川陈皮素对非酒精性脂肪性肝病大鼠肝细胞凋亡的影响

The Effect of Nobiletin on Hepatocyte Apoptosis in Rat Model of Non-alcoholic Fatty Liver Disease

目的从肝细胞凋亡途径研究川陈皮素对非酒精性脂肪性肝病(nonalcoholic fatty liver disease,NAFLD)大鼠的保护作用。方法适应性喂养2周后,72只SD大鼠随机分成6组,分别为正常对照组、模型组、阳性药物水飞蓟素片治疗组(14 mg/kg),以及川陈皮素低(100 mg/kg)、中(200 mg/kg)、高剂量(400 mg/kg)组,高脂高糖饮食8周构建NAFLD模型,治疗6周后,HE染色观察肝脏病理变化,计算肝指数,取血清检测ALT、AST、TC、TG、LDL-C、HDL-C、IL-1a、IL-1b和IL-6的含量,Western blot和qRT-PCR法测定肝组织TNF-α、TNFR-1、Fas、FasL、Caspase-8、Caspase-3、Bcl-2和Bax的蛋白及mRNA表达水平。结果正常组大鼠精神活跃,模型组在造模成功后精神萎靡,水飞蓟素片治疗组以及川陈皮素低、中、高剂量组相较于模型组有一定好转。与正常组相比,模型组大鼠肝湿重、肝指数、ALT、AST、TG、TC、LDL-C、IL-1a、IL-1b、IL-6明显升高(P<0.05),HDL-C明显降低(P<0.05);相较于模型组,水飞蓟素片治疗组及川陈皮素低、中、高剂量组肝湿重、肝指数、ALT、AST、TG、TC、LDL-C、IL-1a、IL-1b、IL-6明显降低(P<0.05),HDL-C明显升高(P<0.05);此外川陈皮素高剂量组肝指数、ALT、AST、IL-1a、IL-1b、IL-6明显低于水飞蓟素片治疗组(P<0.05);川陈皮素中剂量和高剂量组TG、TC、LDL-C明显低于水飞蓟素片治疗组(P<0.05),中剂量和高剂量组HDL-C明显高于水飞蓟素片治疗组(P<0.05),体重各组间没有明显差异。与正常组相比,模型组TNF-α、TNFR-1、FasL、Fas、Bax、Caspase-8、Caspase-3蛋白和mRNA表达水平明显升高(P<0.05),Bcl-2蛋白和mRNA表达明显降低(P<0.05);与模型组相比,水飞蓟素片治疗组、川陈皮素低、中、高剂量组TNF-α、TNFR-1、FasL、Fas、Caspase-8、Caspase-3蛋白和mRNA表达明显降低(P<0.05),水飞蓟素片治疗组、川陈皮素低、中、高剂量组Bcl-2表达明显升高(P<0.05)。结论川陈皮素对高脂饮食诱导的非酒精性脂肪性肝病大鼠肝脏具有明显的保护作用,其作用途径可能与川陈皮素调节凋亡相关蛋白表达,降低炎症反应有关。

Objective To study the protective effect of nobiletin on hepatocyte apoptosis in rat model of nonalcoholic fatty liver disease(NAFLD).Methods 72 SD rats were randomly divided into 6 groups:the control group,model group,positive drug silymarin treatment group(14 mg/kg),and nobiletin low dose(100 mg/kg),medium dose(200 mg/kg)and high dose(400 mg/kg)treatment groups.NAFLD rat model was established by high-fat diet for 8 weeks followed by 6 weeks of treatment.After scarification the liver pathology was evaluated by HE staining,liver index was calculated,and serum levels of ALT,AST,TC,TG,LDL-C,HDL-C,IL-1a,IL-1b and IL-6 were measured.Protein expression and mRNA levels of TNF-α,TNFR-1,Fas,FasL,caspase-8,caspase-3,Bcl-2 and Bax in liver tissue were detected by Western Blot and qRT-PCR.Results Compared with the control group,rats in model group were apathetic,and rats in the silymarin group,low dose,medium dose and high dose nobiletin groups were obviously alleviated.In the model group,increase of the liver wet weight,liver index,ALT,AST,TG,TC,LDL-C,IL-1a,IL-1b and IL-6,also decrease of HDL-C(P<0.05)was observed.Compared with the model group,the liver wet weight,liver index,ALT,AST,TG,TC,LDL-C,IL-1a,IL-1b and IL-6 in the silymarin group and the low dose,medium dose and high dose nobiletin groups were significantly decreased(P<0.05)along with significant increase of HDL-C(P<0.05).In addition,the liver index,ALT,AST,IL-1a,IL-1b and IL-6 in the high dose nobiletin group were significantly lower than that of the silymarin group(P<0.05),and the TG,TC and LDL-C in the middle dose and high dose nobiletin groups were significantly lower than that of the silymarin group(P<0.05),while HDL-C in the middle dose and high dose nobiletin groups were significantly higher than that of the silymarin group(P<0.05).There were no significant difference in the body weight.In the model group,TNF-α,TNFR-1,FasL,Fas,Bax,caspase-8 and caspase-3 protein and mRNA levels were significantly increased(P<0.05),and Bcl-2 protein and mRNA levels were significantly declined(P<0.05).Compared with the model group,the TNF-α,TNFR-1,FasL,Fas,caspase-8 and caspase-3 protein and mRNA levels were significantly decreased in the silymarin and the low dose,medium dose and high dose nobiletin groups(P<0.05),and the expression of Bcl-2 in the silymarin treatment group and the low dose,medium dose and high dose nobiletin groups were significantly increased(P<0.05).Conclusion The nobiletin has obvious protective effect on the NAFLD rats induced by high-fat diet,and the mechanism of action was attribute to the inhibition of nobiletin in apoptosis-related proteins expression and inflammatory response.