瓜蒌皮提取物基于PI3K/Akt/NO信号通路保护缺氧/复氧损伤心肌细胞的机制

Mechanism of Trichosanthis Pericarpium Aqueous Extract in Protecting H9c2 Cardiomyocytes from Hypoxia/Reoxygenation Injury via PI3K/Akt/NO Signaling Pathway

目的:研究磷脂酰肌醇3-激酶/蛋白激酶B/一氧化氮(PI3K/Akt/NO)信号通路在瓜蒌皮提取物保护心肌细胞缺氧/复氧损伤中的作用。方法:以2.5 mmol·L-1Na2S2O4诱导心肌细胞建立缺氧/复氧损伤模型,实验分为正常组、模型组、瓜蒌皮提取物组及抑制剂组。采用噻唑蓝(MTT)比色法检测细胞活力;酶联免疫吸附测定(ELISA)检测心肌细胞中一氧化氮(NO)的释放量及内皮型一氧化氮合酶(e NOS),诱导型一氧化氮合酶(i NOS)的活性水平;实时荧光定量聚合酶链式反应(Real-time PCR)检测细胞中Akt,e NOS,i NOS mRNA表达水平;蛋白免疫印迹法(Western blot)测定Akt,磷酸化Akt(p-Akt),e NOS,i NOS蛋白表达水平。结果:与正常组比较,模型组心肌细胞活力明显下降(P<0.01),心肌细胞NO释放量减少(P<0.01),心肌细胞中p-Akt,Akt及e NOS mRNA和蛋白表达显著降低(P<0.01),而i NOS mRNA和蛋白表达显著升高(P<0.01);与模型组比较,瓜蒌皮提取物预处理24 h后能改善心肌细胞形态,明显提高心肌细胞活力(P<0.01)。瓜蒌皮提取物能促进p-Akt,Akt及e NOS mRNA和蛋白表达(P<0.01),抑制i NOS mRNA和蛋白的表达(P<0.01),增加心肌细胞NO的释放量(P<0.01)。PI3K抑制剂LY294002与瓜蒌皮提取物联合处理后,逆转了瓜蒌皮提取物对心肌细胞的上述作用,导致细胞存活率下降。结论:瓜蒌皮提取物可能通过激活PI3K/Akt信号通路上调e NOS,下调i NOS的表达,增加内源性NO的基础含量水平,发挥抗凋亡作用,从而改善缺氧/复氧损伤诱导的心肌细胞损伤。

Objective:To investigate the effect of Trichosanthis Pericarpium aqueous extract(TPAE)in protecting H9c2 cells from hypoxia/reoxygenation(H/R)injury by activating phosphatidylionsitol-3-kinase/protein kinase B/nitric oxide(PI3K/Akt/NO)signaling pathway.Method:The 2.5 mmol·L-1Na2S2O4 was used to induce the model of H9c2 cardiomyocytes H/R injury in the experiments.The cultured H9c2 cardiomyocytes were randomly divided into normal group,H/R group(model group)and inhibition group(LY294002,10μmol·L-1).In the H/R+TPAE group,50 mg·L-1TPAE was added to the cultures at 24 h before H/R exposure.Cell viability was measured by methyl thiazolyl tetrazolium(MTT)assay.The amounts of NO,endothelial nitric oxide synthase(e NOS),and induced nitric oxide synthase(i NOS)were tested by enzyme linked immunosorbent assay(ELISA)kits.Reverse transcription-quantitative real-time polymerase chain reaction(Real-time PCR)was performed to analyze relative mRNA expressions of Akt,e NOS and i NOS.Western blot was used to detect the expressions of Akt,p-Akt(Ser 473),e NOS,and i NOS.Result:Compared with the normal control group,the cell viability significantly decreased in the model control group(P<0.01),the release of NO was obviously downregulated(P<0.01),the mRNA and protein expressions of p-Akt,Akt,e NOS were remarkably decreased(P<0.01),while those of i NOS were up-regulated(P<0.01).Compared with the H/R group,the pretreatment with TPAE remarkably improved the morphological lesion of cardiomyocytes,enhanced cell viability(P<0.01),increased the expressions of Akt,p-Akt(Ser 473)and e NOS(P<0.01),decreased the expression of i NOS(P<0.01),and increased the release of NO(P<0.01).The PI3K inhibitor LY294002 was used.Obviously,cardioprotection of Trichosanthis Pericarpium aqueous extract was blocked by co-treatment LY294002,and cell viability was correspondingly reversed.Conclusion:TPAE can protect H9c2 cardiomyocytes from H/R injury by activating PI3K/Akt signaling pathway,which might be related to the up-regulation of the mRNA and protein expressions of e NOS,the down-regulation of the level of i NOS,and the increase of the production of physiological amounts of NO.