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目的:研究莪术含药血清调节瘦素活化的大鼠肝星状细胞(hepatic stellate cells,HSC)中刺猬(hedgehog,Hh)信号通路锌指转录因子-1(glioma-associated oncogene homolog-1,Gli1)和Wnt信号通路关键因子β-连环蛋白(β-catenin)表达的机制。方法:按随机数字表法将30只清洁级sprague-dawley(SD)大鼠均分3组,分别给予莪术水煎剂、秋水仙碱、生理盐水灌胃取血制备含药血清。经高效液相色谱法(HPLC)检测血清中莪术成分。体外培养大鼠HSC,分为8组:空白组、模型组、Hh通路抑制剂组、莪术组、Hh通路抑制剂+莪术组、Hh通路激动剂组、Hh通路激动剂+莪术组、秋水仙碱组。除空白组外,其余各组均给予100 ng/m L瘦素诱导后,各组再给予相应药物干预24 h,MTT法检测HSC增殖情况,RT-PCR法检测Gli1 m RNA和β-catenin m RNA表达,Western blot及免疫荧光法检测Gli1和β-catenin蛋白表达。结果:与空白组比较,经瘦素活化的大鼠HSC中Gli1、β-catenin的m RNA和蛋白表达均明显增高(均P=0.000)。与模型组比较,Hh通路抑制剂、莪术含药血清分别干预及莪术含药血清与Hh通路抑制剂协同干预后,Gli1、β-catenin的m RNA和蛋白表达均明显降低(均P=0.000);秋水仙碱含药血清干预后,Gli1 m RNA和蛋白明显降低(P=0.000,P=0.007),β-catenin m RNA和蛋白表达均明显降低(均P=0.000);Hh通路激动剂干预后,Gli1、β-catenin的m RNA和蛋白表达均明显增高(均P=0.000)。与Hh通路激动剂组比较,用莪术含药血清干预Hh通路激动剂组后,Gli1、β-catenin的m RNA和蛋白表达均明显降低(均P=0.000)。结论:莪术含药血清可通过下调瘦素诱导活化的大鼠HSC中Gli1的表达,参与Hh信号通路抑制HSC的活化与增殖,并能通过下调Gli1的表达而下调Wnt信号通路关键因子β-catenin的表达,抑制HSC活化与增殖,从而抑制肝纤维化。
Objective: To study the effects of curcumin-containing serum on regulating the expression of hedgehog (Hh) signaling pathway zinc-finger transcription factor-1 (Gli1) in leptin-activated rat hepatic stellate cells ) And the key factor of Wnt signaling pathway β-catenin (β-catenin). Methods: Thirty clean sprague-dawley (SD) rats were randomly divided into three groups according to random number table method. The rats were treated with Rhizoma Curcumae decoction, colchicine and normal saline, respectively. The content of Curcuma in serum was detected by high performance liquid chromatography (HPLC). The rat HSCs were cultured in vitro and divided into 8 groups: blank group, model group, Hh pathway inhibitor group, Curcuma group, Hh pathway inhibitor + Curcuma group, Hh pathway agonist group, Hh pathway agonist + Ezhu group, Alkali group. Except for the blank group, all the other groups were given 100 ng / ml leptin, and each group was given the appropriate drug intervention 24 h, MTT assay HSC proliferation, RT-PCR method to detect Gli1 m RNA and β-catenin m RNAi, Western blot and immunofluorescence were used to detect the expression of Gli1 and β-catenin. Results: Compared with the blank group, the m RNA and protein expressions of Gli1 and β-catenin in HSC were significantly increased (all P = 0.000). Compared with the model group, the mRNA and protein expressions of Gli1 and β-catenin were significantly decreased after the intervention of Hh pathway inhibitor and Curcuma medicine-containing serum respectively and the intervention of Curcuma medicine containing serum and Hh pathway inhibitor (all P = 0.000) (P = 0.000, P = 0.007), the mRNA and protein expression of β-catenin were significantly decreased (all P = 0.000); Hh pathway agonist intervention After, Gli1, β-catenin m RNA and protein expression were significantly increased (both P = 0.000). Compared with the Hh pathway agonist group, the mRNA and protein expressions of Gli1 and β-catenin were significantly decreased after intervention with the Hh pathway agonist group (all P = 0.000). CONCLUSION: Curcumin-containing serum can down-regulate the expression of Gli1 in HSC induced by leptin, and participate in the activation and proliferation of HSC through Hh signaling pathway. It can down-regulate the expression of Gli1 and down-regulate the expression of key factor β-catenin The inhibition of HSC activation and proliferation, thereby inhibiting liver fibrosis.