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目的:研究莪术含药血清调控大鼠肝星状细胞(hepatic stellate cells,HSCs)中Hedgehog(Hh)信号通路分泌性信号糖蛋白配体(Sonic Hedgehog,Shh)和Wnt信号通路负调节因子分泌型卷曲相关蛋白1(Secreted Frizzled-Related Proteins-1,SFRP1)表达的机制。方法:大鼠50只随机分为空白组、莪术1组(3.75 g/kg)、莪术2组(6.25 g/kg)、莪术3组(8.75 g/kg)、秋水仙碱组,分别制备含药血清。MTT法预实验筛选莪术最佳大鼠灌胃剂量及莪术含药血清最佳作用浓度,根据预实验结果进行正式实验。正式实验中,体外培养的大鼠HSCs分为8组:空白组、模型(瘦素)组、Hh通路抑制剂(环巴胺)组、莪术最佳剂量浓度组、环巴胺+莪术最佳剂量浓度组、Hh通路激动剂(Purmorphamine)组、Purmorphamine+莪术最佳剂量浓度组、秋水仙碱组。除空白组外,其余各组经瘦素(0.1μg/ml)诱导及相应含药血清干预24 h后,MTT法检测HSCs增殖,RT-PCR法检测Shh mRNA和SFRP1 mRNA表达,Western-blot及免疫荧光法检测Shh和SFRP1蛋白表达。结果:预实验得出莪术最佳大鼠灌胃剂量为6.25 g/kg,莪术含药血清最佳作用浓度为10%。正式实验结果提示,用瘦素活化HSCs后,Shh mRNA和蛋白表达显著上调,SFRP1 mRNA和蛋白表达显著下调。环巴胺(20μmol/L)、10%莪术含药血清(6.25 g/kg)、秋水仙碱(100μg/kg)干预后,Shh mRNA和蛋白表达显著下调,SFRP1 mRNA和蛋白表达显著上调。莪术含药血清与环巴胺协同干预后,Shh mRNA和蛋白表达显著下调,SFRP1 mRNA和蛋白表达显著上调。Purmorphamine(1μmol/L)干预后,Shh mRNA和蛋白表达显著上调,SFRP1 mRNA和蛋白表达显著下调。用莪术含药血清干预Purmorphamine组后,Shh mRNA和蛋白表达显著下调,SFRP1 mRNA和蛋白表达显著上调。结论:Shh和SFRP1涉及Hh和Wnt通路的异常调控,在瘦素诱导活化的HSCs中存在负相关关系;莪术含药血清可通过调控瘦素诱导活化的HSCs中Shh和SFRP1的表达,参与Hh和Wnt信号通路抑制HSCs的活化和增殖。
Objective: To study the effects of curcumin-containing serum on the regulation of Hedgehog (Hh) signaling pathway secretory signal glycoprotein ligand (ShH) and the negative regulator of Wnt signaling pathway in rat hepatic stellate cells (HSCs) Mechanism of Secreted Frizzled-Related Proteins-1 (SFRP1) Expression. Methods: Fifty rats were randomly divided into three groups: control group (3.75 g / kg), Curcuma 2 (6.25 g / kg), Curcuma 3 (8.75 g / kg) and colchicine group Serum. MTT pre-experimental screening of the best rat intragastric dose of Curcuma and the best concentration of drug-containing serum, according to pre-experimental results of a formal experiment. Formal experiments, HSCs cultured in vitro were divided into 8 groups: blank group, model (leptin group), Hh pathway inhibitor (cyclopamine) group, the best dose concentration Curcuma, cyclopamine + Curcuma best Dose concentration group, Hh pathway activator (Purmorphamine) group, Purmorphamine + Curcuma optimal dose concentration group, colchicine group. Except for the blank group, the other groups were induced by leptin (0.1μg / ml) and corresponding serum-containing serum for 24 h. The proliferation of HSCs was detected by MTT assay. The expression of Shh mRNA and SFRP1 mRNA was detected by RT- Immunofluorescence method was used to detect Shh and SFRP1 protein expression. Results: The best experimental rat Curcuma Gavage dose of 6.25 g / kg, Curcuma containing serum best concentration of 10%. The results of the formal experiment suggest that the expression of Shh mRNA and protein is significantly up-regulated and the expression of SFRP1 mRNA and protein are significantly down-regulated after leukemic activation of HSCs. The mRNA and protein expression of Shh were significantly down-regulated and the expression of SFRP1 mRNA and protein were significantly up-regulated after treated with 20μmol / L of cyclophosphamide, 6.25 g / kg of curcumin and 10μg / kg of colchicine. Curcumin containing serum and cyclopamine co-intervention, Shh mRNA and protein expression was significantly reduced, SFRP1 mRNA and protein expression was significantly increased. After the intervention of Purmorphamine (1μmol / L), the expression of Shh mRNA and protein were significantly up-regulated and the expression of SFRP1 mRNA and protein were significantly down-regulated. After the Purmorphamine group was treated with curcumin, the expression of Shh mRNA and protein were significantly down-regulated and the expression of SFRP1 mRNA and protein were significantly up-regulated. CONCLUSIONS: Shh and SFRP1 are involved in the abnormal regulation of Hh and Wnt pathways, and have a negative correlation with leptin-induced HSCs. Curcumin-containing serum can regulate the expression of Shh and SFRP1 in activated HSCs by leptin, Wnt signaling pathway inhibits the activation and proliferation of HSCs.