The aim of the present study was to investigate the effect of lipoxin A4 （LXA4） pretreatment on cognitive function of aged rats after global cerebral ischemia reperfusion, and to explore its possible mechanism. Thir...The aim of the present study was to investigate the effect of lipoxin A4 （LXA4） pretreatment on cognitive function of aged rats after global cerebral ischemia reperfusion, and to explore its possible mechanism. Thirty-six aged male Sprague-Dawley rats were randomly divided into three groups （n=12 each）： sham-operation group （S group）, global cerebral ischemia reperfusion group （I/R group） and LXA4-pretreatment group （L group）. The rat model of global cerebral ischemia reperfusion was established by occlusion of the bilateral common carotid artery with hypotension. The cognitive function of rats was determined by a step-down type passive avoidance test and Morris Water Maze test on the third day after reperfusion. Rats were sacrificed after Water Maze test and the pathological changes ofhippocampal CA1 region were observed and the related inflammatory mediators were determined. As compared with S group, the escape latency in I/R group was prolonged from the first day to the fifth day, while that in L group was prolonged from the first day to the third day. The retention time in I/R group and L group in the first quadrant was shortened. The reaction time, frequency of reaction mistake and frequency of escape mistake in I/R group increased, and the latent period shortened. The frequency of escape mistake in L group increased, and the damage in the hippocampal CAI region of I/R group and L group was obvious. The levels of S-10013, TNF-α, IL-1β, IL-10 and NF-κB in I/R group and L group increased. As compared with I/R group, the escape latency in L group was shortened from the first day to the fifth day, and the retention time in the first quadrant prolonged. The reaction time, frequency of reaction mistake and frequency of escape mistake in L group decreased, and the latent period prolonged. The damage in the hippocampal CA1 region of L group was alleviated as well. The levels of S-10013, TNF-α, IL-1β and NF-κB in L group decreased, and those of IL-10 increased. It can be concluded that LXA4 pretreatment can improve the cognitive function in aged rats after global cerebral ischemia reperfusion probably by inhibiting the inflammatory reaction.展开更多
This study examined in vitro effect of lipoxin A 4 (LXA 4) on interleukin-1β (IL-1β) production of monocytes and its possible mechanism in severe preeclampsia (PE).Peripheral venous blood was drawn from 15 patients ...This study examined in vitro effect of lipoxin A 4 (LXA 4) on interleukin-1β (IL-1β) production of monocytes and its possible mechanism in severe preeclampsia (PE).Peripheral venous blood was drawn from 15 patients with severe preeclampsia (PE group) and 20 normal pregnant women (control group) to prepare monocytes which were then treated with LXA 4 at different concentrations of 0,10,100 nmol/L respectively.IL-1β level in the supernatant of monocytes was detected by enzyme linked immunoassay.The [Ca 2+ ] i of monocytes was measured by laser scanning confocal microscopy.The results showed that the IL-1β level and the [Ca 2+ ] i of monocytes in the PE group were significantly higher than those in the control group.LXA 4 significantly decreased the generation of IL-1β in a dose-dependent manner in the PE group.After treatment with 100-nmol/L LXA 4,in the PE group,the [Ca 2+ ] i concentration of monocytes was significantly reduced.It was concluded that LXA 4 may inhibit the IL-1β production of monocytes from severe preeclampsia women by inhibiting extracellular calcium influx.展开更多
Objective: To examine whether lipoxin A 4 (LXA 4) has an antagonistic effect on IL-1β-induced synthesis of IL-6 in glomerular mesangial cells, and to explore the molecular mechanisms of signal pathway in LXA 4 ...Objective: To examine whether lipoxin A 4 (LXA 4) has an antagonistic effect on IL-1β-induced synthesis of IL-6 in glomerular mesangial cells, and to explore the molecular mechanisms of signal pathway in LXA 4 actions. Methods: The glomerular mesangial cells of rat were cultured and treated with IL-1β, with or without preincubation with LXA 4 at different concentrations. The amount of IL-6 in the supernatant of cells was analyzed by enzyme-linked immunosorbent assay(ELISA). The expressions of mRNA of IL-6 were determined by RT-PCR. The expressions of Src homology 2(SH 2) containing protein-tyrosine phosphatase 2(Shp-2) were assessed by immunoprecipitation and immunoblotting. Activities of DNA-binding of nuclear factor-kappa B(NF-κB) were measured by electrophoretic mobility shift assay(EMSA). Results: IL-1β-stimulated secretion of protein and expression of mRNA of IL-6 in mesangial cells were inhibited by LXA 4 in a dose-dependent manner. LXA 4 antagonizes the phosphorylation of Shp-2 and activities of NF-κB induced by IL-1β. Conclusion: LXA 4 antagonists IL-1β-induced synthesis of IL-6 in glomerular mesangial cells through the mechanism of Shp-2/NF-κB pathway-dependent signal transduction.展开更多
