祛湿化瘀方对非酒精性脂肪性肝炎大鼠组织蛋白酶B和肿瘤坏死因子α表达的影响

Effects of Qushi Huayu Decoction on cathepsin B and tumor necrosis factor-α expression in rats with non-alcoholic steatohepatitis

目的:研究祛湿化瘀方防治大鼠非酒精性脂肪肝的作用机制。方法:除正常对照组5只外,Wistar雄性大鼠30只运用四氯化碳(carbon tetrachloride,CCl4)皮下注射联合高脂低蛋白饮食建立非酒精性脂肪肝模型。自造模2周后,随机分为模型组、祛湿化瘀方组和甘乐(复方二氯醋酸二异丙胺)组,每组10只,分别予祛湿化瘀方和甘乐10ml/kg灌胃。用药2周后取材,检测血清丙氨酸氨基转移酶(alanine aminotransferase,ALT)活性和肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)含量、肝组织甘油三酯(triglyceride,TG)和游离脂肪酸(free fatty acid,FFA)含量;并分析其指标间的相关性。蛋白印迹法检测肝组织组织蛋白酶B(cathepsin B,Ctsb)、磷酸化κB抑制蛋白(phospho-inhibitor kappa B,P-IκB)和TNF-α蛋白表达。免疫组织化学染色检测肝组织中Ctsb表达。结果:祛湿化瘀方可显著降低模型大鼠肝组织TG(P<0.05)、FFA(P<0.01)含量和血清ALT活性(P<0.05),而对照药物甘乐只显示有降低ALT活性(P<0.05)作用;模型组肝组织Ctsb、P-IκB、TNF-α蛋白表达均高于正常对照组,同时血清TNF-α水平显著上升;而祛湿化瘀方组的上述蛋白表达则低于模型组。肝组织TG与FFA含量、血清ALT活性呈正相关;血清TNF-α与肝组织FFA含量、血清ALT活性呈正相关。结论:祛湿化瘀方抑制肝脏脂肪沉积和炎症作用可能与其抑制FFA以减轻其下游"FFA-Ctsb-TNF-α"肝脂毒性通路有关。

Objective： To study the mechanism of Qushi Huayu Decoction （QHD）, a compound of traditional Chinese herbal medicine, in prevention and treatment of non-alcoholic steatohepatitis （NASH）. Methods. Thirty-five Wistar male rats were randomly divided into normal group, untreated group, QHD and Ganle （diisopropylamine dichloroacetate） group. The rats except those in normal group group were subcutaneously injected with carbon tetrachloride （CCl4） for 4 weeks （twice per week） and simultaneously fed with high-fat and low-protein diet for 2 weeks to induce NASH. Then, the rats were administrated with QHD, Ganle, or distilled water for 2 weeks, respectively. After harvest, alanine aminotransferase （ALT） activity and tumor necrosis factor-α （TNF-α） content in serum as well as triglyceride （TG） and free fatty acid （FFA） in liver tissue were evaluated, and relativity analysis among these parameters was performed. Cathepsin B （Ctsb）, phospho-inhibitor kappa B （P-boB）, TNF-α protein expressions in liver tissue were assayed with western-blot. The expression and distribution of ctsb in liver tissue were observed with immunohistochemical method. Results： The contents of TG, FFA and activity of ALT were significantly decreased in QHD group. While in the Ganle group, only the activity of ALT in serum was decreased significantly. Expressions of Ctsb, P-lkB and TNF-a proteins in liver tissues and serum TNF-α level were all enhanced in untreated group which, however, were significantly inhibited in the QHD group. And as expected, there were significant relativities among contents of TG in liver tissues and the content of FFA in liver tissue and activity of ALT in serum, content of TNF-α in serum and content of FFA in liver tissue and activity of ALT in serum. Conclusion： The inhibiting effects of QHD on fat deposition and inflammation in liver are related with its inhibition on the ＂ FFA-Ctsb-TNF-α＂ pathway of lipo-toxicity.