Oridonin对急性肝衰竭小鼠肝细胞凋亡的影响及其机制研究

Suppression of TNF-α and JNK-related pro-apoptotic signaling expression of oridonin in mice with LPS/D-Gal-induced acute liver failure

目的探讨冬凌草甲素(oridonin)对脂多糖/D-氨基半乳糖氨(LPS/D-Gal)联合诱导的急性肝衰竭(ALF)小鼠肝细胞凋亡的影响,并探讨其可能的机制。方法取25只小鼠,随机分成5组,每组5只。采用LPS/D-Gal腹腔注射建立小鼠ALF模型,设生理盐水对照组、LPS/D-Gal诱导模型组、LPS/D-Gal诱导和不同剂量oridonin干预组及oridonin处理组。采用末端转移酶介导的缺口末端标记法(TUNEL)检测肝细胞凋亡,采用real-time PCR法检测肝组织TNF-αm RNA水平,采用Western blot法检测凋亡相关蛋白的变化。结果模型组小鼠肝细胞凋亡率为(36.4±1.8)%,显著高于两个oridonin干预组的[(19.4±3.3)%和(11.4±0.3)%,P<0.01];模型组小鼠肝组织TNF-αm RNA水平显著高于正常对照组(P<0.01),而两个oridonin干预组肝组织TNF-αm RNA水平显著低于模型组(P<0.01);模型组小鼠线粒体凋亡通路相关的促凋亡蛋白c-Jun氨基末端激酶(JNK)、bax、细胞色素C、cleaved caspase9/3活化水平显著高于两个oridonin干预组(P<0.01),模型组小鼠抗凋亡蛋白bcl-xl水平显著低于两个oridonin干预组(P<0.01),模型组小鼠死亡受体凋亡通路相关的促凋亡蛋白caspase8活化水平与两个oridonin干预组并无明显差异(P>0.01)。结论 Oridonin可抑制LPS/D-Gal诱导的ALF小鼠肝细胞凋亡,其机制可能与下调促凋亡细胞因子TNF-α水平和抑制JNK介导的线粒体凋亡信号通路有关。

Objective To investigate the anti-apoptosis effect of oridonin in mice with lipopolysaccharide （LPS）/D-galactosamine（D-Gal）-induced acute liver failure （ALF）. Methods 25 mice were randomly divided into five groups （5 in each）,e.g.,normal,model,oridonin-intervened and oridonin-intervened at different doses,and oridonin for 12 days. ALF model was established in C57BL/6 mice by intraperitoneal injection of LPS/D-Gal. TUNEL,real-time PCR and Western blot were applied to related detection. Results Hepatocyte apoptosis rate in mice in model group was （36.4±1.8）%,significantly higher than that in oridonin-intervened groups [（19.4±3.3）%and （11.4±0.3）%,respectively,P〈0.01];administration of oridonin significantly decreased hepatic TNF-α mRNA level in model group （P〈0.01）;the expressions of mitochondrial-dependent pro-apoptotic protein such as JNK, bax,cytochrome C,cleaved caspase8/9/3 were upregulated in the model group as compared to in the control group;Pretreatment with oridonin significantly reversed the changes of apoptosis signal pathway induced by LPS/D-Gal, the expression of caspase 8 was not significantly changed and the expression of anti-apoptotic protein bcl-xl increased. Conclusion Oridonin has an anti-apoptosis effect on LPS/D-Gal-induced ALF in mice,and its mechanism may be related to the suppression of pro-apoptotic cytokine TNF-α and JNK-related pro-apoptotic signaling.