AIM:Excessive dissolve of corneal tissue induced by MMPs which were activated by cytokins and chemokines will lead to corneal ulcer. The molecular mechanism of Lipoxin A4 (LXA4) on corneal collagen degradation in thre...AIM:Excessive dissolve of corneal tissue induced by MMPs which were activated by cytokins and chemokines will lead to corneal ulcer. The molecular mechanism of Lipoxin A4 (LXA4) on corneal collagen degradation in three dimensions was investigated. ·METHODS:Rabbit corneal fibroblasts were harvested and suspended in serum -free MEM. Type I collagen, DMEM, collagen reconstitution buffer and corneal fibroblast suspension were mixed on ice. The resultant mixture solidified in an incubator, after which test reagents and plasminogen was overlaid and the cultures were returned to the incubator. The supernatants from collagen gel incubations were collected and the amount of hydroxyproline in the hydrolysate was measured. Immunoblot analysis of MMP-1,-3 and TMMP-1,-2 was performed. MMP-2, -9 was detected by the method of Gelatin zymography. Cytotoxicity assay was measured. RESULTS:LXA4 inhibited corneal collagen degradation in a dose and time manner. LXA4 inhibited the IL -1β induced increases in the pro-MMP-1, -2, -3, -9 and active MMP -1,-2,-3,-9 in a concentration dependent manner. LXA4 also inhibited the IL-1β induced increases in TIMP-1, -2. CONCLUSION:As a potent anti-inflammation reagent, LXA4 can inhibit corneal collagen degradation induced by IL-1β in corneal fibroblasts thus inhibiting corneal dissolving pathology process.展开更多
Objective: To examine whether lipoxin A 4 (LXA 4) induces apoptosis of renal interstitial fibroblasts and explore the mechanisms of signal pathway of LXA 4. Methods: Rat renal inte rstitial fibroblasts (NRK-49F ...Objective: To examine whether lipoxin A 4 (LXA 4) induces apoptosis of renal interstitial fibroblasts and explore the mechanisms of signal pathway of LXA 4. Methods: Rat renal inte rstitial fibroblasts (NRK-49F cells) were exposed to LXA 4 at different concen trations. Prior to the experiment, the cells were transfected with Smac or calpa in 10 antisense oligodeoxynucleotide (ODN), or treated with calcium channel inhi bitor SK&F96365. Apoptosis of cells was recognized by double staining using acri dine orange and ethidium bromide, observed in laser scanning confocal microscope , and counted by a flow cytometer. Caspase-3 activities were measured by colori metric assay. The levels of free cytosolic calcium ( i) were anal yzed in fura-2-loaded cells by laser scanning confocal microscopy. Expression of calpain 10 mRNA was determined by RT-PCR. Expres sions of Smac protein and threonine phosphorylated Akt 1 proteins at 308 site w ere determined by a Western blotting analysis. Activity of signal transducers an d activators of transcription-3 (STAT 3) was determined by electrophoretic mob ility shift assay. Results: LXA 4 at the concentrations of 0.1 and 1 μmol/L induced 9.83% and 33.82% apoptosis of NRK-49F cel ls respectively, reduced at S and G 2-M phase and increased the cells at G 0 -G 1 phase in a dose-dependent manner. Treatment of the cells with LXA 4 inc reased the expressions of calpain 10 and Smac, the levels of i a nd activity of caspase-3. It also down-regulated the DNA-binding activity of STAT 3 and expression of threonine phosphorylated Akt 1. Transfection of the c ells with calpain 10 antisense ODN inhibited the LXA 4-induced apoptosis, acti vity of caspase-3 and expression of calpain 10, and ameliorated the decreased a ctivity of STAT 3. Transfection of the cells with Smac antisense ODN inhibited the LXA 4-induced apoptosis, activity of caspase-3 and expression of Smac. Pr etreatment of the cells with SK & F96365 inhibited the LXA 4-induced apoptosis , levels of i, expression of calpain 10 and Smac. Conclu sion: LXA 4 at high concentration induced apoptosis of rat renal inters titial fibroblasts via i-dependent up-regulation of calpain 10 and Smac expressions.展开更多
In order to investigate the regulatory role of connective tissue growth factor （CTGF） on production of RANTES （regulated on activation, normal T cell expressed and secreted） in rat glomerular mesangial cells, and ...In order to investigate the regulatory role of connective tissue growth factor （CTGF） on production of RANTES （regulated on activation, normal T cell expressed and secreted） in rat glomerular mesangial cells, and the modulatory effect of lipoxin A4 （ LXA4 ） on action of CTGF, and to explore the mechanisms of action of CTGF and LXA4, cultured rat mesangial cells were treated with CTGF, with or without preincubation with LXA4. Expression of mRNA was analyzed by RT-PCR. Protein of RANTES in the supematants was determined by ELISA. Monocyte transmigration was assessed by in vitro chemotaxis assay. Expression of p42/44 mitogen-activated protein kinase （MAPK）, phosphoinositide 3-kinase （ PI3- K） and protein kinase B （PKB） was assessed by Western blotting. DNA-binding activity of nuclear factor-roB （NF-kB） was determined by electrophoretic mobility shift assay （EMSA）. To observe whether transfection of LXA4 receptor homologue gene （LRHg） into mesangial cells intensified these modulatory effects of LXA4, mesangial cells were transfected with pcDNA3.1/LRHG vector. The results showed that CTGF enhanced the mRNA expression and protein release of RANTES, and the expression of phospho （P）-p42/44 MAPK, P-PI3-K, P-PKB and NF-kB. P-p42/44 MAPK blockade inhibited the CTgF-induced expression of P-p42/44 MAPK and partially decreased the level of RANTES in supematants. P- PI3-K blockade downregulated the CTGF-stimulated expression of P-PI3-K, P-PKB and NF-kB, and partially decreased the release of RANTES. NF-kB blockade abrogated the CTGF-activated NF-kB and partially decreased the secretion of RANTES. LXA4 dose-dependently inhibited the CTGF-stimulated above action. Transfection of LRHG into mesangial cells intensified these inhibitory effects of LXA4 on CTGFinduced release of RANTES and expression of the P-p42/44 MAPK. In conclusion, LXA4 inhibits CTGFinduced production of RANTES via PI3-K/PKB/NF-kB and p42/44 MAPK-dependent signal pathway, which is mediated by LRHG in rat mesangial cells.展开更多
