desensitization by BMY-7378;while RS-100329 and prazosin partially mitigated desensitization. In another set of experiments, isolated aorta-derived smooth muscle cells were exposed to Ang II and α<sub>1</sub>-ARs proteins were evaluated. α<sub>1D</sub>-AR increased at 30 and 60 min post Ang II exposure, the α<sub>1A</sub>-AR diminished from 1 to 4 h, while α<sub>1B</sub>-AR remained unchanged over 24 h of Ang II exposure. Ang II induced an increase of α<sub>1D</sub>-AR at short times, and BMY-7378 protected α<sub>1D</sub>-AR from desensitization.
基金This work was kindly supported by Na-tional Natural Science Foundation of China(No.39670308)
文摘To investigate whether the expression of exogenous heme oxygenase-1 (HO-1) gene within vascular smooth muscle cells (VSMC) could protect the cells from free radical attack and inhibit cell proliferation, we established an in vitro transfection of human HO-1 gene into rat VSMC mediated by a retroviral vector. The results showed that the profound expression of HO-1 protein as well as HO activity was 1.8- and 2.0-fold increased respectively in the transfected cells compared to the non-transfected ones. The treatment of VSMC with different concentrations of H2O2 led to the remarkable cell damage as indicated by survival rate and LDH leakage. However, the resistance of the HO-1 transfected VSMC against H2O2 was significantly raised. This protective effect was dramatically diminished when the transfected VSMC were pretreated with ZnPP-IX, a specific inhibitor of HO, for 24 h. In addition, we found that the growth potential of the transfected cells was significantly inhibited directly by increased activity of HO-1, and this effect might be related to decreased phosphorylation of MAPK. These results suggest that the overexpression of introduced hHO-1 is potentially able to reduce the risk factors of atherosclerosis, partially due to its cellular protection against oxidative injury and to its inhibitory effect on cellular proliferation.
基金supported by National Natural Science Foundation of China(30700798)
文摘Objective： To investigate the differences of primary culture, purification and biological characteristics between endothelial cells and smooth muscle cells from rat aorta. Methods： Endothelial cells were obtained using the vascular ring adherence, collagenase digestion method and an improved vascular ring adherence method, while smooth muscle cells were separated from tissue sections of rat aorta. Clones of endothelial cells were selected by limiting dilution assay. Both cell types were identified using specific cell immunofluorescent markers, and phase contrast microscopy was used to observe the morphological disparity between endothelial cells and smooth muscle cells at the single cell and colony level. Cell proliferation was determined by the cell counting kit-8. Differences between endothelial cells and smooth muscle cells were evaluated in trypsin digestion time, attachment time and recovery after cryopreservation. Results： Endothelial cells were obtained by all three methods. The improved vascular ring method provided the most reproducible results. Cells were in good condition, and of high purity. Smooth muscle cells were cultured successfully by the tissue fragment culture method. Clonal expansion of single endothelial cells was attained. The two cell types expressed their respective specific markers, and the rate of proliferation of smooth muscle cells exceeded that of endothelial cells. Endothelial cells were more sensitive to trypsin digestion than smooth muscle cells. In addition, they had a shorter adherence time and better recovery following cryopreservation than smooth muscle cells. Conclusion： The improved vascular ring method was optimal for yielding endothelial cells. Limiting dilution is a novel and valid method for purifying primary endothelial cells from rat aorta. Primary rat endothelial cell and vascular smooth muscle cell cultures exhibited different morphological characteristics, proliferation rate, adherence time, susceptibility to trypsin digestion and recovery after cryopreservation. Our research can be a good foundation for further application in the regeneration of blood vessel.
文摘BACKGROUND Epstein-Barr virus associated smooth muscle tumor(EBV-SMT)is a rare oncological entity.However,there is an increasing incidence of EBV-SMTs,as the frequency of organ transplantation and immunosuppression grows.EBV-SMT diagnosis relies on histopathology and immunochemical staining to distinguish it from post-transplant lymphoproliferative disorder(PTLD).There is no clear consensus on the treatment of EBV-SMTs.However,surgical resection,chemotherapy,radiation therapy,and immunosuppression reduction have been explored with varying degrees of success.CASE SUMMARY Our case series includes six cases of EBV-SMTs across different age groups,with different treatment modalities,adding to the limited existing literature on this rare tumor.The median latency time between immunosuppression and disease diagnosis is four years.EBV-SMTs present with variable degrees of aggressiveness and seem to have worse clinical outcomes in patients with tumor multiplicity and worse immunocompetency.CONCLUSION It is imperative to continue building on this knowledge and keeping EBV-SMTs on the differential in immunocompromised individuals.
