姜黄素激活自噬干预非酒精性脂肪肝病模型大鼠氧化应激及炎症反应

Curcumin ameliorates inflammatory reaction and oxidative stress through activation of autophagy in experimental non-alcoholic fatty liver disease rats

背景:自噬、氧化应激及炎症反应在非酒精性脂肪肝病中扮演重要角色,姜黄素具有调节自噬、氧化应激及炎症反应等生物活性。目的:探讨姜黄素对非酒精性脂肪肝病大鼠模型保护作用以及机制研究。方法:高糖高脂饮食8周建立非酒精性脂肪肝病大鼠模型,将40只SD大鼠分成对照组、模型组、姜黄素治疗组、姜黄素+3-甲基腺嘌呤组。在第8周末开始干预,对照组和模型组给予PBS灌胃,姜黄素组给予姜黄素500 mg/(kg·d)灌胃,姜黄素+3-甲基腺嘌呤组给予姜黄素500 mg/(kg·d)灌胃及2 mg/(kg·d)自噬抑制剂3-甲基腺嘌呤腹腔注射,干预8周。检测大鼠血清丙氨酸氨基转移酶、天冬氨酸氨基转移酶及总三酰甘油、总胆固醇、空腹血糖浓度;油红O染色观察各组大鼠肝内细胞脂滴分布,透射电镜观察肝细胞线粒体超微结构;采用硫代巴比妥酸法及黄嘌呤氧化酶法测定丙二醛及超氧化物歧化酶活性;Western blot检测自噬及炎症相关蛋白P62、LC3Ⅱ、Beclin-1、NF-κB的表达。实验方案经武汉大学人民医院动物实验伦理委员会批准(批准号2018-541)。结果与结论:①大鼠血清丙氨酸氨基转移酶、天冬氨酸氨基转移酶及总三酰甘油、总胆固醇水平:模型组均高于对照组(P<0.05),姜黄素治疗组显著低于模型组(P<0.05),姜黄素+3-甲基腺嘌呤组高于姜黄素治疗组但低于模型组(均<0.05);②细胞脂质沉积:模型组明显多于对照组;姜黄素治疗组明显少于模型组,姜黄素+3-甲基腺嘌呤组明显高于姜黄素治疗组但低于模型组;③透射电镜观察发现对照组未见明显线粒体损伤,模型组线粒体明显水肿,嵴断裂或消失等,姜黄素可显著缓解线粒体损伤,姜黄素+3-甲基腺嘌呤组对线粒体损失在模型组及姜黄素之间;④肝组织内超氧化物歧化酶水平:模型组显著低于对照组,姜黄素治疗组显著高于模型组,姜黄素+3-甲基腺嘌呤组显著低于姜黄素治疗组但高于模型组;⑤肝组织丙二醛水平:模型组显著高于对照组,姜黄素治疗组显著低于模型组,姜黄素+3-甲基腺嘌呤组显著高于姜黄素治疗组但低于模型组;⑥Western blot检测:与对照组相比,模型组肝组织P62、NF-κB蛋白表达均增加,而Beclin-1、LC3Ⅱ/LC3Ⅰ均降低(P均<0.05),经姜黄素治疗后P62、NF-κB蛋白表达均降低,而Beclin-1、LC3Ⅱ/LC3Ⅰ增加;姜黄素+3-甲基腺嘌呤组P62、NF-κB蛋白表达均低于模型组但高于姜黄素组,而Beclin-1、LC3Ⅱ/LC3Ⅰ高于模型组但低于姜黄素组,差异均有显著性意义(P均<0.05);⑦结果说明,姜黄素通过激活自噬调节炎症反应及氧化应激而改善非酒精脂肪肝病大鼠模型脂肪变性。

BACKGROUND:Autophagy,oxidative stress and inflammatory reactions play an important role in non-alcoholic fatty liver disease.Curcumin has biological activities such as regulating autophagy,oxidative stress and inflammatory reaction.OBJECTIVE:To explore the effect and underlying mechanism of curcumin on experimental non-alcoholic fatty liver disease rats.METHODS:Non-alcoholic fatty liver model was established in rats fed 8-week high-fat diets.Forty healthy SPF male Sprague-Dawley rats were randomly divided into control group,model group,curcumin treatment group,and curcumin and 3-methyladenine(3-MA,an autophagic flux blocker)treatment group.At the end of 8 weeks of high-fat diet,control and model groups were given PBS intragastrically,curcumin treatment group given curcumin 500 mg/kg per day intragastrically,and curcumin+3-MA given curcumin 500 mg/kg per day intragastrically and 3-MA 2 mg/kg per day intraperitoneally.The interventions in each group were given for 8 continuous weeks.The biochemical parameters including serum alanine aminotransferase,aspartate aminotransferase,total triglyceride,total cholesterol,fasting blood glucose level were measured in rats.Oil red O staining was used to characterize the change of hepatic pathology.The ultrastructure of mitochondria was examined by transmission electron microscopy.The hepatic malondialdehyde level and superoxide dismutase activity were measured by thiobarbituric acid method and xanthine oxidase method,respectively.Western blot assay was performed to detect the expression level of autophagic molecular signals including P62,Beclin,LC3B and nuclear factor-κB.The experimental protocol was approved by the Animal Ethic Committee of Renmin Hospital of Wuhan University(approval No.2018-541).RESULTS AND CONCLUSION:The serum alanine aminotransferase,aspartate aminotransferase,total triglyceride,and total cholesterol levels were significantly higher in the model group than the control group(P<0.05).Compared with the model group,curcumin significantly decreased the levels of serum alanine aminotransferase,aspartate aminotransferase,total triglyceride,and total cholesterol(P<0.05),but this effect was partly inhibited by 3-MA(P<0.05).There was more cellular lipid deposition in the model group than the control group.Compared with the model group,curcumin significantly decreased cellular lipid deposition,but the decrease was partly inhibited by 3-MA.Compared with the control group,mitochondrial edema and cristae rupture(or even completely disappearing)were easily found in the model group.Curcumin significantly attenuated mitochondrial injury,which was partly inhibited by 3-MA.Hepatic superoxide dismutase activity in the model group was significantly lower than that in control group,and it significantly increased after curcumin treatment.The hepatic superoxide dismutase activity in the curcumin+3-MA group was higher than that in model group but lower than that in the curcumin group.Hepatic malondialdehyde level in the model group was higher than that in the control group,and it significantly decreased after curcumin treatment.Whereas the hepatic malondialdehyde level in the curcumin+3-MA group was lower than that in model group but higher than that in the curcumin group.Compared with the control group,the model group showed significantly increased expressions of P62,nuclear factor-κB but decreased expressions of Beclin-1 and LC3II/LC3I(all P<0.05).Curcumin significantly decreased the expressions of P62 and nuclear factor-κB and increased the expressions of Beclin-1 and LC3II/LC3I;however,these changes were partly inhibited by 3-MA(all P<0.05).Therefore,curcumin can effectively prevent hepatic steatosis in experimental non-alcoholic fatty liver disease rats by regulating inflammatory reaction and oxidative stress via activation of autophagy.