线粒体通透性转换孔道在脂多糖致H9c2心肌细胞凋亡中的作用

Effect of mitochondria permeability transition pore on H9c2 myocardial cell apoptosis induced by lipopolysaccharide

目的·观察脂多糖(LPS)刺激下心肌细胞线粒体通透性转换孔道(mPTP)开放状态、心肌细胞凋亡情况及环孢霉素A(CsA)和兰尼定(Rya)联合应用的抗凋亡作用。方法·将H9c2细胞分为对照组、LPS组、LPS+CsA组、LPS+Rya组、LPS+CsA+Rya组;分别检测mPTP的开放状态、细胞内及线粒体Ca^(2+)浓度、线粒体膜电位(ΔΦm),以及反映细胞凋亡的Hoechst染色,Bax、Bcl-2的mRNA表达,caspase 3活性,Bax及Bcl-2蛋白水平的变化。结果·LPS作用心肌细胞24 h后mPTP开放,可使细胞质Ca^(2+)荧光强度增高298%,线粒体Ca^(2+)荧光强度增高231%,约1/3细胞凋亡,caspase 3活性增加了约1倍,上调Bax mRNA表达(P=0.008);联合使用CsA与Rya可有效阻断由LPS引起的心肌细胞mPTP开放,降低LPS所致的细胞质和线粒体的Ca^(2+)浓度增高的幅度,维持正常的ΔΦm,减轻LPS所致的细胞凋亡,抑制caspase 3活性,可显著抑制LPS对心肌细胞Bax mRNA表达的上调作用。结论·mPTP在LPS所致心肌细胞凋亡中发挥重要作用,而CsA与Rya联合应用可有效缓解LPS所致的心肌细胞凋亡。

Objective · To observe mitochondria permeability transition pore （mPTP） opening and apoptosis of H9c2 myocardial cell stimulated by lipopolysaccharide （LPS）, and to explore the anti-apoptotic effect of combined application of cyclosporine A （CsA） and ryanodine （Rya）. Methods · The H9c2 cells were divided into Control group, LPS group, LPS＋CsA group, LPS＋Rya group, and LPS＋CsA＋Rya group. The mPTP opening state, Ca2 ＋ concentration within cell and mitochondrial, mitochondrial membrane potential （ΔΦm）, cell apoptosis, expression of Bax and Bcl-2 at mRNA and protein levels, and activity of caspase 3 were determined respectively. Results · mPTP opened after being stimulated by LPS for 24 h, which increased the fluorescence intensity for Ca2＋ in cytosolic and mitochondria by 298% and 231% respectively, induced about 1/3 cell apoptosis, improved the activity of caspase 3 approximately twice, and enhanced expression of Bax mRNA （P=0.008）. The combined use of CsA and Rya effectively inhibited mPTP opening, increased the enhancement of fluorescence intensity for Ca2＋ in both cytosolic and mitochondria, maintained normal ΔΦm, reduced LPS-induced apoptosis, inhibited the activity of caspase 3, and decreased Bax mRNA expression level induced by LPS in the myocardial cells. Conclusion · mPTP plays an important role in in LPS-induced myocardial apoptosis, whereas the combination of CsA and Rya can alleviate it effectively.