基金Supported by the TCM Leader Talent Funds on Health Revitalization Project of Jiangsu Province,Health Financial Plan of Jiangsu Province,No.2007/158
文摘Objective： To determine the effect of different concentrations of Radix Saposhnikoviae （RS） on the contraction of smooth muscle strips and the Ca2＋ mobilization of cultured smooth muscle ceils of rat colon and its possible mechanism of action. Methods： Strips of rat colon longitudinal muscle were prepared and smooth muscle cells from rat colon were isolated and cultured. In the experiments, in vitro muscle strips were suspended in an organ bath and the contraction of the strips was recorded. In the cell- experiments, intracellular Ca2＋ was assessed using fluorescent intensity （FI） of smooth muscle cells loaded with Fluo-4/AM, measured with a laser scanning confocal microscope and related software. Results： In the in vitro experiment, RS （0.02, 0.2, 2, 20 g/L） inhibited contraction of muscle strips in a concentration-dependent manner, and this inhibition was significant for the three higher RS concentrations （P 〈 0.01） for both Peak （the maximal contraction amplitude） and Area （the area under curves）. Similarly, RS inhibited Ach-induced contraction. In these experiments the inhibition of the Peak values in the RS 2 and 20 g/L groups was significant （P 〈 0.01）, as was the inhibition of the Area values in all RS groups （P 〈 0.05）. Naloxone and propranolol did not significantly affect the inhibitory effect of RS on smooth muscle contractility, while phentolamine significantly reduced the inhibitory effect （P 〈 0.01）. In experiments using primary smooth muscle cell cultures in Ca2＋ - containing buffer, the post-treatment fluorescence of cells in the RS 0.2, 2 and 20 g/L groups differed significantly from pre-treatment values （P 〈 0.05）, and the percent inhibition of fluorescence in the RS 2 g/L and 20 g/L groups was significant （P 〈 0.01）. However, in Ca2＋-free buffer, FS had no significant effect on cell fluorescence. Conclusion： RS inhibited both the spontaneous and Ach-stimulated contraction of rat colonic smooth muscle strips. This RS effect appeared to involve α -adrenoceptors, but not β -adrenoceptors or opioid receptors. In cultured primary smooth muscle cells, RS reduced the mobilization of Ca2＋ from extracellular sources, but may had no effect on the release of Ca2＋ from sarcoplasmic reticulum and endoplasmic reticulum.
文摘AIM:To investigate the pathway(s)mediating rat antral circular smooth muscle contractile responses to the cholinomimetic agent,bethanechol and the subtypes of muscarinic receptors mediating the cholinergic contraction. METHODS:Circular smooth muscle strips from the antrum of Sprague-Dawley rats were mounted in muscle baths in Krebs buffer.Isometric tension was recorded.Cumulative concentration-response curves were obtained for(+)-cis- dioxolane(cD),a nonspecific muscarinic agonist,at 10^(-8)- 10^(-4)mol/L,in the presence of tetrodotoxin(TTX,10^(-7)mol/L). Results were normalized to cross sectional area.A repeat concentration-response curve was obtained after incubation of the muscle for 90 min with antagonists for M1(pirenzepine), M2(methoctramine)and M3(darifenadn)muscarinic receptor subtypes.The sensitivity to PTX was tested by the ip injection of 100 mg/kg of PTX 5 d before the experiment.The antral circular smooth muscles were removed from PTX-treated and non-treated rats as strips and dispersed smooth muscle cells to identify whether PTX-linked pathway mediated the contractility to bethanechol. RESULTS:A dose-dependent contractile response observed with bethanechol,was not affected by TTx.The pretreatment of rats with pertussis toxin decreased the contraction induced by bethanechol.Lack of calcium as well as the presence of the L-type calcium channel blocker,nifedipine,also inhibited the cholinergic contraction,with a reduction in response from 2.5±0.4 g/mm^2 to 1.2±0.4 g/mm^2(P<0.05).The dose- response curves were shifted to the right by muscarinic antagonists in the following order of affinity:darifenacin (M_3)>methocramine(M_2)>pirenzepine(M_1). CONCLUSION:The muscarinic receptors-dependent contraction of rat antral circular smooth muscles was linked to the signal transduction pathway(s)involving pertussis-toxin sensitive GTP-binding proteins and to extracellular calcium via L-type voltage gated calcium channels.The presence of the residual contractile response after the treatment with nifedipine,suggests that an additional pathway could mediate the cholinergic contraction.The involvement of more than one muscarinic receptor(functionally predominant type 3 over type 2)also suggests more than one pathway mediating the cholinergic contraction in rat antrum.