Objective： To examine whether lipoxin A4 （LXA4） has an inhibitory effect on tumor necrosis factor-α（TNF-α-induced DNA synthesis of glomerular mesangial cells of rat, and explore the molecular mechanisms of LXA4 ...Objective： To examine whether lipoxin A4 （LXA4） has an inhibitory effect on tumor necrosis factor-α（TNF-α-induced DNA synthesis of glomerular mesangial cells of rat, and explore the molecular mechanisms of LXA4 action. Methods： Glomerular mesangial cells of rat were cultured and preincubated with LXA4 at different concentrations, and then treated with TNF-α（ 10 ng/ml）. DNA synthesis was assessed by the incorporation of [^3H]-thymidine in mesangial cells. Expression of cyclin E protein was determined by Western blotting analysis. Activities of signal transducers and activators of transcription-3 （STAT3） were analyzed by electrophoretic mobility shift assay （EMSA）. Results： TNF-α-stimulated DNA synthesis of mesangial cells, upregulafion of cyclin E protein and STAT3 activities were inhibited by LXA4 in a dose-dependent manner. Conclusion： TNF-α-induced DNA synthesis of mesangial cells can be inhibited by TXA4 probably through the mechanism of Jak1/STAT3 pathway-dependent signal transduction.展开更多
Objective:To investigate the effect of lipoxin receptor agonist BML-111 on the NLRP3 inflammasome after traumatic brain injury in rats.Methods:Sixty male Sprague-Dawley rats,weighing 280~340 g,were randomly divided in...Objective:To investigate the effect of lipoxin receptor agonist BML-111 on the NLRP3 inflammasome after traumatic brain injury in rats.Methods:Sixty male Sprague-Dawley rats,weighing 280~340 g,were randomly divided into 4 groups(n=15):the sham operation group(group Sham),the traumatic brain injury group(group TBI),the BML-111 treatment group(group BML-111),and the BOC-2 treatment group(group BOC-2).The TBI model was prepared by craniocerebral collision,while the rats in group Sham underwent only craniotomy without collision.Acute traumatic brain injury model was prepared in group TBI,BML-111 and BOC-2.The rats in group BOC-2 were intraperitoneally injected with 50μg/kg of BOC-230 min prior to trauma.Then the rats in group BOC-2 and BML-111 were injected intraperitoneally with 1 mg/kg of BML-111 immediately and 24 hours after trauma.The neurological severity scores(NSS)were evaluated at 3 and 7 days after brain trauma.The protein expression levels of NLRP3,Caspase-1-p20 and active Caspase-3 were determined by Western blot.The content of IL-1βand IL-18 was detected by ELISA assays.The apoptotic cells were analyzed by the TUNEL method.Results:Compared with group Sham,the brain water content and NSS scores in group TBI were increased,and the protein levels of NLRP3,Caspase-1-p20,activated Caspase-3,IL-1βand IL-18 as well as TUNEL-positive cells in the cortex were elevated significantly(P<0.05);compared with group TBI,the brain water content and NSS scores in group BML-111 were reduced,and the protein levels of NLRP3,Caspase-1-p20,activated Caspase-3,IL-1βand IL-18 as well as TUNEL-positive cells in the cortex were decreased(P<0.05);Compared with group BML-111,the brain water content and NSS scores in group BOC-2 were increased,and the protein levels of NLRP3,Caspase-1-p20,activated Caspase-3,IL-1βand IL-18 as well as TUNEL-positive cells in the cortex were up-regulated(P<0.05).Conclusions:The lipoxin receptor agonist BML-111 might attenuate traumatic brain injury in rats by inhibiting NLRP3 inflammasome activation.展开更多
In order to investigate whether lipoxin A4 （LXA4） has an antagonistic effect on lipopolysaccharide （LPS）-induced synthesis of interleukin （IL）-β3, IL-6 and IL-8 in rat pulmonary microvascular endothelial cells ...In order to investigate whether lipoxin A4 （LXA4） has an antagonistic effect on lipopolysaccharide （LPS）-induced synthesis of interleukin （IL）-β3, IL-6 and IL-8 in rat pulmonary microvascular endothelial cells （PMVEC）, and to explore the molecular mechanisms of signal pathway in LXA4 actions, cultured PMVEC were treated with LPS, with or without preincubation with LXA4. Proteins of IL-β3, IL-6 and IL-8 in supernatant were analyzed by enzyme-linked immunosorbent assay （ELISA）. Expressions of mRNA of IL-β3, IL-6 and IL-8 were determined by RT-PCR. Expressions of phosphorylation of phosphoinositide 3-kinase （PI3-K） and myeloid differentiation factor 88 （MyD88） were analyzed by Western blot. Activities of DNA-binding of nuclear factor-kappa B （NF-κB） and activator protein-1 （AP-1） were measured by electrophoretic mobility shift assay （EMSA）. The results showed that LPS induced production of IL-β3, IL-6 and IL-8 in rat PMVEC via MyD88/PI3-K/NF-κB and AP-1 pathway-dependent signal transduction. LPS-stimulated expression of PI3-K, activities of NF-κB and AP-1, secretion of protein and expression of mRNA of IL-β3, IL-6 and IL-8 but not MyD88 expression in PMVEC were inhibited by LXA4 in a dose-dependent manner. In conclusion, LXA4 inhibits synthesis of IL-β3, IL-6 and IL-8 by down-regulation of PI3-K/NF-κB and AP-1 signal pathway in PMVEC.展开更多
Background Lipoxins （LXs）, endogenous anti-inflammatory and pro-resolving eicosanoids generated during various inflammatory conditions, have novel immunomodulatory properties. Because dendritic cells （DCs） play cr...Background Lipoxins （LXs）, endogenous anti-inflammatory and pro-resolving eicosanoids generated during various inflammatory conditions, have novel immunomodulatory properties. Because dendritic cells （DCs） play crucial roles in the initiation and maintenance of immune response, we determined whether LXs could modulate the maturation process of DCs and investigated the effects of lipoxin A4 （LXA4） on lipopolysaccharide （LPS）-induced differentiation of RAW264.7 cells into dendritic-like cells. Methods RAW264.7 cells were cultured in vitro with 1 pg/ml LPS in the absence or presence of LXA4 for 24 hours, and cellular surface markers （MHC-II, CD80 （B7-1）, CD86（B7-2）） were measured by flow cytometry （FCM）. Mixed lymphocyte reaction was performed to evaluate the allostimulatory activity. Cytoplastic IKB degradation and nuclear factor kappa B （NF-KB） translocation were detected by Western blotting. Luciferase reporter plasmid was transiently transfected into RAW264.7 cells, and luciferase activity was determined to measure the transcriptional activity of NF-KB. Results LXA4 reduced the ratio of LPS-treated RAW264.7 cells to DCs with morphological characteristics and inhibited the expression of MHC I1. LPS-induced up-regulation of CD86 was moderately suppressed by LXA4 but no obvious change of CD80 was observed. Moreover, LXA4 weakened the aUostimulatory activity of LPS-treated RAW264.7 cells. These alterations of LPS＋LXA4-treated cells were associated with a marked inhibition of IKB degradation, NF-KB translocation and then the transcriptional activity of NF-KB. Conclusions LXA4 negatively regulates LPS-induced differentiation of RAW264.7 cells into dendritic-like cells.This activity reveals an undescribed mechanism of LXA4 to prevent excessive and sustained immune reaction by regulating maturation of DCs.展开更多
Background： Lipoxin A4 （LXA4） can alleviate lipopolysaccharide （LPS）-induced acute lung injury （ALl） and acute respiratory distress syndrome through promoting epithelial sodium channel （ENaC） expression in lu...Background： Lipoxin A4 （LXA4） can alleviate lipopolysaccharide （LPS）-induced acute lung injury （ALl） and acute respiratory distress syndrome through promoting epithelial sodium channel （ENaC） expression in lung epithelial cells. However, how LXA4 promote ENaC expression is still largely elusive. The present study aimed to explore genes and signaling pathway involved in regulating ENaC expression induced by LXA4. Methods： A549 cells were incubated with LPS and LXA4, or in combination, and analyzed by quantitative real-time polymerase chain reaction （qRT-PCR） of ENaC-α/γ. Candidate genes affected by LXA4 were explored by transcriptome sequencing ofA549 cells. The critical candidate gene was validated by qRT-PCR and Western blot analysis ofA549 cells treated with LPS and LXA4 at different concentrations and time intervals. LXA4 receptor （ALX） inhibitor BOC-2 was used to test induction of candidate gene by LXA4. Candidate gene siRNA was adopted to analyze its influence on A549 viability and ENaC-α expression. Phosphoinositide 3-kinase （PI3K） inhibitor LY294002 was utilized to probe whether the PI3K signaling pathway was involved in LXA4 induction of candidate gene expression. Results： The A549 cell models of ALl were constrticted and subjected to transcriptome sequencing. Among candidate genes, N-myc downstream- regulated gent- 1 （NDRG 1 ） was validated by real-time-PCR and Western blot. NDRG 1 mRNA was elevated in a dose-dependent manner of LXA4, whereas BOC-2 antagonized NDRG 1 expression induced by LXA4. NDRG I siRNA suppressed viability of LPS-treated A549 cells （treatment vs. control, 0.605± 0.063 vs. 0.878 ± 0.083, P = 0.040） and ENaC-α expression （treatment vs. control, 0.458 ± 0.038 vs. 0.711 ± 0.035, P = 0.008）. LY294002 inhibited NDRG 1 （treatment vs. control, 0.459 ± 0.023 vs. 0.726 ± 0.020, P 0.001 ） and ENaC-α （treatment vs. control, 0.236 ± 0.021 vs. 0.814 ±0.025, P 〈 0.001 ） expressions and serum- and glucocorticoid-inducible kinase I phosphorylation （treatment vs. control, 0.442± 0.024 vs. 1.046 ± 0.082, P = 0.002）, indicating the PI3K signaling pathway was involved in regulating NDRG 1 expression induced by LXA4. Conclusion： Our research uncovered a critical role of NDRG1 in LXA4 alleviation of LPS-induced A549 cell injury through mediating PI3K signaling to restore ENaC expression.展开更多
该文旨在探讨脂氧素受体激动剂BML-111对H22细胞的影响及机制。利用小鼠肝癌细胞株H22,构建肿瘤相关体内外模型,并给予脂氧素受体激动剂BML-111和脂氧素受体阻断剂BOC-2分别作为治疗组和阻断剂组。体内实验,测定小鼠皮下肿瘤质量、体积...该文旨在探讨脂氧素受体激动剂BML-111对H22细胞的影响及机制。利用小鼠肝癌细胞株H22,构建肿瘤相关体内外模型,并给予脂氧素受体激动剂BML-111和脂氧素受体阻断剂BOC-2分别作为治疗组和阻断剂组。体内实验,测定小鼠皮下肿瘤质量、体积。体外实验,CCK8法检测细胞活力,试剂盒检测细胞乳酸含量、葡萄糖含量、ATP含量、谷氨酰胺代谢情况,Western blot检测磷酸酶张力蛋白同源物基因(phosphatase and tensin homolog deleted on chromosome ten,PTEN)的蛋白含量,qRT-PCR法检测PTEN mRNA表达情况。治疗组较CON组和阻断剂组皮下肿瘤质量和体积均有所减小。与CON组相比,BML-111可以抑制肝癌细胞活力,可明显降低H22细胞葡萄糖含量、乳酸含量以及谷氨酰胺含量(P<0.05)。同时BML-111可以提高PTEN的表达水平,而脂氧素受体阻滞剂BOC-2可以逆转所有这些现象。BML-111通过降低肝癌细胞糖酵解及谷氨酰胺代谢水平,抑制肝癌细胞的增殖,降低肝癌细胞的能量代谢水平,其作用机制可能与调控PTEN的表达相关。展开更多
Objective:To examine whether lipoxin A 4(LXA 4) has an inhibitory effect on tumor necrosis factor-α(TNF-α)-induced proliferation of glomerular mesangial cells of rat, and explore the molecular mechanisms of signal...Objective:To examine whether lipoxin A 4(LXA 4) has an inhibitory effect on tumor necrosis factor-α(TNF-α)-induced proliferation of glomerular mesangial cells of rat, and explore the molecular mechanisms of signal pathway in LXA 4 actions. Methods: Glomerular mesangial cells of rat were cultured and treated with TNF-α(10 ng/ml), with or without preincubation with LXA 4 at different concentrations. Cell proliferation was evaluated by monotetrazolium (MTT) colorimetric assay. The expression of cyclin E mRNA was measured by RT-PCR. Phosphorylated Akt1(Thr308) and p27 kip1 were analyzed by Western blotting. Results: TNF-α-stimulated proliferation of mesangial cells was inhibited by LXA 4 in a dose-dependent manner. The marked increments in cyclin E mRNA expression induced by TNF-α during proliferation of mesangial cells were down-regulated by LXA 4. Threonine phosphorylated Akt1 proteins at 308 site stimulated by TNF-α was reduced by LXA 4. TNF-α-induced decrements in expression of p27 kip1 proteins was ameliorated by LXA 4 in a dose-dependent manner. Conclusion: TNF-α-induced proliferation of rat mesangial cells can be inhibited by TXA 4 through the mechanism of Akt 1/p27 kip1 pathway-dependent signal transduction.展开更多
基金This study was supported by grants from the Innovation Foundation of Health and Family Planning Commission of Hubei Province （No. WJ2017M036） and the National Natural Science Foundation of China （No. 81471858）.
