利拉鲁肽降低2型糖尿病伴非酒精性脂肪性肝病大鼠肝脏磷酸化c-Jun氨基末端激酶的蛋白表达

Liraglutide ameliorates hepatic expression of c-Jun N-terminal protein kinase in type 2 diabetic rats with nonalcoholic fatty liver disease

目的：探讨c-Jun氨基末端激酶（JNK）信号通路与2型糖尿病伴非酒精性脂肪性肝病（NAFLD）的关系及利拉鲁肽的干预作用。方法选用24只雄性Wistar大鼠，按随机数字表法分为正常对照组（NC组，n=6）和实验组（n=18），实验组采用高糖高脂饲料喂养联合腹腔注射小剂量链脲佐菌素建立2型糖尿病动物模型。将成模糖尿病大鼠（n=14）依随机数字表法分为2型糖尿病组（DM组，n=7）及利拉鲁肽干预组（LIR组，n=7）。LIR组给予腹腔注射利拉鲁肽100μg/kg，每日2次，NC组和DM组给予等量的蒸馏水皮下注射；各组均干预8周。实验结束后处死大鼠，取血清测定大鼠血清中肿瘤坏死因子α（TNF-α）、核因子κB（NF-κB）、血脂和肝酶水平，留取肝组织行病理染色，并采用免疫组织化学法及Western blotting法检测各组大鼠肝组织中JNK、磷酸化JNK（p-JNK）的蛋白表达水平。数据采用SNK-q、Spearman相关分析进行统计学处理。结果实验结束时，与NC组相比，DM组大鼠肝脏组织呈弥漫脂肪变性，并伴炎症浸润，p-JNK/JNK显著升高[1.3（0.8,2.0）比1（0，1），Z=-3.055，P〈0.05]。干预8周后，LIR组大鼠肝组织脂肪变性、炎症活动程度较DM组明显改善，肝脏组织p-JNK/JNK蛋白表达水平较DM组明显降低（Z=-2.320，P〈0.05）。与NC组相比，DM组大鼠肝脏p-JNK/JNK蛋白表达是NC组的（2.60±0.03）倍，而LIR组肝脏p-JNK/JNK蛋白表达为NC组的（2.10±0.05）倍。Spearman相关分析显示，肝脏组织p-JNK/JNK和血清TNF-α、NF-κB的表达均呈显著正相关（r=0.623、0.722，均P〈0.05）。结论 JNK的过度激活参与了2型糖尿病NAFLD的发生发展，利拉鲁肽可通过降低JNK的磷酸化，减轻炎症反应，改善糖尿病NAFLD。

Objective To explore the role of hepatic c-Jun N-terminal protein kinase（JNK） signaling pathway in diabetic rat with nonalcoholic fatty liver disease（NAFLD）, and the intervention effect of liraglutide. Methods Twenty-four male Wistar rats （4-week old） were randomly divided into control group （NC, n=6） and type 2 diabetic model group（n=18） with random number table. The model group were given high-fat high-glucose feeding and abdominal injection of little dosage of streptozotocin （30 mg/kg） to set up type 2 diabetic models. And the successfully-established 14 model rats were divided into diabetic group（DM, n=7） and diabetic＋liraglutide group（LIR, n=7） with random number table. Control rats were fed with standard rodent chow diet. LIR group were injected intraperitoneally with liraglutide 100μg/kg twice daily for 8 weeks, while rats in NC and DM groups were injected with distilled water for 8 weeks. Body weight was measured weekly. Blood samples were collected after rats were terminated and serum levels of lipids, tumor necrosis factor-α（TNF-α）, nuclear factor kappa B（NF-κB）, alanine aminotransferase（ALT） and aspartate aminotransferase（AST） were all detected. Liver tissue samples were collected for histological analysis, and hepatic protein expression of JNK and phosphorylated JNK（p-JNK） was measured by immunohistochemistry and Western blotting methods. Data were analyzed by one-way analysis of variance, SNK-q, nonparametric tests and Spearman correlation analysis. Results Histological staining showed liver steatosis and inflammation in DM rats while these pathological alterations were improved in LIR group. Hepatic p-JNK/JNK ratio was significantly higher in DM group than that in NC group（1.3（0.8,2.0） vs 1（0, 1）, Z=-3.055, P〈0.05）, while it was decreased remarkably by liraglutide treatment（Z=-2.320,P〈0.05）. Compared with that in NC group, the hepatic p-JNK/JNK protein expression in DM group was elevated for （2.60±0.03） folds, while it was elevated for （2.10 ± 0.05） folds in LIR group. Serum levels of TNF-α and NF-κB were positively correlated with hepatic p-JNK/JNK ratio with Spearman correlation analysis（r=0.623, 0.722, both P〈0.05）. Conclusions JNK pathway activation may play an important role in the development of NAFLD in type 2 diabetic rats. Liraglutide may improve diabetic NAFLD through decreasing JNK phosphorylation and reducing liver inflammation response.