基于TLR4/MyD88/NF-κB信号通路探讨毒消肝清丸对肝硬化内毒素血症大鼠肝组织炎症反应的影响

Effect of Duxiao Ganqing Pill on liver inflammation in rats with cirrhotic endotoxemia: An analysis based on the TLR4/MyD88/NF-κB signaling pathway

目的通过CCl4复合造模制备肝硬化内毒素血症模型,探讨毒消肝清丸对大鼠肝脏炎性损伤的保护机制。方法:66只大鼠造模过程中有17只死亡,取3只大鼠检测内毒素和观察肝脏结构,确定造模成功,剩余随机分成正常组(n=6),模型组(n=8)、乳果糖组(n=8)、毒消肝清丸高、中、低剂量组(223.4 mg/kg、111.7 mg/kg、58.9 mg/kg,n=8)。采用CCl4复合造模法造模8周,制备肝硬化内毒素血症动物模型。正常组和模型组给予蒸馏水,乳果糖组予乳果糖,毒消肝清丸药物组按上述剂量每天给药1次,连续8周。测定内毒素含量变化、HE染色观察肝脏组织变化情况、采用ELISA检测肝脏组织TNFα、IL-1β、IL-6的含量;Western Blot和RT-PCR技术检测TLR4、MyD88、NF-κB蛋白及其mRNA的表达。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法。结果各组间大鼠血浆内毒素水平差异有统计学意义(F=26.011,P<0.001);模型组内毒素水平较正常组显著升高(P<0.01);给药后乳果糖组、毒消肝清丸不同剂量组大鼠血清内毒素含量较模型组均显著降低(P值均<0.01)。各组间大鼠血清IL-6、IL-1β以及TNFα水平差异均有统计学意义(F值分别为35.390、38.271、40.241,P值分别为0.002、0.001、<0.001);与正常组比较,模型组大鼠血清IL-1β、IL-6以及TNFα显著升高(P值均<0.01);给药后乳果糖组及毒消肝清丸不同剂量药物组大鼠血清IL-1β、IL-6以及TNFα水平较模型组均显著降低(P值均<0.01)。各组间大鼠肝组织TLR4、MyD88、NF-κB mRNA及其蛋白表达差异均有统计学意义(F值分别为24.483、29.547、19.242、18.752、22.146、15.834,P值均<0.01);与正常组大鼠相比,模型组大鼠肝脏TLR4、MyD88、NF-κB mRNA及其蛋白相对表达量均显著升高(P值均<0.01);乳果糖和毒消肝清丸不同剂量治疗后大鼠肝组织TLR4、MyD88、NF-κB mRNA及其蛋白相对表达量较模型组均显著下降(P值均<0.01)。与低剂量治疗组比较,中高剂量组大鼠肝脏TLR4、MyD88、NF-κB mRNA及其蛋白相对表达量下降尤为显著(P值均<0.01)。结论药物组可能通过下调肝脏组织TLR4/MyD88/NF-κB信号通路来抑制炎症信号的转导,减少了细胞因子TNFα、IL-1β、IL-6的表达,进而缓解内毒素血症并对肝脏组织起到保护作用。

Objective To establish a rat model of cirrhotic endotoxemia by CCl4 compound modeling,and to investigate the protective mechanism of Duxiao Ganqing Pill against liver inflammatory injury.Methods A total of 66 rats were selected,among which 17 died during modeling.Among the remaining 49 rats,3 were selected to measure endotoxin and observe liver structure to determine whether the model was successfully established,and the other 46 rats were randomly divided into normal group with 6 rats,model group with 8 rats,lactulose group with 8 rats,and high-,middle-,and low-dose Duxiao Ganqing Pill( 223.4,111.7,and 58.9 mg/kg) groups with 8 rats in each group.CCl4 compound modeling was performed for 8 weeks to establish a rat model of liver cirrhosis.The rats in the normal group and the model group were given distilled water,those in the lactulose group were given lactulose,and those in the Duxiao Ganqing Pill groups were given the drug according to the above doses,once a day for 2 consecutive months.The change in the content of endotoxin was measured.HE staining was used to observe the change in liver tissue,ELISA was used to measure the content of tumor necrosis factor-α( TNF-α),interleukin-1β( IL-1β),and interleukin-6( IL-6) in liver tissue,and Western blot and RT-PCR were used to measure the protein and mRNA expression of TLR4,MyD88,and NF-κB.A one-way analysis of variance was used for data comparison between groups,and the least significant difference t-test was used for further comparison of data with homogeneity of variance between two groups.Results There was a significant difference in serum endotoxin level between these groups( F = 26.011,P<0.001);the model group had a signifi-cantly higher endotoxin level than the normal group( P<0.01);after treatment,the lactulose group and high-,middle-,and low-dose Duxiao Ganqing Pill groups had significantly lower serum endotoxin levels than the model group( P<0.01).There were significant differences in serum levels of IL-6,IL-1β,and TNF-α between these groups( F = 35.390,P = 0.002;F = 38.271,P = 0.001;F =40.241,P<0.001);the model group had significantly higher serum levels of IL-6,IL-1β,and TNF-α than the normal group( all P<0.01);after treatment,the lactulose group and high-,middle-,and low-dose Duxiao Ganqing Pill groups had significantly lower serum levels of IL-6,IL-1β,and TNF-α than the model group( all P<0.01).There were significant differences in the mRNA and protein expression of TLR4,MyD88,and NF-κB in liver tissue between these groups( F = 24.483,29.547,19.242,18.752,22.146,and 15.834,all P<0.01);the model group had significantly higher mRNA and protein expression of TLR4,MyD88,and NF-κB in liver tissue than the normal group( all P<0.01);after treatment,the lactulose group and high-,middle-,and low-dose Duxiao Ganqing Pill groups showed significant reductions in the mRNA and protein expression of TLR4,MyD88,and NF-κB in liver tissue( all P<0.01),and the middle-and high-dose Duxiao Ganqing Pill groups had significantly greater reductions than the low-dose Duxiao Ganqing Pill group( all P<0.01).Conclusion Duxiao Ganqing Pill can inhibit the transduction of inflammatory signals by downregulating the TLR4/MyD88/NF-κB signaling pathway in liver tissue,reduce the expression of the cytokines TNF-α,IL-1β,and IL-6,and thus alleviate endotoxemia and exert a protective effect on liver tissue.