文摘The aim of the present study was to investigate the effect of lipoxin A4 （LXA4） pretreatment on cognitive function of aged rats after global cerebral ischemia reperfusion, and to explore its possible mechanism. Thirty-six aged male Sprague-Dawley rats were randomly divided into three groups （n=12 each）： sham-operation group （S group）, global cerebral ischemia reperfusion group （I/R group） and LXA4-pretreatment group （L group）. The rat model of global cerebral ischemia reperfusion was established by occlusion of the bilateral common carotid artery with hypotension. The cognitive function of rats was determined by a step-down type passive avoidance test and Morris Water Maze test on the third day after reperfusion. Rats were sacrificed after Water Maze test and the pathological changes ofhippocampal CA1 region were observed and the related inflammatory mediators were determined. As compared with S group, the escape latency in I/R group was prolonged from the first day to the fifth day, while that in L group was prolonged from the first day to the third day. The retention time in I/R group and L group in the first quadrant was shortened. The reaction time, frequency of reaction mistake and frequency of escape mistake in I/R group increased, and the latent period shortened. The frequency of escape mistake in L group increased, and the damage in the hippocampal CAI region of I/R group and L group was obvious. The levels of S-10013, TNF-α, IL-1β, IL-10 and NF-κB in I/R group and L group increased. As compared with I/R group, the escape latency in L group was shortened from the first day to the fifth day, and the retention time in the first quadrant prolonged. The reaction time, frequency of reaction mistake and frequency of escape mistake in L group decreased, and the latent period prolonged. The damage in the hippocampal CA1 region of L group was alleviated as well. The levels of S-10013, TNF-α, IL-1β and NF-κB in L group decreased, and those of IL-10 increased. It can be concluded that LXA4 pretreatment can improve the cognitive function in aged rats after global cerebral ischemia reperfusion probably by inhibiting the inflammatory reaction.
文摘This study examined in vitro effect of lipoxin A 4 (LXA 4) on interleukin-1β (IL-1β) production of monocytes and its possible mechanism in severe preeclampsia (PE).Peripheral venous blood was drawn from 15 patients with severe preeclampsia (PE group) and 20 normal pregnant women (control group) to prepare monocytes which were then treated with LXA 4 at different concentrations of 0,10,100 nmol/L respectively.IL-1β level in the supernatant of monocytes was detected by enzyme linked immunoassay.The [Ca 2+ ] i of monocytes was measured by laser scanning confocal microscopy.The results showed that the IL-1β level and the [Ca 2+ ] i of monocytes in the PE group were significantly higher than those in the control group.LXA 4 significantly decreased the generation of IL-1β in a dose-dependent manner in the PE group.After treatment with 100-nmol/L LXA 4,in the PE group,the [Ca 2+ ] i concentration of monocytes was significantly reduced.It was concluded that LXA 4 may inhibit the IL-1β production of monocytes from severe preeclampsia women by inhibiting extracellular calcium influx.
文摘Objective: To examine whether lipoxin A 4 (LXA 4) has an antagonistic effect on IL-1β-induced synthesis of IL-6 in glomerular mesangial cells, and to explore the molecular mechanisms of signal pathway in LXA 4 actions. Methods: The glomerular mesangial cells of rat were cultured and treated with IL-1β, with or without preincubation with LXA 4 at different concentrations. The amount of IL-6 in the supernatant of cells was analyzed by enzyme-linked immunosorbent assay(ELISA). The expressions of mRNA of IL-6 were determined by RT-PCR. The expressions of Src homology 2(SH 2) containing protein-tyrosine phosphatase 2(Shp-2) were assessed by immunoprecipitation and immunoblotting. Activities of DNA-binding of nuclear factor-kappa B(NF-κB) were measured by electrophoretic mobility shift assay(EMSA). Results: IL-1β-stimulated secretion of protein and expression of mRNA of IL-6 in mesangial cells were inhibited by LXA 4 in a dose-dependent manner. LXA 4 antagonizes the phosphorylation of Shp-2 and activities of NF-κB induced by IL-1β. Conclusion: LXA 4 antagonists IL-1β-induced synthesis of IL-6 in glomerular mesangial cells through the mechanism of Shp-2/NF-κB pathway-dependent signal transduction.
