SIRT1通过调节FoxO3a/BIM通路减轻心肌细胞高糖缺氧复氧损伤

Role of SIRT1 in alliviating hypoxia-reoxygenation injury in diabetic myocardiocytes by modulating FoxO3a/BIM pathway

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.