活化胆碱能抗炎通路对非酒精性脂肪性肝炎炎症反应的抑制作用及其机制

Effects and mechanisms of the inflammatory reaction related to NASH and induced by activation of the cholinergic anti-inflammatory pathway

目的 探讨活化胆碱能抗炎通路对非酒精性脂肪性肝炎（NASH）炎症反应的抑制作用及其机制. 方法 建立高脂高糖饮食诱导的NASH小鼠模型,在正常小鼠和NASH小鼠给予烟碱活化胆碱能抗炎通路,通过肝脏病理切片和细胞因子水平观察肝脏炎症情况;体外培养肝脏巨噬细胞系Raw264.7细胞,给予脂多糖处理后加入不同浓度的烟碱,观察细胞上清液中细胞因子肿瘤坏死因子（TNF）α的水平,通过Western blot观察烟碱对信号通路NF-κB和IκB的影响.多组间比较采用单因素方差分析. 结果 通过肝脏病理检查和肝功能生物化学指标检测确定造模成功.NASH小鼠给予烟碱激活胆碱能抗炎通路后,肝组织炎症程度下降;血清中TNFα水平,烟碱治疗组[（21.95±0.8）pg/ml]较等渗盐水组[（38.07±1.7） pg/ml]显著下降（P＜0.05）.Raw264.7细胞给予脂多糖处理后加入不同浓度的烟碱后检测上清液中TNFα水平,发现加入5 mmol/L以上浓度烟碱后能明显降低脂多糖引起的TNFα增高（P＜0.05）.内毒素刺激后RAw264.7细胞内p-NF-κB水平增加,I-κB水平降低,表明NF-κB通路激活,不同剂量的烟碱能明显下调p-NF-κB水平,升高I-κB水平,并表现出剂量依赖. 结论 活化胆碱能抗炎通路对NASH炎症反应有抑制作用,其作用机制可能为通过NF-κB信号通路抑制炎症反应.

Objective To investigate the effects and mechanisms of the inflammatory reaction related to nonalcoholic steatohepatitis （NASH） and induced by activation of the cholinergic anti-inflammatory pathway.Methods A mouse model of NASH was established by feeding a high-fat and high-sugar diet.Activation of the cholinergic anti-inflammatory pathway was achieved by nicotine administration to the NASH modeled mice and normal controls.Liver biopsies were taken and the concentrations of cytokines were measured.Isolated liver primary Kupffer cells and RAw264.7 cells were cultured,pre-treated or not with lipopolysaccharide （LPS） and exposed to nicotine,after which the supematant concentrations of IL-6 and TNFα were determined by ELISA.The protein expression levels of phosphorylated （p）-NF-κ B and I κ B were detected in primary cultured Kupffer cells by western blotting.Results The mouse model of NASH was successfully established,as evidenced by findings from liver biopsy and serum liver function tests.The degree of liver inflammation in the NASH mice decreased after nicotine administration,and the level of serum TNFα also significantly decreased.The levels of serum TNFα were 21.95 ± 0.8 pg/mL in nicotine-treated mice and 38.07 ± 1.7 pg/mL in the non-nicotine-treated NASH mice （P ＜ 0.05）.The nicotine treatment also significantly reduced the concentration of TNFα in the culture supematants of Kupffer cells after LPS stimulation; moreover,the supematant level of TNFα decreased significantly after the nicotine treatment （P＜ 0.05）.LPS stimulation of the RAw264.7 cells led to an increased level ofp-NF-κ B and a reduced level ofI-κ B,suggesting that the NF-κ B pathway had been activated; different doses of nicotine pre-treatment led to down-regulation of the p-NF-κ B level and up-regulation of the I-κ B level,both in dose-dependent manners.Conclusion Activating the cholinergic anti-inflammatory pathway inhibits the NASH-related inflammatory reaction,and the mechanism for this inhibition involves the NF-κ B signaling pathway.