基金Jilin University Basic Scientific Research Operating Expenses Fund, China (Research Fund of the Bethune B Plan of Jilin University, 2012 No.2012230)Research Fund of Jilin Provincial Science and Technology Department, China (international cooperation item, No.20120726)
文摘AIM:Excessive dissolve of corneal tissue induced by MMPs which were activated by cytokins and chemokines will lead to corneal ulcer. The molecular mechanism of Lipoxin A4 (LXA4) on corneal collagen degradation in three dimensions was investigated. ·METHODS:Rabbit corneal fibroblasts were harvested and suspended in serum -free MEM. Type I collagen, DMEM, collagen reconstitution buffer and corneal fibroblast suspension were mixed on ice. The resultant mixture solidified in an incubator, after which test reagents and plasminogen was overlaid and the cultures were returned to the incubator. The supernatants from collagen gel incubations were collected and the amount of hydroxyproline in the hydrolysate was measured. Immunoblot analysis of MMP-1,-3 and TMMP-1,-2 was performed. MMP-2, -9 was detected by the method of Gelatin zymography. Cytotoxicity assay was measured. RESULTS:LXA4 inhibited corneal collagen degradation in a dose and time manner. LXA4 inhibited the IL -1β induced increases in the pro-MMP-1, -2, -3, -9 and active MMP -1,-2,-3,-9 in a concentration dependent manner. LXA4 also inhibited the IL-1β induced increases in TIMP-1, -2. CONCLUSION:As a potent anti-inflammation reagent, LXA4 can inhibit corneal collagen degradation induced by IL-1β in corneal fibroblasts thus inhibiting corneal dissolving pathology process.
基金135 Medical Emphasis grant from Government of Jiangsu Province (2002 45)
文摘Objective: To examine whether lipoxin A 4 (LXA 4) induces apoptosis of renal interstitial fibroblasts and explore the mechanisms of signal pathway of LXA 4. Methods: Rat renal inte rstitial fibroblasts (NRK-49F cells) were exposed to LXA 4 at different concen trations. Prior to the experiment, the cells were transfected with Smac or calpa in 10 antisense oligodeoxynucleotide (ODN), or treated with calcium channel inhi bitor SK&F96365. Apoptosis of cells was recognized by double staining using acri dine orange and ethidium bromide, observed in laser scanning confocal microscope , and counted by a flow cytometer. Caspase-3 activities were measured by colori metric assay. The levels of free cytosolic calcium ( i) were anal yzed in fura-2-loaded cells by laser scanning confocal microscopy. Expression of calpain 10 mRNA was determined by RT-PCR. Expres sions of Smac protein and threonine phosphorylated Akt 1 proteins at 308 site w ere determined by a Western blotting analysis. Activity of signal transducers an d activators of transcription-3 (STAT 3) was determined by electrophoretic mob ility shift assay. Results: LXA 4 at the concentrations of 0.1 and 1 μmol/L induced 9.83% and 33.82% apoptosis of NRK-49F cel ls respectively, reduced at S and G 2-M phase and increased the cells at G 0 -G 1 phase in a dose-dependent manner. Treatment of the cells with LXA 4 inc reased the expressions of calpain 10 and Smac, the levels of i a nd activity of caspase-3. It also down-regulated the DNA-binding activity of STAT 3 and expression of threonine phosphorylated Akt 1. Transfection of the c ells with calpain 10 antisense ODN inhibited the LXA 4-induced apoptosis, acti vity of caspase-3 and expression of calpain 10, and ameliorated the decreased a ctivity of STAT 3. Transfection of the cells with Smac antisense ODN inhibited the LXA 4-induced apoptosis, activity of caspase-3 and expression of Smac. Pr etreatment of the cells with SK & F96365 inhibited the LXA 4-induced apoptosis , levels of i, expression of calpain 10 and Smac. Conclu sion: LXA 4 at high concentration induced apoptosis of rat renal inters titial fibroblasts via i-dependent up-regulation of calpain 10 and Smac expressions.
文摘In order to investigate the regulatory role of connective tissue growth factor （CTGF） on production of RANTES （regulated on activation, normal T cell expressed and secreted） in rat glomerular mesangial cells, and the modulatory effect of lipoxin A4 （ LXA4 ） on action of CTGF, and to explore the mechanisms of action of CTGF and LXA4, cultured rat mesangial cells were treated with CTGF, with or without preincubation with LXA4. Expression of mRNA was analyzed by RT-PCR. Protein of RANTES in the supematants was determined by ELISA. Monocyte transmigration was assessed by in vitro chemotaxis assay. Expression of p42/44 mitogen-activated protein kinase （MAPK）, phosphoinositide 3-kinase （ PI3- K） and protein kinase B （PKB） was assessed by Western blotting. DNA-binding activity of nuclear factor-roB （NF-kB） was determined by electrophoretic mobility shift assay （EMSA）. To observe whether transfection of LXA4 receptor homologue gene （LRHg） into mesangial cells intensified these modulatory effects of LXA4, mesangial cells were transfected with pcDNA3.1/LRHG vector. The results showed that CTGF enhanced the mRNA expression and protein release of RANTES, and the expression of phospho （P）-p42/44 MAPK, P-PI3-K, P-PKB and NF-kB. P-p42/44 MAPK blockade inhibited the CTgF-induced expression of P-p42/44 MAPK and partially decreased the level of RANTES in supematants. P- PI3-K blockade downregulated the CTGF-stimulated expression of P-PI3-K, P-PKB and NF-kB, and partially decreased the release of RANTES. NF-kB blockade abrogated the CTGF-activated NF-kB and partially decreased the secretion of RANTES. LXA4 dose-dependently inhibited the CTGF-stimulated above action. Transfection of LRHG into mesangial cells intensified these inhibitory effects of LXA4 on CTGFinduced release of RANTES and expression of the P-p42/44 MAPK. In conclusion, LXA4 inhibits CTGFinduced production of RANTES via PI3-K/PKB/NF-kB and p42/44 MAPK-dependent signal pathway, which is mediated by LRHG in rat mesangial cells.
