葛根素联合有氧运动对非酒精性脂肪肝大鼠SIRT1介导肝细胞自噬的保护作用研究

Study on the protective effects of puerarin combined with aerobic exercise on SIRT1-mediated hepatocyte autophagy in rats with non-alcoholic fatty liverdisease

目的研究葛根素联合有氧运动对非酒精性脂肪肝(NAFLD)大鼠沉默信息调节因子2相关酶1(SIRT1)介导肝细胞自噬的保护作用。方法将53只SD大鼠随机分为空白组10只,造模组43只。造模组大鼠予高脂饲料饮食;空白组大鼠予普通饲料饮食。均喂养10周。最后1次喂养结束后,从造模组大鼠中随机选取3只处死验证造模成功。将造模组剩余40只大鼠按照随机数字表法分为模型组、葛根素组、运动组及葛根素+运动组,各10只。葛根素组、葛根素+运动组大鼠予葛根素250 mg/kg(葛根素以0.5%羟甲基纤维素钠溶解为25 mg/mL混悬液),灌胃容积为10 mL/kg,每日上午灌胃给药;空白组、模型组、运动组大鼠予等容积的双蒸水灌胃,每日1次。均灌胃4周。运动组、葛根素+运动组大鼠于第1、2、3 d在水温28~32℃有氧运动游泳箱内分别适应性游泳30、60、90 min,以后运动方案固定,每日固定时间游泳90 min,每周运动5 d,持续4周。空白组、模型组、葛根素组大鼠关在笼中不运动。末次干预结束后,检测各组大鼠血清甘油三酯(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)、丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)、白细胞介素-1β(IL-1β)、肿瘤坏死因子α(TNF-α);苏木素-伊红(HE)染色观察肝组织病理形态变化,透射电镜观察肝细胞线粒体及自噬体超微结构;蛋白质印迹(Western Blot)法检测肝细胞LC3Ⅱ/LC3Ⅰ、Beclin-1、p62、SIRT1蛋白表达。结果治疗前后模型组、葛根素组、运动组、葛根素+运动组大鼠体质量均高于空白组(P<0.05)。治疗后葛根素组、运动组、葛根素+运动组大鼠体质量均低于模型组(P<0.05);葛根素+运动组大鼠体质量均低于葛根素组、运动组(P<0.05);葛根素组与运动组大鼠体质量比较差异无统计学意义(P>0.05)。模型组TC、LDL-C均高于空白组(P<0.05),HDL-C低于空白组(P<0.05);葛根素组、运动组、葛根素+运动组TG、TC、LDL-C均低于模型组(P<0.05),HDL-C虽高于模型组但比较差异无统计学意义(P>0.05)。运动组TC高于葛根素组(P<0.05),葛根素+运动组TC、LDL-C均低于葛根素组(P<0.05)。葛根素+运动组TC、LDL-C均低于运动组(P<0.05)。与空白组比较,模型组AST、ALT、IL-1β、TNF-α水平均明显升高(P<0.05);与模型组比较,干预各组AST、ALT、IL-1β水平均明显下降(P<0.05),葛根素+运动组TNF-α水平明显下降(P<0.05);与葛根素组比较,运动组AST、ALT、IL-1β、TNF-α水平均上升但无统计学意义(P>0.05);葛根素+运动组AST、ALT、IL-1β、TNF-α水平均低于葛根素组、运动组(P<0.05)。模型组大鼠肝组织LC3Ⅱ/LC3Ⅰ、Beclin-1、p62、SIRT1表达均高于空白组(P<0.05)。葛根素组、运动组、葛根素+运动组大鼠肝组织LC3Ⅱ/LC3Ⅰ均低于模型组(P<0.05),葛根素组、葛根素+运动组大鼠肝组织Beclin-1均高于模型组(P<0.05),葛根素组、葛根素+运动组大鼠肝组织p62均低于模型组(P<0.05),葛根素组、运动组、葛根素+运动组大鼠肝组织SIRT1均高于模型组(P<0.05)。结论葛根素联合有氧运动对NAFLD大鼠肝功能、血脂、炎症因子等指标有很好的改善作用,其作用机制可能是通过SIRT1介导p62激活自噬相关蛋白Beclin-1、LC3Ⅱ/LC3Ⅰ调控大鼠肝细胞的自噬水平。

