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OBJECTIVES@#Peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α) controls mitochondrial biogenesis, but its role in cardiovascular diseases is unclear. The purpose of this study is to explore the effect of PGC1α on myocardial ischemia-reperfusion injury and the underlying mechanisms.@*METHODS@#The transverse coronary artery of SD rat was ligated for 30 minutes followed by 2 hours of reperfusion. Triphenyltetrazolium chloride (TTC) staining was performed to measure the area of myocardial infarction. Immunohistochemistry and Western blotting were used to detect the PGC1α expression in myocardium. The rat cardiomyocyte H9C2 was subjected to hypoxia/reoxygenation (H/R) with the knockdown of PGC1α or hypoxia- inducible factor 1α (HIF-1α), or with treatment of metformin. Western blotting was used to detect the expression of PGC1α, HIF-1α, p21, BAX, and caspase-3. CCK-8 was performed to detect cell viability, and flow cytometry was used to detect apoptosis and mitochondrial superoxide (mitoSOX) release. RT-qPCR was used to detect the mRNA expression of PGC1α and HIF-1α. Besides, chromatin immunoprecipitation (ChIP)-qPCR and luciferase reporter gene assay were applied to detect the transcriptional regulation effect of HIF-1α on PGC1α.@*RESULTS@#After I/R, the PGC1α expression was increased in infarcted myocardium. H/R induced H9C2 cell apoptosis (@*CONCLUSIONS@#After I/R, HIF-1α up-regulates the expression of PGC1α, leading to an increase in ROS production and aggravation of injury. Metformin can inhibit the accumulation of HIF-1α during hypoxia and effectively protect myocardium from ischemia/reperfusion injury.
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Animals , Rats , Apoptosis , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Myocardial Reperfusion Injury/genetics , Myocytes, Cardiac/metabolism , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/metabolism , Rats, Sprague-Dawley , Reperfusion InjuryABSTRACT
Objective: To observe the regulatory effects of miRNA-31 (miR-31) on LATS2 and cardiomyocyte hypertrophy via down-regulating miR-31 expression in rat's cardiomyocytesin vitro. Methods: Rat's cardiomyocytes were isolated and cultured for 10 daysin vitro, according to different intervention methods, the cells were divided into 4 groups:①Blank control group,②AngII intervention group,③Lentivirus with miR-31 inhibitor infection group,④Negative lentivirus infection group. On day-8, gene expressions of MiR-31, LATS2, cardiac hypertrophy ANP and β-MHC were examined by qRT-PCR; on day-10, cell morphology was observed by fluorescence staining. LATS2 protein expression was examined by Western blot analysis. Dual luciferase reporter plasmids were transfected into 293T cells, then luciferase activity was detected to identify the targeting effect of miR-31 on LATS2. Results: Compared with Blank control group, AngII intervention group showed increased gene expressions of miR31, cardiac hypertrophy ANP and β-MHC,P<0.05, enlarged cardiomyocyte surface,P<0.05; while decreased gene and proteinexpressions of LATS2,P<0.05. Compared with AngII intervention group, Lentivirus with miR-31 inhibitor infection group had down-regulated expressions of miR31, cardiac hypertrophy ANP and β-MHC,P<0.05, reduced cardiomyocyte surface, P<0.05; while slightly increased LATS2 gene expression and obviously increased protein expression,P<0.05. Dual luciferase reporter assay presented that relative luciferase activity of TRAF6-3' UTR+miR-146b was significantly decreased than TRAF6-3' UTR+miR-NC,P<0.01 and relative luciferase activity of LATS2-3' UTR+ miR-31 was signiifcantly reduced than LATS2-3' UTR-NC+miR-31,P<0.01. Conclusion: Cardiomyocytes hypertrophy could be reversed at certain degree by down-regulating miR-31; the targeting effect of miR-31 on LATS2 was involved in cardiomyocyte hypertrophyregulation.