文摘Objective： To examine whether lipoxin A4 （LXA4） has an inhibitory effect on tumor necrosis factor-α（TNF-α-induced DNA synthesis of glomerular mesangial cells of rat, and explore the molecular mechanisms of LXA4 action. Methods： Glomerular mesangial cells of rat were cultured and preincubated with LXA4 at different concentrations, and then treated with TNF-α（ 10 ng/ml）. DNA synthesis was assessed by the incorporation of [^3H]-thymidine in mesangial cells. Expression of cyclin E protein was determined by Western blotting analysis. Activities of signal transducers and activators of transcription-3 （STAT3） were analyzed by electrophoretic mobility shift assay （EMSA）. Results： TNF-α-stimulated DNA synthesis of mesangial cells, upregulafion of cyclin E protein and STAT3 activities were inhibited by LXA4 in a dose-dependent manner. Conclusion： TNF-α-induced DNA synthesis of mesangial cells can be inhibited by TXA4 probably through the mechanism of Jak1/STAT3 pathway-dependent signal transduction.
文摘Objective:To investigate the effect of lipoxin receptor agonist BML-111 on the NLRP3 inflammasome after traumatic brain injury in rats.Methods:Sixty male Sprague-Dawley rats,weighing 280~340 g,were randomly divided into 4 groups(n=15):the sham operation group(group Sham),the traumatic brain injury group(group TBI),the BML-111 treatment group(group BML-111),and the BOC-2 treatment group(group BOC-2).The TBI model was prepared by craniocerebral collision,while the rats in group Sham underwent only craniotomy without collision.Acute traumatic brain injury model was prepared in group TBI,BML-111 and BOC-2.The rats in group BOC-2 were intraperitoneally injected with 50μg/kg of BOC-230 min prior to trauma.Then the rats in group BOC-2 and BML-111 were injected intraperitoneally with 1 mg/kg of BML-111 immediately and 24 hours after trauma.The neurological severity scores(NSS)were evaluated at 3 and 7 days after brain trauma.The protein expression levels of NLRP3,Caspase-1-p20 and active Caspase-3 were determined by Western blot.The content of IL-1βand IL-18 was detected by ELISA assays.The apoptotic cells were analyzed by the TUNEL method.Results:Compared with group Sham,the brain water content and NSS scores in group TBI were increased,and the protein levels of NLRP3,Caspase-1-p20,activated Caspase-3,IL-1βand IL-18 as well as TUNEL-positive cells in the cortex were elevated significantly(P<0.05);compared with group TBI,the brain water content and NSS scores in group BML-111 were reduced,and the protein levels of NLRP3,Caspase-1-p20,activated Caspase-3,IL-1βand IL-18 as well as TUNEL-positive cells in the cortex were decreased(P<0.05);Compared with group BML-111,the brain water content and NSS scores in group BOC-2 were increased,and the protein levels of NLRP3,Caspase-1-p20,activated Caspase-3,IL-1βand IL-18 as well as TUNEL-positive cells in the cortex were up-regulated(P<0.05).Conclusions:The lipoxin receptor agonist BML-111 might attenuate traumatic brain injury in rats by inhibiting NLRP3 inflammasome activation.
文摘In order to investigate whether lipoxin A4 （LXA4） has an antagonistic effect on lipopolysaccharide （LPS）-induced synthesis of interleukin （IL）-β3, IL-6 and IL-8 in rat pulmonary microvascular endothelial cells （PMVEC）, and to explore the molecular mechanisms of signal pathway in LXA4 actions, cultured PMVEC were treated with LPS, with or without preincubation with LXA4. Proteins of IL-β3, IL-6 and IL-8 in supernatant were analyzed by enzyme-linked immunosorbent assay （ELISA）. Expressions of mRNA of IL-β3, IL-6 and IL-8 were determined by RT-PCR. Expressions of phosphorylation of phosphoinositide 3-kinase （PI3-K） and myeloid differentiation factor 88 （MyD88） were analyzed by Western blot. Activities of DNA-binding of nuclear factor-kappa B （NF-κB） and activator protein-1 （AP-1） were measured by electrophoretic mobility shift assay （EMSA）. The results showed that LPS induced production of IL-β3, IL-6 and IL-8 in rat PMVEC via MyD88/PI3-K/NF-κB and AP-1 pathway-dependent signal transduction. LPS-stimulated expression of PI3-K, activities of NF-κB and AP-1, secretion of protein and expression of mRNA of IL-β3, IL-6 and IL-8 but not MyD88 expression in PMVEC were inhibited by LXA4 in a dose-dependent manner. In conclusion, LXA4 inhibits synthesis of IL-β3, IL-6 and IL-8 by down-regulation of PI3-K/NF-κB and AP-1 signal pathway in PMVEC.
基金the grants from the National Nature Science Foundation of China (No. 30570726 and No. 30100226)
文摘Background Lipoxins （LXs）, endogenous anti-inflammatory and pro-resolving eicosanoids generated during various inflammatory conditions, have novel immunomodulatory properties. Because dendritic cells （DCs） play crucial roles in the initiation and maintenance of immune response, we determined whether LXs could modulate the maturation process of DCs and investigated the effects of lipoxin A4 （LXA4） on lipopolysaccharide （LPS）-induced differentiation of RAW264.7 cells into dendritic-like cells. Methods RAW264.7 cells were cultured in vitro with 1 pg/ml LPS in the absence or presence of LXA4 for 24 hours, and cellular surface markers （MHC-II, CD80 （B7-1）, CD86（B7-2）） were measured by flow cytometry （FCM）. Mixed lymphocyte reaction was performed to evaluate the allostimulatory activity. Cytoplastic IKB degradation and nuclear factor kappa B （NF-KB） translocation were detected by Western blotting. Luciferase reporter plasmid was transiently transfected into RAW264.7 cells, and luciferase activity was determined to measure the transcriptional activity of NF-KB. Results LXA4 reduced the ratio of LPS-treated RAW264.7 cells to DCs with morphological characteristics and inhibited the expression of MHC I1. LPS-induced up-regulation of CD86 was moderately suppressed by LXA4 but no obvious change of CD80 was observed. Moreover, LXA4 weakened the aUostimulatory activity of LPS-treated RAW264.7 cells. These alterations of LPS＋LXA4-treated cells were associated with a marked inhibition of IKB degradation, NF-KB translocation and then the transcriptional activity of NF-KB. Conclusions LXA4 negatively regulates LPS-induced differentiation of RAW264.7 cells into dendritic-like cells.This activity reveals an undescribed mechanism of LXA4 to prevent excessive and sustained immune reaction by regulating maturation of DCs.
