吡格列酮对非酒精性脂肪性肝炎小鼠NLRP3炎症小体的影响

Effect of pioglitazone on NLRP3 inflammasome in mice with Non-alcoholic Steatohepatitis

目的通过观察吡格列酮对非酒精性脂肪性肝炎(NASH)小鼠肝脏核苷酸结合寡聚化结构域样受体3(NLRP3)炎症小体信号通路的影响,探讨吡格列酮防治NASH的机制。方法18只6~7周龄SPF级健康雄性C57BL/6J小鼠随机分为正常对照组(NC组)、NASH模型组(HFD组)及吡格列酮干预组(PIO组),每组6只。采用各自的喂食方案饲养16周,随后PIO干预组给予PIO灌胃,NC组和HFD组灌胃给予等体积生理盐水作为安慰剂,连续干预6周。检测血清空腹血糖(FBG)、总胆固醇(TC)、甘油三酯(TG)、丙氨酸氨基转移酶(ALT)和天冬氨酸氨基转移酶(AST)水平,HE及油红O染色观察肝组织病理变化,实时荧光定量PCR(RT-PCR)检测肝组织NLRP3、凋亡相关斑点样蛋白(ASC)的mRNA水平,免疫组化和蛋白免疫印迹(Western blot)检测肝组织NLRP3、含半胱氨酸的天冬氨酸蛋白水解酶-1(caspase-1)、白细胞介素-1β(IL-1β)的蛋白表达。结果与NC组比较,HFD组小鼠的血清FBG、TC、TG、ALT、AST含量明显升高(均P<0.05),肝细胞脂质沉积明显,肝组织Nlrp3、Asc mRNA表达水平明显增加,NLRP3、caspase-1、IL-1β的蛋白水平亦增加(均P<0.05);经吡格列酮干预后,PIO组小鼠的血清FBG、TC、TG、ALT、AST含量较HFD组明显降低(均P<0.05),肝细胞脂质沉积显著改善,肝组织Nlrp3、Asc mRNA水平明显下降,NLRP3、caspase-1、IL-1β的蛋白表达明显下调(均P<0.05)。结论吡格列酮可有效减轻高脂饮食诱导的NASH小鼠肝脏炎症,其机制可能与抑制肝脏NLRP3炎症小体激活及IL-1β的产生有关。

Objective To investigate the mechanism of pioglitazone(PIO)for preventing and treating non-alcoholic steatohepatitis(NASH)via observing the effect on the NLRP3 inflammsome signaling pathway in liver of mice with NASH.Methods Eighteen SPF healthy male C57BL/6J mice were randomly divided into control group,high fat diet(HFD)model group and PIO group.A HFD was given for 16 weeks to establish the NASH models,Then the PIO group was treated with PIO iva gavage for 6 weeks,and the control group and HFD model group were given normal saline as placebo.Serum levels of fasting blood glucose(FBG),total cholesterol(TC),triglyceride(TG),alanine transaminase(ALT)and aspartic transaminase(AST)were detected,and liver histopathological changes were observed by HE and oil-red O staining.The mRNA expression of nucleotide binding oligomeric domain like receptor 3(NLRP3)and apoptosis-associated speck-like protein(ASC)in liver tissues were detected by Realtime PCR.The protein levels of NLRP3,caspase-1 and interleukin-1β(IL-1β)in liver were detected by immunohistochemistry and Western blot.Results Compared with the normal control group,the serum levels of FBG,TC,TG,ALT and AST in the HFD model group were significantly elevated(all P<0.05),and hepatic steatosis was more obvious.In addition,the mRNA expression levels of Nlrp3 and Asc in the liver tissues were significantly increased in the HFD model group,and the protein levels of NLRP3,caspase-1 and IL-1βwere also increased(all P<0.05).However,contrast to the HFD model group,pioglitazone treatment significantly decreased the serum levels of FBG,TC,TG,ALT and AST(all P<0.05),improved hepatic lipid deposition,reduced the mRNA levels of Nlrp3 and Asc,and decreased the protein levels of NLRP3,caspase-1 and IL-1βin the liver tissues(all P<0.05).Conclusion Pioglitazone can effectively reduce the liver inflammation in HFD-induced NASH mice,which may be associated with the inhibition of NLRP3 inflammasome activation and IL-1βproduction in the liver.