卡维地洛对肝纤维化动物模型TLR4-MyD88-NF-κB信号通路的影响及其抗肝纤维化的作用机制

The effect of carvedilol on the TLR4-MyD88-NF-κB signaling pathway in an animal model of liver fibrosis and its anti-liver fibrosis mechanism

目的探讨卡维地洛对肝纤维化动物模型TLR4-MyD88-NF-κB信号通路的影响及其抗肝纤维化的作用机制。方法将60只SD大鼠作为研究对象,随机分为空白对照组、模型组以及低、中、高剂量卡维地洛组,每组各12只。采用胆总管结扎法建立模型组和卡维地洛组大鼠的肝纤维化模型,并于造模成功后48 h开始,低、中、高剂量卡维地洛组每天分别灌胃给药药液0.5、1.0、1.5 mg/kg;空白对照组和模型组灌胃等量蒸馏水。干预4 w后比较各组大鼠血清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、白蛋白(ALB)水平、炎症因子[白细胞介素-1(IL-1)、白细胞介素-6(IL-6)及肿瘤坏死因子α(TNF-α)]水平以及肝纤维化血清指标[羟脯氨酸(Hyp)、层黏连蛋白(LN)、III型前胶原肽(PIIINP)]水平。并取出每组大鼠的肝脏左叶组织进行切片,采用常规HE染色和Masson三色染色后于显微镜下观察对比肝脏纤维化程度;采用Westernblot法检测各组大鼠肝组织中NF-κB与TLR4/MyD88蛋白表达水平;采用实时定量PCR法检测NF-κB与TLR4/MyD88 mRNA水平。结果HE及Masson三色染色结果显示模型组大鼠肝纤维模型造模成功,与空白对照组比较,模型组IL-1、IL-6、TNF-α、ALT、AST以及Hyp、LN、PIIINP水平明显升高(P<0.05),ALB水平水平明显下降(P<0.05);干预4 w后,低、中、高剂量卡维地洛组大鼠血清ALT、AST、炎症因子IL-1、IL-6、TNF-α水平以及Hyp、LN、PIIINP相比模型组明显下降(P<0.05),ALB水平明显上升(P<0.05),且随着给药剂量增加,各因子水平改善更显著。模型组大鼠TLR4、MyD88和NF-κB蛋白及mRNA表达水平相比空白对照组明显升高(P<0.05)。不同剂量卡维地洛干预后,随着剂量增加,TLR4、MyD88和NF-κB蛋白及mRNA表达水平逐渐降低(P<0.05)。结论卡维地洛可能通过调控TLR4-MyD88-NF-κB信号通路调控TLR4、MyD88以及NF-κB蛋白表达,发挥抑制肝脏炎症反应和抗纤维作用,从而改善肝纤维化程度。

Objective To investigate the effect of carvedilol on the TLR4-MyD88-NF-κB signaling pathway in an animal model of liver fibrosis and its anti-liver fibrosis mechanism.Methods 60 SD rats were taken as the research objects and randomly divided into blank control group,model group and low,medium and high dose carvedilol groups,with 12 rats in each group.The common bile duct ligation method was used to establish a rat liver fibrosis model,and the low,medium and high dose carvedilol groups were given intragastrically with carvedilol 0.5 mg/kg/day,1.0mg/kg/day,1.5mg/kg/day starting 48 hours after the successful modeling,and the blank control group and model group were given the same amount of distilled water.After 4 weeks of intervention,After 4 weeks of intervention,the serum alanine aminotransferase(ALT),aspartate aminotransferase(AST),albumin(ALB)levels,inflammatory factors[interleukin-1(interleukin-1,IL-1),IL-6 and tumor necrosis factor-α(tumor necrosis factor-α,TNF-α)levels and serum indicators of liver fibrosis[hydroxyproline(Hydroxyproline,Hyp),laminin(Laminin,LN),Type III procollagen peptide(PIIINP)levels are compared.The left lobe of the liver of each group of rats was taken out and sliced,and the degree of liver fibrosis was compared under a microscope after conventional HE staining and Masson trichrome staining;In addition,Westernblot method was used to determine the protein expression levels of NF-κB and TLR4/MyD88 in the liver tissues of rats in each group;Real-time quantitative PCR was used to detect the levels of NF-κB and TLR4/MyD88 mRNA.Results The results of HE and Masson trichrome staining showed that the model group of rat liver fiber model was successfully established.Compared with the blank control group,the IL-1,IL-6,TNF-α,ALT,AST,Hyp,LN,and PIIINP levels in the model group were increased(P<0.05),and the ALB level was decreased(P<0.05);After 4 weeks of intervention,the levels of serum ALT,AST,inflammatory factors IL-1,IL-6,TNF-α,Hyp,LN,and PIIINP in the low,medium,and high dose carvedilol groups significantly decreased compared with the model group(P<0.05),the level of ALB increased significantly(P<0.05),and as the dose increased,the level of each factor improved more significantly.The protein and mRNA expression levels of TLR4,MyD88 and NF-κB in the model group were significantly higher than those in the blank control group(P<0.05).After the intervention of different doses of carvedilol,as the dose increased,the protein and mRNA expression levels of TLR4,MyD88 and NF-κB gradually decreased,showing a significant dose dependence(P<0.05).Conclusion Carvedilol may regulate the expression of TLR4,MyD88 and NF-κB protein by regulating the TLR4-MyD88-NF-κB signaling pathway,which can inhibit liver inflammation and antifibrosis,thereby improving liver fibrosis.