硫化氢对脂多糖诱导的大鼠肺组织细胞凋亡的影响

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。

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

Aim To study the effect of hydrogen sulfide(H2S) on pulmonary apoptosis in the lipopolysaccharide(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+NaHS Low-dose group,LPS+NaHS Middle-dose group,LPS+NaHS High-dose group and LPS+PPG group.Model of ALI was reproduced by injection of LPS(5 mg·kg-1) via sublingual vein in LPS group,LPS+NaHS Low,Middle,High dose groups and LPS+PPG group.Different doses of sodium hydrosulfide(NaHS) or DL-propargylglycine(PPG) were administered at 3 h after administering LPS in LPS+NaHS 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 detected by immunohistochemisty(IHC),and the expressions 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 expressions 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+NaHS Middle and High dose groups,Bcl-2 protein expression was significantly increased and Caspase-3,9 protein expressions were significantly decreased in LPS+NaHS High dose group,Bax protein expression was significantly decreased in LPS+NaHS Middle and High dose groups,Bcl-2/Bax was significantly increased in LPS+NaHS Low,Middle and High dose groups(P<0.05 or P<0.01).Compared with those of model group,apoptosis 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.