扶正化瘀方对实验性急性肝损伤肝细胞凋亡的影响

目的观察扶正化瘀方对内毒素诱导的急性肝损伤小鼠肝细胞凋亡的影响与部分机制。方法雄性BABL／c小鼠30只．随机分为3组，正常组10只，模型组10只，治疗组10只。治疗组小鼠给予扶正化瘀方灌胃，每日2次，连续3d，第4日灌胃后1h开始造模；正常组与模型组给予等量生理盐水灌胃。模型组与治疗组小鼠以内毒素10μg／Kg、D-氨基半乳糖900mg／Kg刑量，腹腔注射，诱导急性肝损伤模型；正常组以等剂量生理盐水腹腔注射作为对照。各组小鼠于造模后6h处死，试剂盒法检测血清ALT、AST活性，肝组织TUNEL染色检测肝细胞凋亡，计算凋亡指数；HE染色观察炎症反应与肝细胞坏死；蛋白免疫印迹法分析肝组织细胞凋亡因子bct-2、bax表达。荧光定量PCR法检测bcl-2和bax的基因表达。结果与正常小鼠相比，模型组小鼠血清ALT、AST活性均明显升高，治疗组小鼠较模型组明显降低。大体病理观察，模型组小鼠肝脏明显肿胀、色泽紫黯、表面见散在点状及片状出血点；治疗组小鼠肝脏肿胀明显减轻、色泽红润、表面未见出血点或少量点状出血点。TUNEL染色显示，正常组小鼠肝组织无或偶见肝细胞凋亡；模型组小鼠肝组织中见大量凋亡肝细胞弥漫性分布于肝小叶各区，肝细胞凋亡指数明显升高；治疗组小鼠肝细胞凋亡指数明显降低。HE染色显示模型组小鼠肝组织小叶结构紊乱，肝窦明显充血，大量炎性细胞浸润，肝细胞灶性坏死；治疗组小鼠肝窦充血、炎性细胞浸润与肝细胞坏死均明显减轻。与正常小鼠比较，模型组小鼠肝组织凋亡调节因子bcl-2基因与蛋白表达明显减少，而bax基因与蛋白表达显著增加；扶正化瘀方能促进bcl-2基因与蛋白表达增加，减少bax基因与蛋白表达。结论扶正化瘀方可抑制LPS诱导急性肝损伤小鼠的肝细胞凋亡，部分机制与调节凋亡因子bcl-2

Effect of Fuzheng huayu decoction on hepatocytic apoptosis and necrosis on acute hepatotoxin due to LPS in mice

Objective To explore effect of Fuzhenghuayu decoction on hepatocytic apoptosis in acute liver injured mice induced by LPS. Methods Thirty male BABL/c mice were randomly designed into 3 groups： normal group （10）, model group （10）, treatment group （10）. The treatment group orally take Fuzheng Huayu recipe （4.6g/Kg）, twicea day for 3 days. The normal and model groups take the same volume of saline. 1 h after administration at the fourth day, the model and treatment groups were intraperitoneally injected with LPS （10 la g/Kg） and GaIN （900mg/Kg）, the normal mice with the same volume of saline. All the mice were sacrificed 6 h after injection. The serum parameters of liver function （ALT, AST） were assayed with kits. Hepatocyte apoptosis and necrosis were checked with TUNEL and HE stain. The gene and protein expressions ofbcl-2 and bax were analyzed with real time- PCR and Western blotting. Results Compared to normal mice, serum ALT and AST activity of model group were obviously increased, while the treatment group had a lower level than the model. At the model group, there were a lot of inflammatory cells infiltration and necrotic hepatocytes in liver, but the lesions became much slighter at the treatment existed in liver at model group, the apoptosis group. TUNEL stain show many apoptotic hepatocytes index is 28.05：7.4 （P〈0.01, vs at normal group）; at thetreatment group, the hepatocytic apoptosis index is 10.3 ±4.3 （P〈0.01, vs model group）. The gene and protein expression of bcl-2 was significantly up-regulated in liver in treatment group （P〈0.01）, but those of bax was down-regulated （P〈0.01）. Conclusion Fuzheng Huayu recipe could protect hepatocytes from apoptosis and necrosis in acute liver injury by LPS in mice, and the mechanisms in part were associated with regulations of aooototie factors--bcl-2 and bax.