下瘀血汤下调NLRP3改善高脂饮食诱导小鼠非酒精性脂肪性肝炎

Xiayuxue Decoction Improved HFD-induced-Nonalcoholic Steatohepatitis Mice by Down-regulating NLRP3

目的探讨下瘀血汤对高脂饮食诱导的非酒精性脂肪性肝炎(NASH)的干预作用及机制。方法24只雄性C57/BL6小鼠随机分为低脂饮食(LFD)组(8只)、高脂饮食(HFD)组(16只),喂养6周。第7周始,HFD组随机分为HFD-water组(简称HFD组,8只)和下瘀血汤干预组(高脂饮食+下瘀血汤治疗组,简称XYX组,8只),灌胃共计16周。22周结束后,检测小鼠血清中丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、甘油三酯(TG)、总胆固醇(TC)含量;采用HE染色、油红O染色,观察小鼠肝脏组织形态、脂滴沉积;实时定量PCR检测肝组织中脂肪代谢途径相关因子固醇调节元件结合蛋白1(SREBP-1)、脂肪酸合成酶(FASN)、乙酰辅酶羧化酶α(ACCα),炎症因子肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)、白细胞介素18(IL-18)、簇分化抗原68(CD68)、单核细胞趋化蛋白-1(MCP-1)及核苷酸结合寡聚化结构域蛋白样受体3(NLRP3)炎性小体相关基因等;Western Blot、免疫组化检测NLRP3表达;天狼猩红染色、免疫组化检测a-SMA观察肝脏纤维化。结果与LFD组比较,HFD组血清ALT活性、TC、TG含量显著升高(P<0.05,P<0.01)。肝脏脂肪变明显,NAS评分显著升高(P<0.05);SREBP-1、FANS、ACCα等脂肪代谢合成相关因子显著上调,CD68、MCP-1等炎症相关因子mRNA表达显著升高(P<0.05);NLRP3炎性小体相关基因的表达显著上调(P<0.01,P<0.05);与HFD组比较,XYX组的肝脏脂肪变改善,NAS评分下调,脂肪代谢合成相关因子、炎症因子的mRNA表达下调,NLRP3炎性小体相关基因下调和NLRP3蛋白表达下调(均P<0.05)。天狼猩红染色及α-SMA染色显示HFD组的染色面积较LFD组增加(P<0.05),XYX组则较HFD组减少(P<0.05)。结论下瘀血汤对HFD诱导小鼠NASH有显著改善作用;抑制NLRP3是下瘀血汤改善NASH的作用机制之一。

Objective To observe the intervention effect and mechanism of Xiayuxue Decoction(XYXD)on nonalcoholic steatohepatitis NASH induced by high fat diet.Methods Twenty-four male C57/BL6 mice were randomly divided into low fat diet(LFD,8 mice)group and high fat diet(HFD,16 mice)group.All were fed for 6 weeks.From the 7th week mice in HFD were further randomly divided into HFD-water group(HFD,n=8)and XYXD group(HFD+XYXD treated group,abbreviated as XYX,n=8).The intragastrical medication lasted for 16 weeks.At week 22 after modeling,contents of alanine aminotransferase(ALT),aspartate aminotransferase(AST),triglyceride(TG),and total cholesterol(TC)were detected.The morphology of liver tissue and lipid droplet deposition were observed by HE staining and oil red O staining.Real-time quantitative PCR were used to detect related factors of fat metabolism pathway sterol regulatory element binding protein-1(SREBP-1),recombinant acid synthase(FASN),acetyl coenzyme a carboxylaseα(ACCα),inflammation factors[tumor necrosis factorα(TNF-α),interleukin 1 B(IL-1 B),interleukin 18(IL-18),differentiation-68(CD68),monocyte chemoattractant protein-1(MCP-1)],and nucleotide-binding oligomerization-domain protein-like receptors protein 3(NLRP3)inflammatory body related genes.Western Blot and immunohistochemistry were used to detect NLRP3 expression,Sirius red staining and SMA immunohistochemical staining were used to observe liver fibrosis.Results Compared with LFD group,the activity of serum ALT,contents of TC and TG,hepatic steatosis occurred obviously,NAS score significantly increased in HFD group.SREBP-1,FANS,ACCα,and so on were significantly up-regulated.mRNA expressions of CD68 and MCP-1 significantly increased(P<0.05).Expressions of NLRP3 related genes were significantly up-regulated(P<0.01,P<0.05).Compared with HFD group,hepatic steatosis was improved,NAS score was down-regulated,mRNA expressions of fat metabolism synthesis related factors and inflammatory factors were significantly down-regulated,NLRP3 inflammatory body related genes and NLRP3 protein were significantly down-regulated in XYX group(P<0.05).Sirius red staining andα-SMA staining showed more staining area in HFD group than in LFD group(P<0.05),less staining area in XYX group than in HFD group(P<0.05).Conclusion XYXD significantly improved HFD induced NASH in mice,and inhibiting NLRP3 might be one of its mechanisms for improving NASH.