茵陈对非酒精性脂肪肝小鼠肝脏脂质沉积及p38 MAPK、p62/LC3表达的影响

Effect of Artemisia capillarisThunb.on hepatic steatosis and the expression of hepatic p-38 MAPK and p62/LC3 in NAFLD mice

目的观察茵陈对高脂诱导的非酒精性脂肪肝小鼠的作用及其可能机制,为茵陈的临床合理应用提供依据。方法从42只C57BL/6J小鼠中随机选取12只作为普食组,给予普通饮食;剩余30只给予高脂饮食(60%kcal脂肪)喂养12周建立非酒精性脂肪肝模型。造模成功后,将小鼠随机分为模型组、茵陈低剂量组、茵陈高剂量组,每组9只。茵陈低剂量组和茵陈高剂量组小鼠在高脂饮食同时分别给予茵陈1.95 g/(kg·d)和7.8 g/(kg·d)灌胃,模型组给予高脂饮食同时给予等量蒸馏水灌胃,普食组给予普通饮食同时给予等量蒸馏水灌胃,均每日1次,连续灌胃16周。每周常规检测小鼠体质量、进食量,16周末收集各组小鼠血样本、肝脏组织,检测血清丙氨酸氨基转移酶(ALT)、门冬氨酸氨基转移酶(AST)、三酰甘油(TG)、总胆固醇(TC)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)水平,应用HE染色、油红O染色观察肝脏病理情况,根据病理结果进行NAS评分(肝脏脂肪变性积分、小叶炎症积分、肝细胞气球样变积分),检测肝脏组织中TG和TC含量,应用Western blot法检测肝脏组织中磷酸化p38(p-p38 MAPK)、总p38(p38 MAPK)、p62、LC3蛋白表达情况。结果与普食组比较,模型组小鼠体质量及血清ALT、AST、TC、HDL-C、LDL-C水平均明显增高(P均<0.05),肝脏实体可见弥漫性肿大,色泽偏黄,表面油腻感,边缘变钝,HE染色可见大量脂滴空泡、部分气球样变性及少量炎性灶,油红O染色脂滴增大、增多,肝脏脂肪变性积分、小叶炎症积分、肝细胞气球样变积分及肝脏组织中TG含量均明显增高(P均<0.05);与模型组比较,茵陈干预组体质量、进食量及血清AST、TG、TC、HDL-C、LDL-C水平无明显变化(P均>0.05),血清ALT水平明显降低(P<0.05),肝脏肿大程度减轻,色泽较模型组红润,且茵陈低剂量组肝脏组织中TG含量、肝脏脂肪变性积分、肝细胞气球样变积分均明显降低(P均<0.05),茵陈高剂量组肝脏组织中TG含量、肝脏脂肪变性积分、小叶炎症积分、肝细胞气球样变积分无明显变化(P均>0.05),HE染色、油红O染色可见肝脏内脂滴减少。模型组小鼠肝脏组织中p-p38 MAPK、p62、LC3蛋白表达水平均明显高于普食组(P均<0.05);茵陈低剂量组p-p38 MAPK、p62蛋白表达水平均明显低于模型组(P均<0.05),LC3蛋白表达水平与模型组比较差异无统计学意义(P>0.05)。结论低剂量茵陈能减少高脂诱导的非酒精性脂肪肝小鼠肝脏TG沉积,保护肝细胞,延缓非酒精性脂肪肝的进展,其作用机制可能与激活p38 MAPK通路,促进自噬有关。

Objective It is to investigate the effect of Artemisia capillarisThunb.(AC)on non-alcoholic fatty liver(NAFD)mice model induced by high fat diet and its possible mechanism to provide a basis for the reasonable clinical application of this Chinese drug.Methods Twelve of 42 C57BL/6J mice were randomly selected as the general diet group and given normal diet;the remaining 30 mice were fed with high-fat diet(60%kcal fat)for 12 weeks to establish NAFD models.After successful modeling,the mice were randomly divided into model group,AC low-dose group,AC high-dose group,9 mice in each group.The mice in the AC low-dose group and the AC high-dose group were given AC 1.95 g/(kg·d)and 7.8 g/(kg·d)respectively by gavage based on high-fat diet at the same time.The model group was given the same amount of distilled water by gavage based on a high-fat diet at the same time,and the general diet group was given the ordinary diet and the same amount of distilled water by gavage,both once a day for 16 weeks.The weight and food intake of mice were routinely checked every week.The blood samples and liver tissues from each group at the end of the 16th week were collected to determined the levels of serum alanine aminotransferase(ALT),aspartate aminotransferase(AST),and triacylamide Glycerol(TG),total cholesterol(TC),high-density lipoprotein cholesterol(HDL-C),low-density lipoprotein cholesterol(LDL-C),HE staining and oil red O staining were used to observe hepatic pathology,NAS scores(liver steatosis score,lobular inflammation score,hepatocyte ballooning degeneration score)according to the pathology,the contents of TG and TC in liver tissue were detected,and Westernblot method was used to detect the expression of phosphorylated p38(p-p38 MAPK)and total p38(p38 MAPK),p62,LC3 protein.Results Compared with the general diet group,the body weight and levels of serum ALT,AST,TC,HDL-C,and LDL-C of the mice in the model group were significantly increased(all P<0.05),and the liver entities showed diffuse swelling with yellow,greasy surface and passivated edge.HE staining showed a large number of lipid droplet vacuoles,some balloon-like degeneration and a small number of inflammatory focus,Oil red O staining showed the number and size of lipid droplets were increased,the scores of liver steatosis,lobular inflammation,and hepatocyte ballooning degeneration and the content of TG in liver tissue were significantly increased(all P<0.05);compared with the model group,the body mass,food intake and levels of serum AST,TG,TC,HDL-C,and LDL-C of the AC intervention group were not significantly changed(P>0.05),serum ALT levels was significantly reduced(P<0.05),liver swelling was reduced,and the color was rudder than the model group,the scores of liver steatosis and hepatocyte ballooning degeneration and the content of TG in liver tissue were significantly were significantly reduced in AC low dose group(all P<0.05),and there were no significant changes in the TG content,liver steatosis score,lobular inflammation score,and hepatocyte ballooning scores in the liver tissue of the AC high-dose group(all P>0.05),HE staining and Oil Red O staining showed a decrease in lipid droplets in the liver.The expression levels of p-p38 MAPK,p62 and LC3 proteins in the liver tissues of the model group were significantly higher than those in the general diet group(all P<0.05);the expression levels of p-p38 MAPK and p62 proteins in the AC low-dose group were significantly lower than those in the model group(all P<0.05),there was no significant difference in the expression level of LC3 protein compared with the model group(P>0.05).Conclusion Low-dose AC can reduce TG deposition in the liver,protect liver cells,and delay the progression of NAFD in NAFD mice induced by high fat.Its mechanism of action may be related to activation of the p38 MAPK pathway and promotion of autophagy.