硫化氢对脂多糖诱导的大鼠肺组织细胞凋亡的影响

Effects of hydrogen sulfide on pulmonary apoptosis induced by lipopolysaccharide in rats

目的探讨硫化氢(H2S)对脂多糖(LPS)诱导的内毒素性急性肺损伤(ALI)大鼠肺组织细胞凋亡的影响。方法♂SD大鼠48只,随机分为6组,每组8只:空白对照组、LPS组、LPS+NaHS低剂量组、LPS+NaHS中剂量组、LPS+NaHS高低剂量组和LPS+PPG组。LPS组、LPS+NaHS低、中、高剂量组和LPS+PPG组舌下静脉注射LPS(5 mg.kg-1),空白对照组注射等容量的生理盐水。LPS+NaHS低、中、高剂量组和LPS+PPG组在注射LPS 3 h时腹腔分别注射低、中、高剂量的氢硫化钠(NaHS)或炔丙基甘氨酸(PPG),空白对照组和LPS组腹腔注射等容量生理盐水。各组大鼠均在舌下静脉注射LPS或生理盐水6 h后处死,取肺组织。应用流式细胞学技术检测各组大鼠肺组织细胞凋亡情况,免疫组化法检测肺组织Bcl-2和Bax的表达,Westernblot检测肺组织天冬氨酸特异性半胱氨酸蛋白酶3和9(Caspase-3、9)的蛋白表达。结果 LPS组与空白对照组比较肺组织细胞凋亡率明显升高,Bcl-2蛋白表达明显减弱,Bax蛋白表达明显增强,Bcl-2/Bax比值明显降低,Caspase-3和Caspase-9蛋白表达明显增强(P<0.05或P<0.01)。与LPS组比较,LPS+NaHS中、高剂量组肺组织细胞凋亡率明显降低,LPS+NaHS高剂量组Bcl-2阳性细胞表达明显增强,LPS+NaHS中、高剂量组Bax阳性细胞表达明显减弱,LPS+NaHS低、中、高剂量组Bcl-2/Bax比值明显增加,LPS+NaHS高剂量组Caspase-3和Caspase-9蛋白表达明显减弱(P<0.05或P<0.01)。与LPS组比较,LPS+PPG组肺组织细胞凋亡率明显升高,Bcl-2阳性细胞表达明显减弱,Bax阳性细胞表达明显增强,Bcl-2/Bax比值明显降低,Caspase-3和Caspase-9蛋白表达明显增强(P<0.05或P<0.01)。结论 LPS诱导的ALI早期肺组织细胞凋亡增加,给予外源性H2S可减少肺组织细胞凋亡,可能与通过下调Bax、Caspase-3、9蛋白表达和上调Bcl-2蛋白表达有关。

Aim To study the effect of hydrogen sulfide （H2S） on pulmonary apoptosis in the lipopolysaccha- ride （ LPS ） -induced acute lung injury （ALI） in rats. Methods Fourty-eight ♂ SD rats were randomly divided into six groups （ n = 8 ） ： Control group, LPS group, LPS ＋ Naris Low-dose group, LPS ＋ Naris Middle-dose group, LPS ＋ NariS High-dose group and LPS ＋ PPG group. Model of ALI was reproduced by in- jection of LPS （5 mg· kg-1） via sublingual vein in LPS group, LPS ＋ NariS Low, Middle, High dose groups and LPS ＋ PPG group. Different doses of sodi- um hydrosulfide （Naris） or DL-propargylglyeine （PPG） were administered at 3 h after administering LPS in LPS ＋ NariS Low, Middle, High dose groups and LPS ＋ PPG group respectively, while normal saline was administered in control group and LPS group. The rats were sacrificed at 6 hours after normal saline or LPS injection via sublingual vein, and the lung tissue was obtained. The apoptosis rate of pulmonary cells was evaluated by flow cytometry （FCM）. Meanwhile, the expressions of Bcl-2 and Bax were respectively de- tected by immunohistochemisty （IHC）, and the ex- pressions of Caspase-3 and Caspase-9 were analysed by Western blot. Results Compared with those of control group, apoptosis rate of pulmonary cells was markably increased, Caspase-3, Caspase-9 and Bax protein ex- pressions were markably increased, and Bcl-2 protein expression and Bcl-2/Bax were significantly decreased in LPS group（P 〈0.05 or P 〈0.01 ）. Compared with those of LPS group, apoptosis rate of pulmonary cells was significantly decreased in LPS ＋ NariS Middle and High dose groups, Bcl-2 protein expression was signifi- cantly increased and Caspase-3, 9 protein expressions were significantly decreased in LPS ＋ NariS High dose group, Bax protein expression was significantly decreased in LPS ＋ NariS Middle and High dose groups, Bcl-2/Bax was significantly increased in LPS ＋ NariS Low, Middle and High dose groups （P 〈 0. 05 or P 〈 0.01 ）. Compared with those of model group, apopto- sis rate of pulmonary cells, Bcl-2 protein expression and Bcl-2/Bax were significantly increased, while Bax and Caspase-3, 9 protein expressions were markably decreased in LPS ＋ PPG group（P 〈 0.05 or P 〈 0. 01 ）. Conclusion Pulmonary cells apoptosis is increased in early ALI induced by LPS. H2S can decrease pulmonary cells apoptosis through down-regulating Bax and Caspase-3, 9 protein expressions, and up-regulating Bcl-2 protein expression.