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Objective To compare the effects of genistein,bosentan,and tadalfil in treatment of monocrotaline-induced pulmonary arterial hypertension (PAH) in rats.Methods Monocrotaline-induced PAH SD-rat model was established by injecting monocrotaline (50 mg/kg) intraperitoneally (n=48),while the rats in control group (n=8)were given normal saline injection.Then all model rats were randomly divided into 6 groups (n=8) according to different drugs given by gavage after 3 weeks:PAH group (PAH rats were treated with normal saline),genistein G30,G60,G120 groups (PAH rats were treated with genistein 30,60,120 mg· kg-1 · d-1,respectively),bosentan group (PAH rats were treated with bosentan 5 mg · kg-1 · d-1),and tadalafil group (PAH rats were treated with tadalafil 0.5 mg · kg-1 · d-1).After 2 weeks of treatment,survival rate,tricupid regurgitation (TR),pulmonary artery diameter (PAD),mean pulmonary arterial pressure (PAP),acetylcholine (Ach)-induced endothelium-dependent relaxation (EDdR),sodium nitroprusside (SNP)-induced endothelium-independent relaxation (EDiR),right ventricular hypertrophy index (RVHI),and lung phathological of rats in each group were compared.Results Compared with the control group,the TR,PAD,mean PAP and RVHI of rats were significantly increased in PAH group (P＜0.05).Compared with the PAH group,the TR and RVHI of rats were significantly decreased in G60 and G-120 groups,PAD was significantly decreased in G120 group,mean PAP was significantly decreased in G30,G-60 and G120 groups,survival rate was significantly increased in G120 group,and endothelium-dependent relaxation was significantly improved in G30,G60 and G120 groups,while pulmonary vascular luminal stenosis,wall thickening and vascular smooth muscle cell proliferation were significantly inhibited in G30,G60 and G120 groups (P＜0.05).However,the above effects of genistein were less than bosentan and tadalafil (P＜0.05).Conclusion Genistein has a certain effect on monocrotaline-induced PAH in rat;although its efficacy is less than that of bosentan and tadalafil,genistein is cheaper,easy to obtain and has less side effects,and is also expected to be a new auxiliary drug for PAH.
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AIM: To investigate the effect and the mechanism of apolipoprotein (a) [apo (a) ] on proliferation of vascular smooth muscle cells ( VSMCs). METHODS: All VSMCs used in experiments were serial subcultured from primary cells and were identified by immunohistochemistry staining of a - actin. Cell growth assay was observed as cell counting and MTT assay. Western blotting was also employed to detect the related mechanism. RESULTS: All cells used in experiments were confirmed as VSMCs. Although apo (a) enhanced VSMCs proliferation, this effect was attenuated by anti -integrin α_vβ_3, LM609.Use these reagents alone had no effect on VSMCs growth. The results of Western blotting demonstrated that focal adhesion kinase (FAK) was activated by apo (a) and the expression of total or phosphorylated transforming growth factor β_1 (TGF -β_1) was also decreased. However, these effects described above were all blocked by LM609.CONCLUSION: Apolipoprotein (a) enhances VSMCs proliferation and this effect is mediated by integrin α_vβ_3, which activates FAK and attenuates TGF - β_1 and phospho -TGF - β_1 expression.