文摘The purpose of this study was to examine the relaxation effect of CY on the vascular smooth muscle （VSM） from rabbits. Experiments were carried out on isolated thoracic aorta of rabbits. CY （3 x 103 mM- 3 mM） could relax the VSM preparations pre-contracted by adrenaline （AD）, noradrenaline （NE）, high-K^＋ solution or BaCl2 with respective EC50 values of （0.3 1±0.11） mM, 0.19±0.03 mM, 0.20±0.04 mM and 0.25±0.04 mM. Moreover, CY （10-2 mM, 0.1 mM and 1 mM） inhibited norepinephrine （NE）, CaCl2 and KCl-induced vasoconstriction in a concentration dependent manner. The phasic contraction produced by NE was concentration dependently attenuated with CY （10^-2 mM, 0.1 mM and 1 mM） in calcium-free medium, similar to that caused by verapamil. The present findings suggest that CY relaxed thoracic aortic rings by blocking voltage-dependent Ca^2＋ channels. The inhibition of intracellular Ca^2＋ release may be one of the main vasorelaxant mechanisms of CY.
基金the National Natural Science Foundation of China(No.81574058,81473760)Shanghai Key Research Program of Shanghai Municipal Commission of Health and Family Planning(No.ZY3-CCCX-3-3005)Shanghai Municipal Commission of Health and Family Planning research project(201640133)
文摘OBJECTIVE: To investigate the influence of serum from asthmatic rat treated with acupuncture(acupuncture serum) on the contractile responses of airway smooth muscle cells(ASMCs).METHODS: OVA-induced asthmatic rats were subject to acupuncture treatment at the points of Dazhui(GV 14), Fengmen(BL 12), and Feishu(BL 13).The resulting serum was collected, as well as serum from normal animals. Contraction of ASMCs was induced with 10 μM acetylcholine(Ach), and acupuncture serum or normal serum was supplemented 15 min later. The effects of the sera on the changes in cell length, width, and surface areawere monitored in real time via a live cell imaging system.RESULTS: The contraction rates of ASMCs 15 min and 30 min after Ach stimulation reached 38.2% ±3.8% and 49.7% ± 4.6%, both of which were significantly higher than unstimulated control cells(P <0.01). Acupuncture serum at the 1∶100 dosage reduced the contraction rate of ASMCs from 40.1% ±5.1% to 17.6% ± 6.5%(P < 0.01). Thus, the treatment significantly inhibited the Ach-induced contractile responses of ASMCs and yielded a relaxation efficiency of 58%, which was greater than the effects obtained with a 1∶250 dosage. Treatment with acupuncture serum at 1∶500 dosage or normal serum displayed no clear effect of suppressing Ach-induced contraction of ASMCs.CONCLUSION: Our data suggest that acupuncture serum has the effect of inhibiting Ach-induced contraction of ASMCs, thereby promoting relaxation of the cells.
