衢枳壳提取物对CCl_(4)诱导的肝纤维化小鼠肝脏炎症及NF-κB/NLRP3炎性体通路的影响

Effect of extract of Quzhou Aurantii Fructus on hepatic inflammation and NF-κB/NLRP3 inflammasome pathway in CCl_(4)-induced liver fibrosis mice

研究衢枳壳提取物(QAF)对四氯化碳(CCl_(4))诱导的肝纤维化小鼠肝脏炎症的影响及其机制。将60只C57BL/6雄性小鼠按照随机数字表法随机分为对照组(蒸馏水灌胃)、模型组(蒸馏水灌胃)、秋水仙碱组(Col,秋水仙碱2 mg·kg^(-1)·d^(-1)灌胃)、QAF低剂量组(QAF-L,QAF 100 mg·kg^(-1)·d^(-1)灌胃)和QAF高剂量组(QAF-H,QAF 300 mg·kg^(-1)·d^(-1)灌胃)。模型组和各给药组用1 mL·kg^(-1)CCl_(4)(CCl_(4)-橄榄油1∶4)腹腔注射,每周2次。给药和造模共6周。末次给药后16 h,处死小鼠,收集血清以及肝脏组织。检测血清谷丙转氨酶(alanine aminotransferase,ALT)和谷草转氨酶(aspartate aminotransferase,AST)水平,并观察小鼠肝功能情况;HE染色法观察各组小鼠肝组织病理学变化;F4/80免疫组化法观察各组小鼠肝脏炎症细胞浸润情况;采用RT-PCR法检测各组小鼠肝脏肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素(interleukin,IL)^(-1)8和IL^(-1)βmRNA表达;采用Western blot法检测各组小鼠肝脏IκBα,p-IKKα/β,p-p65,NLRP3,caspase^(-1)和cleaved caspase^(-1)表达情况。结果显示,不同剂量QAF显著缓解肝损伤程度,并降低肝纤维化小鼠肝功能指标血清ALT和AST水平。免疫组化结果显示,QAF显著减少肝纤维化小鼠肝脏炎症细胞浸润。RT-PCR和Western blot结果显示,QAF显著降低肝纤维化小鼠肝组织TNF-α,IL^(-1)8和IL^(-1)β基因表达,并抑制肝脏IκBα蛋白降解,以及p-IKKα/β,p-p65,NLRP3和cleaved caspase^(-1)蛋白表达。综上所述,QAF具有改善CCl_(4)诱导的小鼠肝纤维化的作用,其机制可能与抑制NF-κB/NLRP3炎性体介导的炎症信号通路有关。

To study the effect and mechanism of extract of Quzhou Aurantii Fructus(QAF)on liver inflammation in CCl_(4)-induced liver fibrosis mice.Totally 60 C57 BL/6 male mice were randomly divided into control group(distilled water,oral),model group(distilled water,oral),colchicines group(Col,colchicines 2 mg·kg^(-1)·d^(-1),oral),low-dose QAF group(QAF-L,QAF 100 mg·kg^(-1)·d^(-1),oral)and high-dose QAF group(QAF-H,QAF 300 mg·kg^(-1)·d^(-1),oral)by random number table method.The model group and each administration group were injected with carbon tetrachloride(CCl_(4))1 mL·kg^(-1)(CCl_(4)-olive oil 1∶4),twice a week,totally 6 weeks.After the last administration,the mice were sacrificed,and serum and liver tissue were collected.Serum ALT and AST levels were measured in each group to observe the liver function of mice.The pathological changes and inflammatory cell infiltration in liver were observed by HE staining and F4/80 immunohistochemical staining.The mRNA expressions of TNF-α,IL^(-1)8 and IL^(-1)βwere detected by RT-PCR.The protein expressions of IκBα,p-IKKα/β,p-p65,NLRP3,caspase^(-1)and cleaved caspase^(-1)were analyzed by Western blot.The results showed that QAF significantly reduced serum ALT and AST levels,and alleviated the degree of liver damage.The results of immunohistochemistry showed that QAF significantly reduced liver inflammatory cell infiltration in liver fibrosis mice.The results of RT-PCR and Western blot showed that QAF significantly inhibited mRNA expressions of TNF-α,IL^(-1)8 and IL^(-1)βin liver of fibrosis mice.QAF also suppressed the degradation of IκBαprotein and reduced p-IKKα/β,p-p65,NLRP3 and cleaved caspase^(-1)protein expressions.In conclusion,QAF improves CCl_(4)-induced liver fibrosis in mice.The mechanism may be related to the inhibition of NF-κB/NLRP3 inflammasome-mediated inflammation signaling pathway.