基于NLRP3基因研究其调节非酒精性脂肪性肝病小鼠炎症反应的作用机制

Research on the Mechanism of NLRP3 Gene’s Regulation on Inflammatory Response in Non-alcoholic Fatty Liver Disease Mice

目的利用NLRP3基因敲除小鼠,研究NLRP3基因在调节高脂高果糖饮食诱导的非酒精性脂肪性肝病(non-alcoholic fatty liver disease,NAFLD)小鼠炎症反应方面的作用机制。方法利用雄性纯合子(NLRP3-/-)小鼠,以高脂高果糖饮食诱导NLRP3基因敲除(knock-out,KO)小鼠和野生型(wild-type,WT)小鼠建立NAFLD模型,分别设为KO高脂高果糖饮食组(KO-HFD组)和WT高脂高果糖饮食组(WT-HFD组),同时设WT组和KO组。通过观察各组小鼠体质量变化,血清和组织样本ALT、AST、TG、TC、MDA、SOD、脂质沉积、细胞凋亡率、IL-1β、IL-18、TNF-α、NF-κB、NLRP3、Caspase-1和ASC等的变化,初步研究NLRP3基因调节NAFLD小鼠炎症反应的作用机制。结果随时间的推移,各组小鼠体质量逐步增加,但KO-HFD组相比WT-HFD组增加较少;各组血清ALT、AST、TG和TC水平逐步升高,但KO-HFD组相比WT-HFD组增幅较小;各组肝组织MDA水平逐步升高,SOD水平逐步降低,但KO-HFD组相比WT-HFD组变化幅度较小;油红O染色和Tunel切片结果显示,各组肝细胞内脂质沉积和细胞凋亡程度均逐渐增大,但KO-HFD组相比WT-HFD组变化较少;各组血清和肝组织炎症因子IL-1β、IL-18、TNF-α、NF-κB水平升高,但KO-HFD组20周相比WT-HFD组20周变化较少,且差异具有统计学意义;WT-HFD组小鼠肝组织NLRP3炎性小体及相关炎症因子蛋白NLRP3、Caspase-1、ASC、IL-1β和IL-18的表达水平相较KO-HFD组升高;WT-HFD组的NLRP3、ASC、Caspase-1、IL-1β、IL-18的mRNA转录水平逐步升高,而KO-HFD组基本未出现变化。结论NLRP3基因在NAFLD小鼠模型中可能被激活,导致NLRP3炎性小体相关蛋白表达的增加,促进下游炎症因子的合成和分泌,造成明显的炎症反应和肝脏损伤,进而推动NAFLD疾病的进展。

OBJECTIVE To study the mechanism of NLRP3 gene’s regulation on inflammatory response in non-alcoholic fatty liver disease(NAFLD)mice induced by high-fat and high-fructose diet using NLRP3 gene knockout mice.METHODS Use male homozygous(NLRP3-/-)mice,and the high-fat and high-fructose diet was used to establish NAFLD model in NLRP3 knockout(KO)mice and wild-type(WT)mice,divided into KO high-fat and high-fructose diet(KO-HFD)group and WT high-fat and high-fructose diet(WT-HFD)group,while the WT and KO groups were also established.The body weight of mice in each group were observed.The changes of ALT,AST,TG,TC,MDA,SOD,lipidosis,apoptosis rate,IL-1β,IL-18,TNF-α,NF-κB,NLRP3,Caspase-1 and ASC in serum and tissue samples were tested to study the mechanism of the NLRP3 gene regulating the inflammatory response in NAFLD mice.RESULTS As time goes on,the mice weight of each group increased gradually,but the KO-HFD group increased less than the WT-HFD group.Each group’s level of ALT,AST,TG,TC in serum increased gradually,but the KO-HFD group increased less than the WT-HFD group.The level of MDA in liver tissues of each group was gradually increased and the level of SOD was gradually decreased,but the change range of KO-HFD group was smaller than that of WT-HFD group.The results of oil red O staining and Tunel section showed that the degree of lipid deposition and apoptosis in hepatocytes increased gradually in all groups,but the changes in KO-HFD group were less than that in WT-HFD group.The levels of serum and liver inflammatory factors IL-1β,IL-18,TNF-αand NF-κB increased in all groups,but the changes of KO-HFD group 20 weeks were less than those of WT-HFD group 20 weeks,and the difference was statistically significant.The expression levels of NLRP3 inflammasomes and related inflammatory cytokines NLRP3,Caspase-1,ASC,IL-1βand IL-18 in liver tissues of WT-HFD group were higher than those of KO-HFD group.The mRNA transcription levels of NLRP3,ASC,Caspase-1,IL-1βand IL-18 in WT-HFD group were gradually increased,while there was no change in KO-HFD group.CONCLUSION The NLRP3 gene may be activated in NAFLD mice model,resulting in increased expression of NLRP3 inflammasome-associated protein,promoting the synthesis and secretion of downstream inflammatory factors,resulting in significant inflammatory response and liver damage,and promoting the progression of NAFLD disease.