摘要
目的探讨SIRT1通过调节FoxO3a/BIM通路在心肌细胞高糖缺氧复氧(hypoxia/reoxygenation,H/R)损伤中的作用。方法正常培养的对数期H9C2心肌细胞,随机分为高糖常氧组(high glucose,HG组)、高糖H/R组和高糖H/R+Srt1720组(H/R+Srt组)。3组H9C2细胞均经高糖培养基(葡萄糖浓度为33mmol/L)培养24h建立高糖模型,高糖H/R组置于37℃、缺氧培养箱(体积分数95%N2+体积分数5%CO2)培养4h,37℃、复氧(体积分数90%O2+体积分数10%CO2)2h建立心肌细胞高糖H/R损伤模型,高糖H/R+Srt组于H/R前24h给予SIRT1激动剂Srt1720(20μmol/L)。高糖H/R结束后,采用ELISA法检测培养液上清乳酸脱氢酶(lactate dehydrogenase,LDH)水平,流式细胞仪检测细胞凋亡率,JC-1染色法检测线粒体膜电位(JC-1单/多聚体比值),Western blot法检测心肌细胞SIRT1、细胞质磷酸化FoxO3a(phosphor-FoxO3a,P-FoxO3a)、细胞核FoxO3a及BIM蛋白表达。结果高糖H/R组、高糖H/R+Srt组细胞凋亡率[(42±7)%、(27±5)%]、JC-1单/多聚体比值(5.2±0.9、3.3±0.6)、培养液上清LDH水平[(485±62)、(394±52)u/L]均高于HG组[(19±4)%、1.7±0.4、(264±42)u/L](P<0.05),且高糖H/R组高于高糖H/R+Srt组(P<0.05);高糖H/R组H9C2细胞SIRT1(0.41±0.07)、细胞质P-FoxO3a(0.34±0.05)表达低于高糖HG组(0.62±0.10、0.51±0.07)(P<0.05),细胞核BIM(0.83±0.13)和FoxO3a(0.57±0.06)表达高于高糖HG组(0.49±0.07、0.33±0.04)(P<0.05);高糖H/R+Srt组H9C2细胞SIRT1(0.75±0.12)、细胞质P-FoxO3a(0.62±0.08)表达高于高糖H/R组,细胞核BIM(0.45±0.05)和FoxO3a(0.26±0.03)表达低于高糖H/R组(P<0.05)。结论 SIRT1可通过抑制FoxO3a/BIM通路的活化来减轻H9C2心肌细胞高糖H/R损伤。
Objective To evaluate the role of SIRT1 in alliviating hypoxia-reoxygenation(H/R)injury in diabetic myocardiocytes by modulating FoxO3 a/BIM pathway.Methods The normally cultured H9 C2 cells in logarithmic phase were randomly divided into high glucose(HG)group,H/R group,and H/R+Srt1720(H/R+Srt)group.The H9 C2 cells in all groups were cultured in HG medium(33 mmol/L)for 24 hto establish HG models.H/R group was exposed to hypoxia(95% N2 and 5% CO2)for 4 hand reoxygenation(90% O2 and 10% CO2)for 2 hto establish HG+H/R models,and H/R+Srt group received Srt1720(20μmol/L)24 hbefore H/R.At the end of H/R,the level of lactate dehydrogenase(LDH)was detected by ELISA method,the cell apoptotic rate was detected by flow cytometer,the changes in mitochondrial membrane potential was detected by JC-1 assay,and the levels of SIRT1,cytoplasm phosphor-FoxO3 a,nucleus FoxO3 aand BIM protein were detected by Western blot.Results The apoptotic rates((42±7)%,(27±5)%),the ratios of JC-1 single to multimer(5.2±0.9,3.3±0.6),and the contents of LDH in culture supernatant((485±62),(394±52)u/L)in H/R group and H/R+Srt group were significantly higher than those in HG group((19±4)%,1.7±0.4,(264±42)u/L)(P〈0.05),and higher in H/R group than those in H/R+Srt group(P〈0.05).The expressions of SIRT1 and phosphor-FoxO3 a(0.41±0.07,0.34±0.05)of H9 C2 cells were significantly lower,and the expressions of BIM and FoxO3 a(0.83±0.13,0.57±0.06)were significantly higher in H/R group than those in HG group(0.62±0.10,0.51±0.07,0.49±0.07,0.33±0.04)(P〈0.05).The expressions of SIRT1 and phosphor-FoxO3 a(0.75±0.12,0.62±0.08)were significantly higher,and the expressions of BIM and FoxO3 a(0.45±0.05,0.26±0.03)were significantly lower in H/R+Srt group than those in H/R group(P〈0.05).Conclusion SIRT1 could relieve H/R injury of H9 C2 cells in diabetic myocardiocytes by inhibiting the activation of FoxO3 a/BIM pathway.
作者
冷燕
吴洋
赵博
李文远
熊永红
王凯
夏中元
LENG Yan;WU Yang;ZHAO Bo;LI Wen-yuan;XIONG Yu-hong;WANG Kai;XIA Zhong-yuan(Department of Anesthesiology, Renmin Hospital of Wuhan University, Wuhan 430000, Chin)
出处
《中华实用诊断与治疗杂志》
2018年第5期431-433,共3页
Journal of Chinese Practical Diagnosis and Therapy
基金
国家自然科学基金(81471844)
国家自然科学基金(81671891)