基金Supported by the Chinese National Natural Science Foundation(30400596)The Jinan University Natural Science Foundation(51204017)The Science and Technology Innovation Project for Undergraduates of Jinan University(CX07080)
文摘The aim of this study was to establish a method of isolating and culturing smooth muscle cells from the ductus deferens of rats. Smooth muscle cells were prepared from ductus deferens by explanting technique after dissection of adventitia and intimae, and cultured in vitro. The identification of the smooth muscle cells were verified by using anti u-smooth muscle actin （a-SMA） immunohistochemistry studies. The result suggested that the cells are multi-morphous, showing long fusiform or star shapes. The apophysis of cells contacted and coalesced to each other, in some regions the cells overlapped in multilayer, while in the other regions they formed monolayer that fluctuated and showed a ＂peak-valley＂ shape. They presented a positive reaction through immunohistochemistry studies. The purity of the cells was more than 99% through this method. The culturing of smooth muscle cells by explanting technique is simple and stable.
文摘Objective: To investigate the effect of intravascular in radiation on thearterial wall smooth muscle cells (SMCs) proliferation and apoptosis after iliac artery bollominjury in figs. Methods: Twenty-seven miniature figs were divided into three groups. All pigsunderwent iliac artery balloon over-stretch. An^(192) Ir source through afterloader was positionedat the injuried segments to give 10 Gy in 9 pigs and 20 Gy in the other 9 pigs, and the rest 9 pigswere, used as control group. The pigs were killed on the 3rd, 10th and 28th days respectively forobservation. The injured segments were processed to examine SMCs proliferation by proliferation cellnuclear antigen (PCNA) and apopto-sis by terminal deoxynucleotidyl transferase-mediated dUTPnick-end labeling (TUNEL). Results: PC-NA index analysis has some that SMCs proliferation inneointima was significantly inhibited in irradiation group on the 10th and 28th days. The value forintimal SMCs apoptosis in control vs 10 Gy and 20 Gy irradiation groups were: (1. 185+-0. 49)% vs(2. 27+-0. 49)%(P>0. 05) and (1. 85+-0. 49)% vs (2. 53+-0. 45)%(P<0. 05), at the 10th day; (1.61+-0. 35)% vs (3. 11+-0. 51)%(P<0. 05), and (1.61+-0. 35)% vs (7. 05+-1. 82)% (P<0. 05), on the28th day. In irradiated arteries, the maximal incidence of intimal SMCs apoptosis was (7. 05+--1.82)% in 20 Gy group vs (3. 11+-0. 51)% in 10 Gy group (P<0. 05), on the 28th day. In the same doseirradiation group, the incidence of intimal SMCs apoptosis was higher on the 28th day than that onthe 10th day. Conclusion: Intra-arterial gamma irradiation can inhibit intimal SMCs proliferationand stimulate SMCs apoptosis in balloon-in jured arteries. These may be contributive to preventionof restenosis of arteries after balloon injury.
文摘The effects of sodium ferulate(SF), a water-soluble element of Chinese medicine Angelica sinensis diels, on cell-mediated oxidative modification of human low density lipoprotein(LDL) and proliferation of rabbit aortic smooth muscle cells(SMCs) were investigated. Using experimental models of proliferation of cultured rabbit aortic SMCs induced by oxidized LDL(ox-LDL), the extent of oxidation was determined by thiobarbituric acid reactive substances(TBARS) method, MTT colorimetry and 3H-thymidine(3H-TdR) incorporation were used to observe proliferation of SMCs. It showed that SF effectively inhibited cell-mediated oxidation induced by Cu2+ in a concentration-dependent manner. At the final concentration of 40, 80, 120 gmL-1, SF could significantly inhibit 3H-TdR incorporation and cell Proliferation in a dose-dependent manner. The results indicated that SF could, in vitro protect LDL against oxidative modification and inhibit the proliferation of SMC, which might be due to its free radical scavenging capacity.