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BACKGROUND:The benefit of cell therapy may be partly due to the secretion of angiogenic and antiapoptotic growth factors.Whether amniotic fluid stem cells (AFS) could secrete some growth factors requires further studies.OBJECTIVE:To isolate and culture AFS cells,and explore the angiogenic or antiapoptotic effect of cytokines secreted by AFS on endothelial cells.DESIGN,TIME AND SETTING:A in vitro cytological experiment was performed at the Institute of Hypertensive Disease,First Affiliated Hospital,Nanchang University from December 2008 to June 2009.MATERIALS:Term amniotic fluid of ten samples,50 mL/case,was obtained following caesarean delivery.The umbilical vein was used to isolate endothelial cells.Written informed content was obtained from all women.METHODS:AFS isolated from human amniotic fluid was cultured and digested by trypsin at confluence of 80%.The third passage of cells at a density of 5×10~8/L were divided into two groups:hypoxia group:the cells were cultured in 2% O_2 + 5% CO_2 +93% N_2;normal group:the cells were cultured in 5% CO_2 + 95% air.Two groups were cultured at 37 ℃ for 24 hours.The supematant of two groups was collected.The second passage of human umbilical vein endothelial cells cultured in vitro was collected and seeded onto 12-well culture plate at a density of 2×10~4 cells/well,and divided into 3 groups:control group was cultured in 2 mL EBM-2 containing 5% fetal bovine serum (FBS);normal group was cultured in 1 mL EBM-2 containing 5% FBS and 1 mL AFS cell culture solution;hypoxia group was cultured in 1 mL EBM-2 containing 5% FBS and 1 mL hypoxia AFS cell culture solution for 3 days,followed by incubation with 10 μg/L tumor necrosis factor (TNF)-α.MAIN OUTCOME MEASURES:AFS surface phenotype was examined by flow cytometry;the secretion level and mRNA expression of vascular endothelial cell growth factor (VEGF) and hepatocyte growth factor (HGF) were examined by ELISA or RT-PCR.The proliferation and apoptotic rates of endothelial cells were examined.RESULTS:AFS cells were long fusiform-shaped and arranged radially after 7 days of culture.The third passage of AFS cells expressed CD29 and CD105 while did not express CD34.AFS cells of normal culture secreted VEGF and HGF;AFS cells of hypoxia condition significantly increased secrete of VEGF (P＜0.01),and VEGF mRNA expression was significantly upregulated (P＜0.05),while HGF and mRNA expression remained unchanged (P＞0.05).Compared with control group,the number of endothelial cells was significantly increased in normal and hypoxia AFS cell groups after 3 days of culture (P＜0.05).After cocultured with TNF-α for 24 hours,the apoptosis rates of endothelial cells in AFS-conditioned medium was significantly decreased (P ＜ 0.05),and the change degree of hypoxia AFS cell group was greater than normal AFS cell group (P ＜ 0.05).CONCLUSION:AFS can secrete cytokines such as VEGF and HGF.Moreover,it significantly promotes endothelial cells proliferation and inhibits apoptosis.Under hypoxia condition,the secretion of VEGF from AFS cells is increased,and the effects on endothelial cells proliferation and apeptosis are enhanced.
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Objective To investigate the effect of the Senlm of patients with congestive heart failure(CHF) on the apoptosis of endothelial progentior eelts(EPCs)and protective role of carvedil01.Method Totally 20 patientswithCHF、
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Objective To investigate the association between plasma lipoprotein (a) [LP (a)] concentra-tion and in-stent restenosis after coronary stent implantation. Methods 152 patients with successful elective coro-nary stont implantation and percutancous transluminal coronary angioplasty (PICA) undergoing foUow-up angiogra-phy were retrospectively analyzed. These patients were divided into restenosis group( n = 29) and no-restenosis group (n = 123 ). The serum LP (a) levels of all patients were also investigated. The general clinical data were analyzed. Multivariate logistic regression was used for statistical analysis. Results We compared the serum Lap (a) levels, smoking and diabet in the two groups, and there was a statisticaLly significant difference between the restenosis group and no-restenosis group(P<0.05). Multivariate logistic regression showed that the elevation of Lap (a) level re-mained as an independent predictor of restenosis (RR =2. 648,95% CI 1. 066-6. 575,P <0. 05). Other risk fac-tors,such as smoking(P =0.023) ,diabet(P =0. 036) and the type of stent(P = 0.011 ) were also correlated with restenosis. Conclusions High plasma LP (a) concentration is an independent predictor of stent restenosis after stent implantation.