尼古丁减轻高脂高果糖诱导的非酒精性脂肪性肝炎小鼠的肝脏炎症

Nicotine alleviates the liver inflammation of non-alcoholic steatohepatitis induced by high-fat and high-fructose in mice

目的:探讨活化胆碱能抗炎通路对非酒精性脂肪性肝炎(non-alcoholic steatohepatitis,NASH)模型小鼠肝脏炎症的抑制作用及其分子机制。方法:60只雄性6周龄的无特定病原体(specific pathogen free,SPF)级C57BL/6J小鼠被随机分为4组:正常饮食小鼠生理盐水注射组、正常饮食小鼠尼古丁注射组、NASH模型小鼠生理盐水注射组和NASH模型小鼠尼古丁注射组,分别给予普通饮食及高脂饮食加高果糖饮水,喂养17周后建立NASH小鼠模型,然后予各组小鼠生理盐水或尼古丁腹腔注射,每天1次,注射量为400μg/kg,注射3周。3周后处死动物进行肝组织病理检查,取小鼠血清行酶联免疫吸附测定(enzyme linked immunosorbent assay,ELISA)检测炎症因子白细胞介素-6(interleukin-6,IL-6)和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α),同时原代分离培养肝巨噬细胞,使用Western blot和荧光共聚焦显微镜检测α7尼古丁型乙酰胆碱能受体(alpha 7 nicotinic acetylcholine receptors,α7n AChR)、Toll样受体-4(Toll-like receptors-4,TLR-4)和磷酸化核转录因子-κB(nuclear factorκB of phosphorylation,p-NF-κB)的蛋白水平。结果:成功建立了NASH小鼠模型。给予小鼠尼古丁治疗后,小鼠肝组织病理结果显示,小鼠肝脏炎症和脂肪变性明显减轻;ELISA结果显示,小鼠血清中炎症因子IL-6、TNF-α水平下降;Western blot和荧光共聚焦显微镜结果显示,尼古丁治疗组小鼠α7n AChR蛋白水平上调,p-NF-κB水平下调。结论:活化胆碱能抗炎通路可以通过抑制NF-κB通路减轻NASH小鼠的肝脏炎症。

Objective： To investigate the anti-inflammation effects by activation of the cholinergic antiinflammatory pathway and its mechanisms in non-alcoholic steatohepatitis（ NASH） model mice. Methods： 6-week-old male C57 BL /6J（ B6） mice were randomly divided into four groups： the first group was normal mice,injected with saline; the second group was normal mice,injected with nicotine; the third group was NASH model mice,injected with saline; the fourth group was NASH model mice,injected with nicotine. The experimental mice were fed with either standard chow（ SC） or high-fat and highfructose（ HFHF） for 17 weeks to generate an NASH model mice. The mice received injection once daily for 3 weeks [nicotine dose,400 μg / kg]. Then,their pathological characteristics and function of the liver were assessed. The expressions of interleukin-6（ IL-6） and tumor necrosis factor-α（ TNF-α） in serum were analyzed by enzyme linked immunosorbent assay（ ELISA）. The expressions of alpha 7 nicotinic acetylcholine receptors（ α7n AChR）,Toll-like receptors-4（ TLR-4） and nuclear factor κB of phosphorylation（ p-NF-κB） in Kupffer cells were determined by Western blot and immunofluorescence assays. Results： We successfully generated NASH model mice by imitating the high-fat and high-fructose dietary style of NASH patients. The results of our investigation demonstrated that nicotine could reduce significantly the levels of IL-6,and TNF-α in serum（ P〈0. 05）. The expression of p-NF-κB protein in the group which was NASH model mice injected with nicotine declined significantly as compared with the group which was NASH model mice injected with saline（ P〈0. 05）. And the expression of α7n AChR protein elevated significantly conversely（ P〈0. 05）. Conclusion： Activation of the cholinergic antiinflammatory pathway could inhibit the release of inflammatory factors as TNF-α and IL-6 in NASH model mice,and the mechanism for the inhibition of inflammatory was mediated by NF-κB pathway.