多烯磷脂酰胆碱联合阿托伐他汀对非酒精性脂肪性肝病小鼠的干预效果

The effect of nucleotide binding oligonucleotide domain like receptor protein 3 expression in mice with nonalcoholic fatty liver disease and the treatment of polyene phosphatidylcholine combined with atorvastatin

目的探究非酒精性脂肪性肝病(NAFLD)小鼠核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)的表达和多烯磷脂酰胆碱(PPC)联合阿托伐他汀(ATO)治疗的干预效果。方法从50只C57BL/6雄性小鼠中随机选取10只饲喂普通饲料(Normal组),40只饲喂甲硫氨酸胆碱缺乏(MCD)饲料用于构建NAFLD模型,实验共设置5组,每组10只:Normal组(生理盐水),Model组(NAFLD+生理盐水),PPC组(NAFLD+PPC),ATO组(NAFLD+ATO),Combine组(NAFLD+PPC+ATO)。采用灌胃给药(给药剂量为PPC:0.1 mL/kg,ATO:4 mg/kg),Normal组和Model组灌胃等量生理盐水,每天1次,连续7周。测定小鼠血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)、血清胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白(LDL)以及高密度脂蛋白(HDL)的表达,使用酶联免疫吸附法(ELISA)测定小鼠血清炎症因子IL-18以及IL-1β水平。采用苏木素-伊红(H&E)染色观察小鼠肝脏组织病理损伤程度,使用Masson染色检测肝脏组织纤维化程度,采用免疫组化染色检测小鼠肝脏组织中NLRP3的表达情况;同时使用Western blot检测NLRP3以及下游蛋白Caspase-1以及凋亡相关斑点样蛋白(ASC蛋白)的表达。结果与Normal组相比,Model组小鼠的ALT、AST、TC、TG以及LDL含量显著增加(P<0.05),HDL含量明显降低(P<0.05),Combine组与Model组相比,ALT、AST、TC、TG以及LDL含量明显降低(P<0.05),HDL含量明显升高(P<0.05);Combine组血清炎症因子显著低于Model组小鼠(P<0.05);H&E以及Masson染色显示与Normal组相比,Model组肝脏组织结构紊乱以及纤维化程度增加,Combine组相比于Model组,肝脏组织结构紊乱以及纤维化程度降低。NLRP3免疫组化染色显示Model组肝脏组织NLRP3表达显著增加,Combine组与Model组相比,肝脏组织NLRP3表达明显降低(P<0.05);Western blot结果显示:Model组NLRP3、Caspase-1以及ASC的表达相比于Normal组显著升高(P<0.05),Combine组相比于Model组显著降低(P<0.05)。结论NAFLD小鼠NLRP3的表达显著增加,多烯磷脂酰胆碱联合阿托伐他汀对于NAFLD的治疗作用可能与降低NLRP3的表达密切相关。

Objective To investigate the expression of nucleotide binding oligonucleotide domain like receptor protein 3(NLRP3)in NAFLD mice and the intervention effect of polyene phosphatidylcholine(PPC)combined with avastatin(ATO).MethodsTen C57BL/6 male mice were randomly selected to be fed Normal diet(Normal group),and the other 40 mice were fed methionine choline deficiency(MCD)diet to construct the NAFLD Model.A total of five groups were set up in this study and 10 mice in each group:Normal group(Normal saline),Model group(NAFLD+saline),PPC group(NAFLD+PPC),ATO group(NAFLD+ATO),and Combine group(NAFLD+PPC+ATO).The drug was administered by intragastric administration(PPC:0.1 mL/kg,ATO:4 mg/kg).Meanwhile,the control group and the model group were given intragastric administration of the same amount of normal saline once a day for 7 weeks.Automatic biochemistry analyzer was employed to measure the alanine aminotransferase(ALT),aspertate aminotransferase(AST),serum cholesterol(TC),triglyceride(TG),low density lipoprotein(LDL),and the expression of high-density lipoprotein(HDL)in mice serum.Besides,enzyme-linked immunosorbent(ELISA)was used to detect the level of inflammatory cytokines IL-18 and IL-1βin serum.Hematoxylin-eosin(H&E)staining was used to observe the degree of pathological damage in the liver tissues of mice.The degree of fibrosis in liver tissues was detected by Masson staining.Immunohistochemical staining was conducted to detect the expression of NLRP3 in the liver tissues of mice.Western blot was used to detect the expression of NLRP3,Caspase-1 and ASC.Results Compared with the Normal group,the levels of ALT,AST,TC,TG and LDL in the Model group were significantly increased(P<0.05)and the levels of HDL were significantly decreased(P<0.05).Meanwhile,compared with the Model group,the levels of ALT,AST,TC,TG and LDL in the Combine group were significantly decreased(P<0.05)and the levels of HDL were significantly increased(P<0.05).Serum inflammatory factors in the Combine group were significantly lower than those in the Model group(P<0.05).H&E staining showed that compared with the Normal group,the degree of liver structure disorder was increased in the Model group,and the degree of liver structure disorder was decreased in the Combine group.NLRP3 immunohistochemical staining showed that the expression of NLRP3 was significantly increased in the Model group,while the expression of NLRP3 was significantly decreased in the Combine group compared with the Model group(P<0.05).Western blot results showed that the expression of NLRP3,Caspase-1 and ASC in the Model group was significantly higher than the Normal group(P<0.05),and the expression of NLRP3,Caspase-1 and ASC in the Combine group was significantly lower than the Model group(P<0.05).Conclusion The expression of NLRP3 was significantly increased in NAFLD mice,and the therapeutic effect of polyene phosphatidylcholine combined with atorvastatin on NAFLD may be closely related to the reduction of NLRP3 expression.