五灵胶囊对脂多糖诱导枯否细胞内核转录因子-κB表达的作用

Effects of Wuling Capsules on expression of the nuclear factor-kappa B in activate kupffer cell by lipopolysaccharider

目的:通过体内、体外实验探讨五灵胶囊(WL)对免疫性肝脏炎症反应的作用机制。方法:制备卡介苗(BCG)+免疫佐剂+脂多糖(LPS)诱导小鼠免疫性肝损伤模型,WL灌胃给药12d,处死小鼠分离血清并制备肝组织蛋白;予大鼠WL 10、6.25g/kg灌胃4d,腹主动脉取血并制备含药血清;另取SD大鼠分离原代肝细胞和原代枯否细胞(KCs);采用Transwell小室下层培养KCs,含药血清-KCs条件培养上清对小室上层肝细胞作用。酶法测定免疫性肝损伤小鼠血清AST、ALT及条件培养上清中AST、ALT和LDH-L活性。取KCs培养成单层并同步化24h,PDTC和WL分别干预KCs 24h,再加入60μg/L的LPS诱导12h,Real-time PCR检测LPS诱导KCs表达MCP-1、IL-1、TNF-αmRNA水平,Western Blot检测免疫性肝损伤肝组织中和体外培养KCs表达TNFRⅠ、NF-κBs亚蛋白及IκKβ、IκB。结果:WL明显降低免疫性肝损伤小鼠血清ALT、AST活性并抑制肝组织内活化NF-κB信号通路;含药血清Ⅰ、Ⅱ组明显抑制活化KCs分泌促炎因子损伤肝细胞,WL抑制LPS诱导KCs表达NF-κBs信号通路蛋白,下调MCP-1、IL-1、TNF-αmRNA表达。结论:WL治疗慢性肝病炎症反应的作用机制与下调活化NF-κBs信号通路蛋白,阻滞KCs释放过量促炎因子所致肝细胞损伤相关。

Objective： To investigate the effects and mechanism of Wuling capsules （WL） on immunity inflammatory response in liver tissue through experiment of in vivo and vitro. Methods： The models of immunity hepatic injury of mouse induced by lipopolysaccharider （LPS） and Bacillus Calmette-Guerin（BCG） were established, Wuling capsules was treated for 12d, soon after the mouse were executed and separated serum and extracted hepatic tissue protein; other taken WL infused intragastricaUy to rats using 10.0 and 6.25g/kg dose for four days, the medicated serum were collected in the no bacterium from abdominal aorta, again taken rats anesthetized, then the liver cells and kupffer cells （KCs） was separated and purified from rat liver, the KCs displace to Transwell chamber grows into monolayer and medicated serum common cultivation for 24h, abandon besides cultivating supernatant, added 60μg/L LPS action on KCs incubating for 3h, again displace the l×109/mL liver cells on the superstratum of Transwell, continuating culture for 8h. The enzymatic activity level of AST, ALT and LDH in serum and cultivating supernatant were determined by enzymes method. To take the KCs of grow well place culture flask grows into monolayer and synchronization for 12h, pyrrolidine dithiocarbamate （PDTC） and WL respectively with KCs incubation 24h, and the KCs was activated to rejoin 601ag/L -1LPS, the expression level of MCP-1, IL-1, TNF-α mRNA in activated KC were determined by Real-time PCR, the expression of NF- κB signaling pathway protein in hepatic tissue and activated KC were determined by Western bloting. Results： Wuling capsules could obviously decreases the level of ALT and AST in serum of immunity liver injury mouse and activation NF-κB signaling pathway in liver tissue, medicated serum Ⅰ, Ⅱ groups decreased obvioussly the AST and ALT enzymes activity level, WL inhibits the expression of NF-κB signaling pathway protein within KCs activated by LPS, and decreased the MCP-1, IL-1, TNF-α mRNA transcribe and synthesize. Conclusion： WL down-regulated protein expression of NF-κB signaling pathway and that inhibits KC release excessive inflammatory cytokines damage liver cells may be one of the mechanisms for chronic hepatitis treatment.