Objective To investigate the protective effect of Puerarin combined with aerobic exercise on hepatocyte autophagy mediated by Regulatory factor-related enzyme 1(SIRT1)、in rats with nonalcoholic fatty liver disease(NAFLD).Methods A Total of 53 SD rats were randomly divided into blank group(n=10)and model group(n=43).Rats in the model group were fed with high-fat diet,while rats in the blank group were fed with normal diet.They were fed for 10 weeks.At the end of the last feeding,3 rats were randomly selected from the model rats and killed to verify the success of the model.The remaining 40 rats were randomly divided into model group,puerarin group,exercise group and puerarin+exercise group,with 10 rats in each group.Rats in puerarin group and puerarin+exercise group were given puerarin 250 mg/kg.Puerarin was dissolved into 25 mg/mL suspension with 0.5%sodium hydroxymethyl cellulose,and the volume of gavage was 10 ml/kg.Puerarin was given by gavage every morning.Rats in blank group,model group and exercise group were gavaged with equal volume of double distilled water once a day.All patients were given for 4 weeks.The rats in the exercise group and puerarin+exercise group were arranged to swim for 30,60 and 90 minutes in the aerobic exercise swimming box at 28~32℃on the first,second and third days respectively.After that,the exercise program was fixed,and the rats in the exercise group and puerarin+exercise group swam for 90 minutes at a fixed time every day,5 days a week for 4 weeks.Rats in blank group,model group and puerarin group were kept in cages,with no exercise.After the last intervention,serum triglyceride(TG),total cholesterol(TC),high density lipoprotein cholesterol(HDL-C),low density lipoprotein cholesterol(LDL-C),alanine aminotransferase(ALT),aspartate aminotransferase(AST),interleukin-1(IL-1β),tumor necrosis factor-or necrosis and hematoxylin eosin(HE)staining were detected.The ultrastructure of mitochondria and autophagosome was observed by transmission electron microscope.The protein expression of LC3Ⅱ/LC3Ⅰ,Beclin-1,p62 and SIRT1 were detected by Western blot(WB).Results Before and after treatment,the body weight of model group,puerarin group,exercise group and puerarin+exercise group was higher than that of blank group(P<0.05).After treatment,the body weight of puerarin group,exercise group and puerarin+exercise group were lower than that of model group(P<0.05).The body weight of puerarin+exercise group was lower than that in puerarin group and exercise group(P<0.05).There was no significant difference in body weight between puerarin group and exercise group(P>0.05).The body weight of Puerarin+exercise group was lower than that inpuerarin group and exercise group(P<0.05);there was no significant difference between puerarin group and exercise group(P>0.05).TC and LDL-C in model group were higher than those in blank group(P<0.05),HDL-C was lower than that in blank group(P<0.05);TG,TC and LDL-C in Puerarin group,exercise group and Puerarin+exercise group were lower than those in model group(P<0.05),although HDL-C was higher than that of model group,the difference was not statistically significant(P>0.05).TC in exercise group was higher than that in puerarin group(P<0.05),TC and LDL-C of Puerarin+exercise group were lower than those in Puerarin group(P<0.05).TC and LDL-C in Puerarin+exercise group were lower than those in exercise group(P<0.05).Compared with the blank group,the levels of AST,ALT,IL-1βand TNF-αin the model group were significantly increased(P<0.05).Compared with the model group,the levels of AST,ALT and IL-1βin the intervention groups were significantly decreased(P<0.05),and the level of TNF-αin the Puerarin+exercise group was significantly decreased(P<0.05).Compared with the Puerarin group,the levels of AST,ALT,IL-1βand TNF-αin the exercise group were increased,but there was no statistical difference(P>0.05).AST,ALT,IL-1βand TNF-αin Puerarin+exercise group were lower than those in Puerarin group and exercise group(P<0.05).The expressions of LC3Ⅱ/LC3Ⅰ,Beclin-1,p62 and SIRT1 in the model group were higher than those in the blank group(P<0.05).LC3Ⅱ/LC3Ⅰin liver tissue of Puerarin group,exercise group and Puerarin+exercise group were lower than that of model group(P<0.05),Beclin-1 in liver tissue of Puerarin group and Puerarin+exercise group were higher than that of model group(P<0.05),p62 in liver tissue of Puerarin group and Puerarin+exercise group were lower than that of model group(P<0.05).The SIRT1 of liver tissue in Puerarin group,exercise group and Puerarin+exercise group was higher than that in model group(P<0.05).Conclusion The intervention of Puerarin combined with aerobic exercise has a good improvement on liver function,blood lipid,inflammatory factor of NAFLD rats,and the mechanism may be that p62 mediates the activation of autophagy-related proteins Beclin-1 and LC3Ⅱ/LC3Ⅰto regulate the autophagy level of rat hepatocytes through SIRT1.