基金National Natural Science Foundation of China(No.30170368)
文摘Aim This study was to evaluate the effect of arsenic trioxide （As2O3） on the transgenic TNF-α promoter activity in cultured vascular smooth muscle cells （VSMCs） and THP-1 monocytes. Methods Human TNF-α promoter was constructed by reporter gene system and was transiently transfected into VSMCs and THP-1 in vitro. The promoter activity was tested by luciferase activity with or without LPS and Ang Ⅱ stimulation, before and after different dosage of As2O3 treatment. Results 1. TNF-α promoter effectively expressed in VSMCs and THP-1 compared with CMV promoter （58.3% and 80.9%, respectively）. Both LPS and Ang Ⅱ significantly up-regulated TNF-α promoter activity （P〈0.05）. 2. As2O3 significantly inhibited, both intact and LPS/Ang Ⅱ stimulated promoter activity, in a dose dependent manner （P〈0.05）, and in both cell type. Conclusion These results manifested that, the inhibition effect of As2O3 on the activity of human TNF-α promoter indicated its potential inhibition on pro-inflammatory cytokine genes expression at transcriptional level and its potential anti-inflammatory property in the cardiovascular system.
文摘Objective: This study aims to investigate the effects of urocortin (Ucn) on the viability of endothelial cells (ECV304) and rat vascular muscle cells (VSMC). Methods: Rat aortic VSMC were isolated from the rats' thoracic aorta. We studied the effect of Ucn on the viability of ECV304 cells and VSMC by using a tetrazolium (MTT) assay.Results: Ucn (10 -7 mol/L) inhibited the viability of ECV304 cells and VSMC. Inhibition rates are 13% and 15%, respectively(P<0.05, compared with Control). This inhibition was not dependent on the affecting time and was not affected by the addition of ATP-sensitive potassium channel (KATP channel) blocker, glybenclamide (Gly, 10 mol/L). Conclusion: Ucn inhibits the viability of ECV304 and VSMC. Our results suggest that Ucn may be a new vasoactive agent and may have a beneficial effect in the process of vascular remodeling (VR).
文摘\ The effects of tetrandrine (Tet) on cytosolic free calcium ([Ca2+]i) in subcultured bovine aortic smooth muscle cells (SMC) were studied by Fura2 and ARCMMIC cation measurement system. Tet (1～100 μmol·L-1) had no effect on the resting [Ca2+]i, but had inhibitory effects on [Ca2+]i elevation induced by high K+, 5HT, ATP, Ang II and NE in the presence of extracellular Ca2+. High concentration of Tet also inhibited Pheinduced [Ca2+]i elevation in absence of extracellular Ca2+. Tet (1～100 μmol·L-1) inhibited KCl (60 mmol·L-1) induced [Ca2+]i elevation in dosedependent manner, the IC50 value was 9.2 (95% confidence limits: 5.7～14.9) mmol·L-1. The results suggested that Tet had blocking effects on both VOC and ROC in bovine aortic SMC. It appears that the mechanisms of blocking effect of Tet on ROC might be primarily due to its Ca2+ entry blocking effects.
基金supported by grants from National Institutes of Health (HL093429 and HL107526 to S.-Y.C.)
文摘Vascular smooth muscle cell （VSMC） differentiation and proliferation are two important physiological proc- esses during vascular development. The phenotypic alteration from differentiated to proliferative VSMC contrib- utes to the development of several major cardiovascular diseases including atherosclerosis, hypertension, resteno- sis after angioplasty or bypass, diabetic vascular complications, and transplantation arteriopathy. Since the VSMC phenotype in these pathological conditions resembles that of developing VSMC during embryonic development, understanding of the molecular mechanisms that control VSMC differentiation will provide fundamental insights into the pathological processes of these cardiovascular diseases. Although VSMC differentiation is usually ac- companied by an irreversible cell cycle exit, VSMC proliferation and differentiation occur concurrently during embryonic development. The molecular mechanisms simultaneously regulating these two processes, however, remain largely unknown. Our recent study demonstrates that cell division cycle 7, a key regulator of cell cycle, promotes both VSMC differentiation and proliferation through different mechanisms during the initial phase of VSMC differentiation. Conversely, Kriappel-like factor 4 appears to be a repressor for both VSMC differentia- tion and proliferation. This review attempts to highlight the novel role of cell division cycle 7 in TGF-β-induced VSMC differentiation and proliferation. The role of K141ppel-like factor 4 in suppressing these two processes will also be discussed.
