延迟给予外源性硫化氢加剧脂多糖引起的大鼠肝细胞损伤

Delayed administration of exogenous hydrogen sulfide exacerbates rat hepatocyte injury induced by lipopolysaccharide

目的:探讨延迟给予外源性硫化氢(hydrogen sulfide,H2S)对脂多糖(lipopolysaccharide,LPS)诱导大鼠肝细胞损伤的影响。方法:培养大鼠肝细胞系BRL细胞,用10μg/mL LPS或不同剂量的H2S供体NaHS(50、100、200μmol/L)单独或共同处理BRL细胞12 h,共同处理时LPS作用细胞1 h后再给予NaHS。锥虫蓝拒染法检测细胞存活率的变化;Hoechst 33258荧光染色法观察细胞凋亡的变化;蛋白免疫印迹法检测内质网应激(endoplasmic reticulum stress,ERS)标志蛋白GRP78及其凋亡相关蛋白CHOP和caspase-12蛋白表达变化。结果:与溶剂对照组相比,LPS引起BRL细胞存活率明显下降(P<0.01),细胞凋亡明显增加(P<0.01),GRP78、CHOP和caspase-12表达上调(P<0.01);与LPS组相比,延迟给予NaHS导致LPS引起的BRL细胞存活率降低(P<0.01),细胞凋亡率增加(P<0.01),GRP78、CHOP和caspase-12蛋白表达上调(P<0.05或P<0.01),NaHS的效应具有量效关系;NaHS单独作用对BRL细胞无明显影响。结论:延迟给予外源性H2S可加重LPS诱导的大鼠肝细胞损伤,机制与上调ERS有关。

Objective： To explore the effect of delayed administration of exogenous hydrogen sulfide（H2S） on rat hepatocyte injury induced by lipopolysaccharide（LPS）. Methods：Rat hepatocyte line BRL cells were cultured and treated with LPS（10μg/mL） and H2S donor sodium hydrosulfide（NaHS, 50, 100, 200 μmol/L） respectively or in a combined manner for 12 h, and 1 h after LPS exposure, NaHS was added into cell medium. The cell viability was evaluated by trypan blue exclusion test. The cyto-nuclear morphological changes of apoptotic cells were detected by the fluorescent dye Hoechst 33258. The protein ex-pressions of GRP78, which is a marker of ERS, and CHOP and caspase-12, which are related with ERS analysed by West-ern Blot. Results： Compared with those of control group, cell viability of BRL cells decreased significantly;apoptosis rate of BRL cells increased markedly;GRP78 , CHOP and caspase-12 protein expressions increased significantly in LPS group （P＆lt;0.01）. Compared with those of LPS group, in LPS with NaHS delayed administration groups, cell viabilities of BRL cells decreased significantly（P＆lt;0.01）;apoptosis rates of BRL cells increased markedly（P＆lt;0.01）;GRP78 , CHOP and caspase-12 protein expressions increased significantly（P＆lt;0.05 or P＆lt;0.01）. And these changes showed certain concentration-dependent on NaHS. NaHS （50, 100, 200 μmol/L） alone had no significant effect on BRL cells. Conclusion： Delayed administration of exogenous H2S can exacerbate hepatocyte injury induced by LPS through the upregulation of ERS.