NLRP3炎性体在胆总管结扎诱导的肝纤维化大鼠模型中的表达特点

Expression of NLRP3 inflammatory body in a rat model of liver fibrosis induced by common bile duct ligation

目的探讨NLRP3炎性体在胆总管结扎(BDL)大鼠肝纤维化过程中的表达特点,探索NLRP3炎性体与肝纤维化的关系。方法将65只SD大鼠随机分为假手术组(n=15)、BDL模型组(n=50)。分别于第3、7、14、21、28天,处死10只模型组大鼠,同时处死3只假手术组大鼠。检测血清ALT、AST、DBil、TBil、TBA、ALP水平,对肝组织行HE、Masson、天狼星红-苦味酸染色,评估各组肝纤维化程度,免疫组化检测肝组织α平滑肌肌动蛋白(αSMA)、TGFβ1的表达水平,Western Blot、qRT-PCR检测肝组织NLRP3炎性体的表达水平,ELISA法检测肝组织炎症IL-1β水平。计量资料多组间比较采用单因素方差分析,进一步两两比较采用LSD-t检验。结果与假手术组比较,各BDL模型组大鼠血清ALT、AST、DBil、TBil、TBA、ALP水平均升高(P值均<0.05),肝组织IL-1β水平均升高(P值均<0.05),并于第3天达到最高,其后有所降低。与假手术组比较,随时间延长,BDL大鼠模型肝纤维化评分明显增加(P值均<0.05),免疫组化显示αSMA、TGFβ1表达逐渐增加(P值均<0.05),Western Blot、qRT-PCR显示肝组织NLRP3炎性体蛋白表达逐渐增加(P值均<0.05),并于第14天后保持稳定。结论BDL大鼠模型肝功能损伤持续存在,肝组织病理学存在肝炎-肝纤维化-肝硬化的动态演变,NLRP3炎性体处于持续活化状态,可能在肝纤维化过程中发挥重要作用。

Objective To investigate the expression of NLRP3 inflammatory body in the process of liver fibrosis in a rat model of common bile duct ligation(BDL)and the association of NLRP3 inflammatory body with liver fibrosis.Methods A total of 65 Sprague-Dawley rats were randomly divided into sham-operation group with 15 rats and BDL model group with 50 rats.On days 3,7,14,21,and 28,10 rats in the model group and 3 rats in the sham-operation group were sacrificed.The serum levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),direct bilirubin(DBil),total bilirubin(TBil),total bile acid(TBA),and alkaline phosphatase(ALP)were measured,and HE staining,Masson staining,and sirius red-picric acid staining were performed for liver tissue to evaluate liver fibrosis degree.Immunohistochemistry was used to measure the expression levels of alpha-smooth muscle actin(α-SMA)and transforming growth factor-β1(TGF-β1)in liver tissue,Western blot and qRT-PCR were used to measure the expression level of NLRP3 inflammatory body,and ELISA was used to measure the level of the inflammatory factor interleukin-1β(IL-1β)in liver tissue.An analysis of variance was used for comparison of continuous data between groups,and the least significant difference t-test was used for further comparison between two groups.Results Compared with the sham-operation group,the BDL model group had significant increases in the serum levels of ALT,AST,DBil,TBil,TBA,and ALP(all P<0.05)and the level of IL-1βin liver tissue(P<0.05),which reached the highest level on day 3 and then decreased.Compared with the sham-operation group over time,the BDL group had a significant increase in liver fibrosis score(P<0.05);immunohistochemistry showed gradual increases in the expression of SMA-αand TGF-β1(P<0.05),and Western blot and qRT-PCR showed a gradual increase in the protein expression of NLRP3 inflammatory body in liver tissue(P<0.05),which remained stable after day 14.Conclusion Liver injury exists persistently in a rat model of BDL,and liver histopathology shows the dynamic evolution of hepatitis,liver fibrosis,and liver cirrhosis.NLRP3 inflammatory body is in a state of continuous activation and may play an important role in the process of liver fibrosis.