基金Acknowledgment We are grateful to Dr Tamotsu Yoshimori for providing the GFP-LC3 plasmid and Dr H. Seimiya for providing the tankyrase 1 plasmid. This study was supported by the National Natural Science Foundation of China （No. 30772285） and Beijing Municipal Commission of Science Technology, China （No. Z080507030808011）.
文摘This study compared tankyrase 1 expression and autophagy quantity between erectile dysfunction （ED） and non-ED rats＇ corpus cavernosum smooth muscle cells （CSMCs）. This study aslo explored the effect and possible mechanism of tankyrase 1 on autophagy and cell proliferation in ageing ED rats＇ CSMCs. The intracavernous pres- sure and mean systemic arterial pressure were measured to investigate erectile function so that eight 24-month-old ED and eight 8-month-old male Wistar rats were choosed respectively. The rat CSMCs were isolated and cultured by enzyme digestion, in which tankyrase 1 expression and autophagy quantity were compared. Tankyrase 1 over-expression was induced with plasmid transfection by Lipofectamine^TM. The effect of tankyrase 1 overexpression on proliferation, autophagy and mTOR pathway in 24-month-old ED rats＇ CSMCs was measured by the cell growth curve in MTT assay, cell cycle analysis in flow cytometry （FCM）, key protein expression in Western blot, autophagy quantity in transmission electron microscopy, monodansylcadaverine staining and GFP-LC3 fluorescence. The primary CSMCs were confirmed by immunofluorescence, and the purity was 99.1% in FCM. Compared with that of 8-month-old rats, tankyrase 1 expression and autophagy quantity significantly decreased in 24-month-old ED rats＇ primary CSMCs （P 〈 0.01）. Tankyrase 1 overexpression significantly increased the growth rate （P 〈 0.05） and increased the S phase of cell cycle （P 〈 0.01）. The autophagosome quantity was remarkably increased （P 〈 0.01）, LC3-Ⅰ/Ⅱ and Beclin 1 were upregulated （P 〈 0.01 and P 〈 0.05）, and p-p70S6K （Thr389） was downregulated in 24-month-old ED rat CSMCs （P 〈 0.05）. In conclusion, Tankyrase 1 and autophagy decrease in the CSMCs from aging rats with ED, and tankyrase 1 may have a positive effect on proliferation by enhancing autophagy and regulating the mTOR signalling pathway.
基金We are grateful to Dr Guan KL （Moore＇s Cancer Center, La Jolla, CA, USA） for the gift of pCMV-MEKca. This study was supported by the National Natural Science Foundation of China （30770787 and 90919035）, the National Basic Research Program of China （2005CB523301）, and the International Cooperation in Science and Technology Projects （2006DFB32460） and the Hebei Province Natural Science Foundation （C2007000831）.
文摘The increased proliferation and migration of vascular smooth muscle cells （VSMCs） are key events in the development of atherosclerotic lesions. Baicalin, an herb-derived flavonoid compound, has been previously shown to induce apoptosis and growth inhibition in cancer cells through multiple pathways. However, the potential role of baicalin in regulation of VSMC proliferation and prevention of cardiovascular diseases remains unexplored. In this study, we show that pretreatment with baicalin has a dose-dependent inhibitory effect on PDGF-BB-stimulated VSMC pro- liferation, accompanied with the reduction of proliferating cell nuclear antigen （PCNA） expression. We also show that baicalin-induced growth inhibition is associated with a decrease in cyclin E-CDK2 activation and increase in p27 level in PDGF-stimulated VSMCs, which appears to be at least partly mediated by blockade of PDGF recep- tor [~ （PDGFR~）-extracellular signal-regulated kinase 1/2 （ERK1/2） signaling. In addition, baicalin was also found to inhibit adhesion molecule expression and cell migration induced by PDGF-BB in VSMCs. Furthermore, using an animal carotid arterial balloon-injury model, we found that baicalin significantly inhibited neointimal hyperplasia. Taken together, our results reveal a novel function of baicalin in inducing growth arrest of PDGF-stimulated VSMCs and suppressing neointimal hyperplasia after balloon injury, and suggest that the underlying mechanism involves the inhibition of cyclin E-CDK2 activation and the increase in p27 accumulation via blockade of the PDGFR^-ERK1/2 signaling cascade.
