摘要
目的 探讨凉血解毒化瘀方对慢加急性肝功能衰竭(acute-on-chronic liver failure,ACLF)模型小鼠的治疗作用及其对受体Toll样受体4(TLR4)/c-Jun氨基末端激酶(JNK)/核因子κB(NF-κB)通路的影响.方法 采用四氯化碳、细菌脂多糖(LPS)和D-氨基半乳糖(D-GalN)联合诱导法构建慢加急肝衰竭小鼠模型;生化分析检测谷丙转氨酶(ALT)、谷草转氨酶(AST)、总胆红素(TBIL)肝功能指标.肝组织病理学检查.荧光定量PCR检测肝组织白介素-6(IL-6)、肿瘤坏死因子α(TNF-α)、白介素-1β(IL-1 β)、TLR4的mRNA表达.CCK8筛选凉血解毒化瘀方对Raw264.7细胞的干预浓度,酶联免疫吸附法检测巨噬细胞上清TNF-α、IL-6、IL-1β含量.蛋白免疫印迹法检测TLR4/JNK/NF-κB通路相关蛋白(TLR4、NF-κB、细胞外调节蛋白激酶(ERK1/2)、p-ERK1/2、p-JNK、JNK)的表达.结果 与ACLF模型组比较,凉血解毒化瘀方可降低血清ALT、AST、TBIL含量,减少肝组织细胞坏死、纤维化、炎症细胞浸润程度,及肝组织TNF-α、IL-6、IL-1β、TLR4的mRNA表达,并降低肝组织TLR4/JNK/NF-κB通路相关蛋白表达.细胞实验进一步发现凉血解毒化瘀方可抑制巨噬细胞TNF-α、IL-6、IL-1β分泌,下调TLR4/JNK/NF-κB通路相关蛋白表达.结论 凉血解毒化瘀方能有效改善ALCF小鼠肝功能、抑制其炎症反应的疗效,其机制与其下调TLR4/JNK/NF-κB通路有关.
Abstract
Objective To investigate the therapeutic effect of Liangxue-jiedu decoction on acute-on-chronic liver failure(ACLF)model mice and the Toll-like receptor 4(TLR4)/c-Jun amino terminal kinase(JNK)/nuclear factor κB(NF-κB)signaling pathway.Methods An ACLF mouse model was established using combined treatment of carbon tetrachloride,lipopolysaccharide,and D-galactosamine.Biochemical analysis was performed to evaluate liver function indicators,including alanine aminotransferase,aspartate aminotransferase,and total bilirubin.Histopathological examination was conducted to assess liver tissue morphological changes.Quantitative PCR was used to detect the mRNA expression of tumor necrosis factor alpha(TNF-α),interleukin(IL)-6,IL-1 β,and TLR4 in liver tissues.A CCK8 assay was used to evaluate the optimal interventional concentration of Liangxue-jiedu decoction on Raw 264.7 cells.Enzyme-linked immunosorbent assay was used to detect the contents of TNF-α,IL-6,and IL-1βin the cell supernatant.Protein immunoblotting was performed to measure the expression of TLR4/JNK/NF-κB signaling pathway-related proteins,including TLR4,NF-κB,p-ERKl/2,ERK1/2,p-JNK,and JNK.Results Compared with the ACLF model group,the Liangxue-jiedu decoction-treated group showed reduced cell necrosis,fibrosis,and inflammatory cell infiltration in liver tissues;decreased serum levels of alanine aminotransferase,aspartate aminotransferase and total bilirubin;and lower expression of TNF-α,IL-6,IL-1β,and TLR4 mRNA.Liangxue-jiedu decoction reduced TLR4/JNK/NF-κB signaling pathway-related protein expression in liver tissues.The in vitro result also showed that Liangxue-jiedu decoction reduced TNF-α,IL-6,and IL-1 β secretion by macrophage cells and down-regulated the expression of TLR4/JNK/NF-κB signaling pathway proteins.Conclusions Liangxue-jiedu decoction effectively improved liver function in ACLF mice in a manner closely related to the downregulation of TLR4/JNK/NF-κB pathway proteins.
基金项目
福建省自然科学基金面上项目(2023J011465)
维普
万方数据