叉头框蛋白O1通过信号传导和转录激活因子3信号通路对高糖高脂诱导的H9c2心肌细胞自噬及凋亡影响的研究

Impacts of forkhead box O1 on autophagy and apoptosis of H9c2 cardiomyocytes induced by high glucose and high fat through signal transducer and activator of transcription 3 signaling pathway

目的 探讨叉头框蛋白O1(FoxO1)通过信号传导和转录激活因子3(STAT3)信号通路对高糖高脂诱导的H9c2心肌细胞自噬及凋亡的影响。方法 H9c2细胞分为正常对照组(Con)、高糖高脂(HG)组、阴性对照(si-NC)组、FoxO1小干扰RNA组(si-FoxO1)、si-FoxO1+STAT3小干扰RNA组(si-FoxO1+si-STAT3)。检测各组细胞增殖、细胞内自噬体、细胞凋亡、活性氧簇(ROS),Western blot法检测各组FoxO1、p-FoxO1、STAT3、p-STAT3、微管相关蛋白1轻链3(LC3)Ⅱ、自噬关键分子酵母Atg6同系物(Beclin1)、B淋巴细胞瘤-2基因(Bcl2)、Bcl2相关X蛋白(Bax)蛋白表达。结果 与Con组比较,HG、si-NC、si-FoxO1、si-FoxO1+si-STAT3组细胞MDC阳性细胞、细胞凋亡率、ROS平均荧光强度升高(P<0.05),OD_(450)降低(P<0.05),HG、si-NC、si-FoxO1+si-STAT3组p-FoxO1/FoxO1、LC3Ⅱ、Beclin1、Bax蛋白表达升高(P<0.05),p-STAT3/STAT3、Bcl2蛋白表达降低(P<0.05),si-FoxO1组LC3Ⅱ蛋白表达升高(P<0.05),FoxO1、Bcl2蛋白表达降低(P<0.05)。与HG、si-NC组比较,si-FoxO1、si-FoxO1+si-STAT3组OD450、p-STAT3/STAT3、Bcl2蛋白表达升高(P<0.05),细胞MDC阳性细胞、细胞凋亡率、ROS平均荧光强度、FoxO1、LC3Ⅱ蛋白表达降低(P<0.05),si-FoxO1组p-FoxO1/FoxO1、Beclin1、Bax蛋白表达降低(P<0.05)。与si-FoxO1组比较,si-FoxO1+si-STAT3组细胞MDC阳性细胞、细胞凋亡率、ROS平均荧光强度、FoxO1、p-FoxO1/FoxO1、LC3Ⅱ、Beclin1、Bax蛋白表达升高(P<0.05),OD450、p-STAT3/STAT3、Bcl2蛋白表达降低(P<0.05)。结论 降低FoxO1可激活STAT3信号通路,以实现对高糖高脂诱导的H9c2心肌细胞自噬和凋亡过度的缓解。

Objective To investigate the effect of forkhead box O1(FoxO1)on autophagy and apoptosis of H9c2 cardiomyocytes induced by high glucose and high fat through signal transducer and activator of transcription 3(STAT3)signaling pathway.Methods H9c2 cells were divided into normal control group(Con),high-glucose and high-fat(HG)group,negative control group(si-NC),FoxO1 small interfering RNA group(si-FoxO1),si-FoxO1+STAT3 small interfering RNA group(si-FoxO1+si-STAT3).Cell proliferation,intracellular autophagosomes,apoptosis,and intracellular reactive oxygen species(ROS)level were detected in each group.The protein expression of FoxO1,p-FoxO1,STAT3,p-STAT3,microtubule associated protein 1 light chain 3( LC3) II, autophagy key molecule yeast Atg6 homolog( Beclin1), B-celllymphoma - 2 (Bcl2) and Bcl2 - associated X protein (Bax) were tested by Western blot assay. ResultsCompared with Con group, MDC positive cells, apoptosis rate and average fluorescence intensity of ROSwere increased( P<0. 05), while OD450 was decreased( P<0. 05) in HG, si-NC,si-FoxO1 and si-FoxO1+si-STAT3 groups;the protein expressions of p-FoxO1 /FoxO1, LC3Ⅱ , Beclin1 and Baxwere increased(P<0. 05), while the protein expressions of p - STAT3/STAT3 and Bcl2 were decreased (P<0. 05)inHG, si-NC and si-FoxO1+si-STAT3 groups;the expression of LC3Ⅱ protein was increased( P<0. 05),while the expression of FoxO1 and Bcl2 protein was decreased (P<0. 05) in si-FoxO1 group. Comparedwith HG and si-NC groups, the protein expressions of OD450, p-STAT3/STAT3 and Bcl2 were increased(P<0. 05), MDC positive cells, apoptosis rate, ROS mean fluorescence intensity, FoxO1, LC3Ⅱ proteinexpressions were decreased (P<0. 05) in si - FoxO1 and si - FoxO1+si - STAT3 groups, and p - FoxO1 /FoxO1, Beclin1, Bax protein expressions were decreased in si-FoxO1 group (P<0. 05). Compared withsi-FoxO1 group, MDC positive cells, apoptosis rate, ROS mean fluorescence intensity, FoxO1, p-FoxO1/FoxO1, LC3Ⅱ, Beclin1 and Bax protein expressions were increased( P<0. 05),OD450,p-STAT3/STAT3and Bcl2 protein expressions were decreased (P<0. 05) in si - FoxO1+si - STAT3 group. ConclusionReducing FoxO1 can activate STAT3 signaling pathway to alleviate the excessive autophagy and excessiveapoptosis of H9c2 cardiomyocytes induced by high glucose and high fat.