丹酚酸A激活AKT/mTOR/4EBP1通路缓解脂多糖诱导的H9c2心肌细胞凋亡和氧化应激

目的探究丹酚酸A(Sal A)对脂多糖(LPS)诱导的氧化应激性损伤H9c2心肌细胞增殖、凋亡,以及蛋白激酶B(AKT)/哺乳动物雷帕霉素靶蛋白(mTOR)/起始因子4E结合蛋白(4EBP1)通路相关蛋白表达的影响。方法用LPS诱导制备H9c2心肌细胞氧化应激性损伤模型,分别用含Sal A 5,10和20 mg·L^-1的DMEM培养基培养细胞24 h,加LPS 1 mg·L^-1继续孵育24 h。用5-乙炔基-2’脱氧尿嘧啶核苷(EdU)染色观察H9c2心肌细胞存活;采用流式细胞术检测H9c2心肌细胞凋亡和线粒体膜电位;用试剂盒检测细胞培养液中乳酸脱氢酶(LDH)和超氧化物歧化酶(SOD)活性及丙二醛(MDA)和谷胱甘肽(GSH)含量;Western印迹法检测胱天蛋白酶3、胱天蛋白酶9、存活蛋白、Bax/Bcl-2,AKT、磷酸化AKT(p-AKT)、mTOR、p-mTOR、4EBP1和p-4EBP1蛋白表达水平。结果与细胞对照组比较,模型组EdU红色标记的细胞减少(P<0.05);与模型组比较,Sal A 5,10和20 mg·L^-1处理组EdU红色标记的细胞增多(P<0.05)。Sal A能降低培养液中LDH活性及细胞内MDA和GSH含量,增加胞内SOD活性(P<0.05),减少心肌细胞的凋亡率(P<0.05),降低心肌细胞线粒体膜电位的下降速度(P<0.05)。Sal A 10和20 mg·L^-1处理后,活化胱天蛋白酶3、活化胱天蛋白酶9和Bax/Bcl-2蛋白水平显著下调(P<0.05),存活蛋白水平显著上调(P<0.05);与细胞对照组比较,模型组AKT/mTOR/4EBP1磷酸化水平均显著降低(P<0.05)。与模型组比较,Sal A 10和20 mg·L^-1组AKT和mTOR磷酸化水平均显著升高(P<0.05)。结论Sal A通过激活AKT/mTOR/4EBP1通路、减少心肌细胞凋亡并降低心肌细胞线粒体膜电位的下降速度,减缓了LPS诱导的H9c2心肌细胞凋亡和氧化应激,表明Sal A对LPS造成的氧化应激损伤心肌细胞具有保护作用。

Salicylic acid A activates AKT/mTOR/4EBP1 pathway to relieve lipopolysaccharide-induced apoptosis and oxidative stress of H9c2 cardiomyocytes

OBJECTIVE To investigate the effects of salvianolic acid A(Sal A)on the proliferation and apoptosis of H9c2 cardiomyocytes induced by lipopolysaccharide(LPS)-induced oxidative stress injury and on the expressions of protein kinase B/mammlain target of Rapamycin/eIF4E-binding protein1(AKT/mTOR/4EBP1)pathway-related proteins.METHODS An oxidative stress injury model of H9c2 cells was prepared via LPS-induced injury.Cells were cultured in serum DMEM medium containing Sal A 5,10 and 20 mg·L^-1 for 24 h before cardiomyocytes were treated with LPS 1 mg·L^-1 for 24 h.Proliferation of H9c2 cells was observed by EdU staining.Flow cytometry was used to detect the apoptosis of H9c2 cells and the changes of the mitochondrial membrane potential.The activities of lactate dehydrogenase(LDH)and superoxide dismutase(SOD)as well as the contents of malondialdehyde(MDA)and glutathione(GSH)in cell culture were detected by the kits.Western blotting was used to detect the expressions of caspase 3,caspase 9,survivin,Bax/Bcl-2,AKT,p-AKT,mTOR,p-mTOR,4EBP1 and p-4EBP1.RESULTS Compared with the cell control group,the number of EdU red labeled cells in the model group decreased.Compared with the model group,the cells of Sal A 10 and 20 mg·L^-1 groups were increased with red markers of EdU.Sal A reduced LDH activity,intracellular MDA and GSH contents,increased intracellular SOD activity(P<0.05),and reduced the rate of cardiomyocyte apoptosis(P<0.05)and the mitochondrial membrane potential of cardiomyocytes(P<0.05).After treatment with Sal A 10 and 20 mg·L^-1,cleaved caspase 3,cleaved caspase 9 and Bax/Bcl-2 protein levels were significantly reduced(P<0.05);while survivin protein levels were significantly increased(P<0.05).The phosphorylation level of AKT/mTOR/4EBP1 in the model group was significantly lower than that in the cell control group(P<0.05).Compared with the model group,the phosphorylation levels of AKT/mTOR/4EBP1 in Sal A 10 and 20 mg·L^-1 groups were significantly increased(P<0.05).CONCLUSION Sal A can reduce the apoptosis rate of cardiomyocytes by activating AKT/mTOR/4EBP1 pathway.The decrease rate of the mitochondrial membrane potential in cardiomyocytes is reduced,and the apoptosis and oxidative stress of H9c2 cells induced by LPS are slowed down,indicating that Sal A can protect cardiomyocytes from the damage caused by LPS.