文摘Background： Lipoxin A4 （LXA4） can alleviate lipopolysaccharide （LPS）-induced acute lung injury （ALl） and acute respiratory distress syndrome through promoting epithelial sodium channel （ENaC） expression in lung epithelial cells. However, how LXA4 promote ENaC expression is still largely elusive. The present study aimed to explore genes and signaling pathway involved in regulating ENaC expression induced by LXA4. Methods： A549 cells were incubated with LPS and LXA4, or in combination, and analyzed by quantitative real-time polymerase chain reaction （qRT-PCR） of ENaC-α/γ. Candidate genes affected by LXA4 were explored by transcriptome sequencing ofA549 cells. The critical candidate gene was validated by qRT-PCR and Western blot analysis ofA549 cells treated with LPS and LXA4 at different concentrations and time intervals. LXA4 receptor （ALX） inhibitor BOC-2 was used to test induction of candidate gene by LXA4. Candidate gene siRNA was adopted to analyze its influence on A549 viability and ENaC-α expression. Phosphoinositide 3-kinase （PI3K） inhibitor LY294002 was utilized to probe whether the PI3K signaling pathway was involved in LXA4 induction of candidate gene expression. Results： The A549 cell models of ALl were constrticted and subjected to transcriptome sequencing. Among candidate genes, N-myc downstream- regulated gent- 1 （NDRG 1 ） was validated by real-time-PCR and Western blot. NDRG 1 mRNA was elevated in a dose-dependent manner of LXA4, whereas BOC-2 antagonized NDRG 1 expression induced by LXA4. NDRG I siRNA suppressed viability of LPS-treated A549 cells （treatment vs. control, 0.605± 0.063 vs. 0.878 ± 0.083, P = 0.040） and ENaC-α expression （treatment vs. control, 0.458 ± 0.038 vs. 0.711 ± 0.035, P = 0.008）. LY294002 inhibited NDRG 1 （treatment vs. control, 0.459 ± 0.023 vs. 0.726 ± 0.020, P 0.001 ） and ENaC-α （treatment vs. control, 0.236 ± 0.021 vs. 0.814 ±0.025, P 〈 0.001 ） expressions and serum- and glucocorticoid-inducible kinase I phosphorylation （treatment vs. control, 0.442± 0.024 vs. 1.046 ± 0.082, P = 0.002）, indicating the PI3K signaling pathway was involved in regulating NDRG 1 expression induced by LXA4. Conclusion： Our research uncovered a critical role of NDRG1 in LXA4 alleviation of LPS-induced A549 cell injury through mediating PI3K signaling to restore ENaC expression.
文摘该文旨在探讨脂氧素受体激动剂BML-111对H22细胞的影响及机制。利用小鼠肝癌细胞株H22,构建肿瘤相关体内外模型,并给予脂氧素受体激动剂BML-111和脂氧素受体阻断剂BOC-2分别作为治疗组和阻断剂组。体内实验,测定小鼠皮下肿瘤质量、体积。体外实验,CCK8法检测细胞活力,试剂盒检测细胞乳酸含量、葡萄糖含量、ATP含量、谷氨酰胺代谢情况,Western blot检测磷酸酶张力蛋白同源物基因(phosphatase and tensin homolog deleted on chromosome ten,PTEN)的蛋白含量,qRT-PCR法检测PTEN mRNA表达情况。治疗组较CON组和阻断剂组皮下肿瘤质量和体积均有所减小。与CON组相比,BML-111可以抑制肝癌细胞活力,可明显降低H22细胞葡萄糖含量、乳酸含量以及谷氨酰胺含量(P<0.05)。同时BML-111可以提高PTEN的表达水平,而脂氧素受体阻滞剂BOC-2可以逆转所有这些现象。BML-111通过降低肝癌细胞糖酵解及谷氨酰胺代谢水平,抑制肝癌细胞的增殖,降低肝癌细胞的能量代谢水平,其作用机制可能与调控PTEN的表达相关。
文摘Objective:To examine whether lipoxin A 4(LXA 4) has an inhibitory effect on tumor necrosis factor-α(TNF-α)-induced proliferation of glomerular mesangial cells of rat, and explore the molecular mechanisms of signal pathway in LXA 4 actions. Methods: Glomerular mesangial cells of rat were cultured and treated with TNF-α(10 ng/ml), with or without preincubation with LXA 4 at different concentrations. Cell proliferation was evaluated by monotetrazolium (MTT) colorimetric assay. The expression of cyclin E mRNA was measured by RT-PCR. Phosphorylated Akt1(Thr308) and p27 kip1 were analyzed by Western blotting. Results: TNF-α-stimulated proliferation of mesangial cells was inhibited by LXA 4 in a dose-dependent manner. The marked increments in cyclin E mRNA expression induced by TNF-α during proliferation of mesangial cells were down-regulated by LXA 4. Threonine phosphorylated Akt1 proteins at 308 site stimulated by TNF-α was reduced by LXA 4. TNF-α-induced decrements in expression of p27 kip1 proteins was ameliorated by LXA 4 in a dose-dependent manner. Conclusion: TNF-α-induced proliferation of rat mesangial cells can be inhibited by TXA 4 through the mechanism of Akt 1/p27 kip1 pathway-dependent signal transduction.
