绿原酸抑制JNK信号通路对非酒精性脂肪性肝病的防治作用

Chlorogenic acid exerts preventive and therapeutic effects on nonalcoholic fatty liver disease by inhibiting the JNK signaling pathway

目的非酒精性脂肪性肝病(NAFLD)与胰岛素抵抗密切相关。绿原酸(CG)能调节糖脂代谢、改善胰岛素抵抗。探讨CG在NAFLD中的作用及其可能的分子基础。方法选取30只SD大鼠,随机分为正常饮食组(NG组)、高脂饮食组(HG组)和绿原酸干预组(CG组)。所有大鼠第1周采用普通饲料适应性喂养,第2周开始NG组喂饲普通饲料,HG组和CG组喂饲高脂饮食。第4周起NG组、HG组给予每周3次生理盐水灌胃,CG组给予每周3次灌胃CG。采用全自动生化仪和放射免疫法检测血清生化指标,采用全波长分光光度计测定超氧化物歧化酶(SOD)、丙二醛(MDA)和游离脂肪酸(FFA)。正常血糖高胰岛素钳夹试验技术测定胰岛素抵抗。Western Blot检测自噬及C-Jun氨基末端激酶(JNK)蛋白。计量资料多组间比较采用单因素方差分析,进一步两两比较采用LSD-t检验。相关分析采用Pearson相关性分析。结果与NG组相比,HG组体质量、肝指数、肝湿重、ALT、AST、TG、TC、TNFα及肝匀浆MDA、FFA均明显升高,而SOD明显低于NG组(P值均<0.05)。HG组与NG组相比,p-JNK1和JNK1的蛋白及自噬相关LC3-Ⅰ、LC3-Ⅱ、Beclin-1、Atg3和Atg5蛋白水平升高(P值均<0.05)。且JNK1蛋白的表达与胰岛素抵抗呈正相关。CG组体质量、肝湿重及肝指数显著低于HG组;ALT、AST、TG、TC、TNFα及肝匀浆MDA、FFA比HG组均明显降低,SOD高于HG组;LC3-Ⅰ、LC3-Ⅱ、Beclin-1、Atg3、Atg5表达均显著低于HG组,差异均有统计学意义(P值均<0.05)。结论 CG在NAFLD大鼠模型中通过JNK途径失活抑制自噬来改善肝损伤和胰岛素抵抗,CG可能为治疗NAFLD的潜在手段。

Objective To investigate the role of chlorogenic acid（CG） in nonalcoholic fatty liver disease（NAFLD） and possible molecular basis,since NAFLD is closely associated with insulin resistance and CG can regulate glucose and lipid metabolism and improve insulin resistance.Methods A total of 30 Sprague-Dawley rats were randomly divided into normal diet group（NG group）,high-fat diet group（HG group）,and chlorogenic acid group（CG group）.All rats were given normal diet in week 1 as adaptive feeding; in weeks 2 and 3,the rats in NG group were given normal diet,and those in HG and CG groups were given high-fat diet; since week 4,the rats in NG and HG groups were given normal saline by gavage three times a week,and those in CG group were given CG by gavage three times a week.An automatic biochemical analyzer and radioimmunoassay were used to measure serum biochemical parameters,and a full-wavelength spectrophotometer was used to measure superoxide dismutase（SOD）,malondialdehyde（MDA）,and free fatty acid（FFA）.A euglycemic-hyperinsulinemic clamp was used to measure insulin resistance.Western blot was used to measure autophagy-related proteins and c-Jun N-terminal kinase（JNK） proteins.A one-way analysis of variance was used for comparison of continuous data between multiple groups,and the least significant difference t-test was used for further comparison between two groups.A Pearson correlation analysis was used for correlation analysis.Results Compared with the NG group,the HG group had significantly higher body weight,liver index,liver wet weight,alanine aminotransferase（ALT）,aspartate aminotransferase（AST）,triglyceride（TG）,total cholesterol（TC）,tumor necrosis factor-α（TNF-α）,and levels of MDA and FFA in liver homogenate and a significantly lower level of SOD（all P〈0.05）.Compared with the NG group,the HG group had significant increases in the levels of p-JNK1 and JNK1 proteins and autophagy-related proteins LC3-Ⅰ,LC3-Ⅱ,Beclin-1,Atg3,and Atg5（all P〈0.05）.The protein expression of JNK1 was positively correlated with insulin resistance.Compared with the HG group,the CG group had significantly lower body weight,liver wet weight,liver index,levels of ALT,AST,TG,TC,and TNF-α,and levels of MDA and FFA in liver homogenate,a significantly higher level of SOD,and significantly lower expression of LC3-Ⅰ,LC3-Ⅱ,Beclin-1,Atg3,and Atg5（all P〈0.05）.Conclusion CG improves liver injury and insulin resistance by inhibiting autophagy via the inactivation of the JNK pathway.CG may be a potential method for the treatment of NAFLD.