硫化氢对大鼠肝窦内皮细胞凋亡影响

目的 观察硫化氢对肝窦内皮细胞(SEC)凋亡的影响.方法 利用胶原酶灌注、Percoll密度梯度离心、选择性贴壁培养去除Kupffer细胞方法分离SEC.原代培养SEC分成为正常对照组、过氧化氢诱导组、硫氢酸钠预处理+过氧化氢组、PPG处理组和PPG预处理+过氧化氢组,各组培养24h再行实验研究.经相应的过氧化氢(200 μmol/L)、硫氢酸钠(200 μmol/L)、PPG(2 mmol/L)处理后,应用流式细胞仪检查各组细胞凋亡、线粒体跨膜电压及easpase-3活化情况.结果 SEC在过氧化氢诱导下,细胞线粒体跨膜电压降低,细胞荧光强度增加(t =5.56,P ＜0.01),caspase-3活化,细胞发生凋亡,并以早期凋亡较为明显(t=4.68、7 26,P＜0.01)；硫氢酸钠预处理后,过氧化氢诱导作用降低,细胞荧光强度降低(t=4.391,P＜0.01),caspase-3活化程度降低,细胞凋亡显著减少(t=3.776、5 626,P＜0.01)；应用PPG抑制内源性硫化氢产生后,caspase-3轻度活化,SEC凋亡增加,且以早期凋亡增加为主(t=5.109,P＜0.01)；加过氧化氢诱导后,细胞线粒体跨膜电压减少,细胞内荧光强度显著增加(t=10.635,P＜0.01),caspase-3活化程度增强,SEC的早、晚期凋亡率均显著增加(t=8.036、9.112,P＜0.01).结论 硫化氢可能通过抑制肝窦内皮细胞的线粒体跨膜电压变化,进而抑制caspase-3的活化而抑制肝窦细胞凋亡.

Role of hydrogen sulfide on the apoptosis of liver sinusoidal endothelial cells

Objective To observe the role of hydrogen sulfide on liver sinusoidal endothelia cell apoptosis. Methods Liver sinusoidal endothelia cells （SEC） were isolated by collagenase perfusion, percoll density gradient centrifugation and selective removal of adherent cultured Kupffer cells. Primary cultured cells were divided into five groups, which contained control group, hydrogen peroxide-induced group, sodium hydrogen sulfide ＋ hydrogen peroxide pretreatment group, PPG pretreatment ＋ hydrogen peroxide group and PPG treatment group. All cells were cultured for 24h before experiments. Following cell treatment of hydrogen peroxide（200μmol/L）, sodium hydrogen sulfide （200 μmol/L）
and PPG （2 mmol/L）, the cell apoptosis and mitochondrial transmembrane voltage momitor were examined by flow cytometry, and the activation of caspase-3 detection was examined by Western Blotting. Results Sinusoidal endothelial cells that induced by hydrogen peroxide reduced mitochondrial transmembrane voltage, increased mitochondria fluores- cence （ t = 5.56, P 〈 0.01） and caspase-3 activation, and resulted to cell apoptosis, especially early apoptosis （ t = 4.68, 7.26, P 〈 0.01）. After sodium hydrogen sulfide pretreatment, the mitochondria fluorescence decreased （t = 4.391, P 〈 0.01）, and resulted to significant reduction of SEC apoptosis （ t = 3.776, 5.626, P 〈 0.01 ）. With the use of PPG, caspase-3 activation slightly increased and SEC apoptosis in early stage increased obviously （ t = 5.109, P 〈 0.01）. After hydrogen peroxide induced, it showed strong increase in mitochondria fluorescence （ t = 10.635, P 〈 0.01 ） and caspase-3 activation, and resulted in significantly high apoptosis rate of SEC in both early and late stages （ t = 8.036, 9.112, P 〈 0.01 ）. Conclusions Hydrogen sulfide probably inhibits the apoptosis of liver sinusoidal cells by in- hibiting mitochondrial membrane voltage changes and the activation of cast）ase-3.