青蒿琥酯抑制肝星状细胞microRNA—154/ β—catenin治疗肝纤维化的机制研究(1)
[摘要] 目的 探讨青蒿琥酯抑制肝星状细胞microRNA-154/β-catenin治疗肝纤维化的机制。 方法 培养大鼠肝星状细胞，用不同浓度(0、5、10、20、30、40 μg/mL)青蒿琥酯作用于大鼠肝星状细胞，采用免疫印迹法(Western blot)检测青蒿琥酯作用后细胞内β-catenin蛋白的表达；实时定量RT-PCR检测青蒿琥酯作用后细胞内的miR-154和β-catenin mRNA的相对表达。 结果 0～40 μg/mL青蒿琥酯作用细胞24 h后，免疫印迹法结果显示，β-catenin蛋白量逐渐降低，并呈剂量相关性(P < 0.05)；实时荧光定量PCR结果显示青蒿琥酯作用后，β-catenin mRNA表达量及miR-154表达量逐渐降低，并呈剂量相关性(P < 0.05)。 结论 青蒿琥酯可能是通过抑制microRNA-154表达作用于Wnt/β-catenin信号通路及来抑制肝纤维化的发生，microRNA-154与Wnt/β-catenin信号通路可能存在一定的相关性。
[关键词] 青蒿琥酯；肝星状细胞；miR-154；Wnt/β-catenin信号通路；肝纤维化
[中图分类号] R914.1 [文献标识码] A [文章编号] 1673-7210(2016)01(a)-0035-04
Mechanism research on Artesunate in the treatment of liver fibrosis by inhibiting miR-154/β-catenin in hepatic stellate cell
ZHANG Ying ZHANG Hong PENG Rui WEI Danyun
Department of Pharmacy， Renmin Hospital of Wuhan University， Hubei Province， Wuhan 430060， China
[Abstract] Objective To investigate the mechanism of Artesunate in the treatment of liver fibrosis by inhibiting miR-154/β-catenin in hepatic stellate cell. Methods HSC-T6 cells were cultured in vitro with different concentrations (0， 5， 10， 20， 30， 40 μg/mL) of Artesunate on rat hepatic stellate cells. Western blot was used to detect the expression of β-catenin protein after drug administration. RT-PCR was used to detect the expression of β-catenin mRNA and microRNA-154 after drug administration. Results After treatment with artesunate for 24 h， the results of Western blot showed that the expression of β-catenin protein significantly decreased in a dose-dependent manner (P < 0.05). The results of RT-PCR showed that the expression of β-catenin mRNA and microRNA significantly decreased in a dose-dependent manner (P < 0.05). Conclusion Artesunate may suppress the occurrence of liver fibrosis through inhibiting the expression of microRNA-154 on Wnt/β-catenin signaling pathway. There may exist a certain correlation between miR-154 and the wnt/β-catenin signaling pathway.
[Key words] Artesunate； Hepatic stellate cell； miR-154； Wnt/β-catenin signaling pathway； Liver fibrosis
肝纤维化是所有慢性肝病发展成肝硬化的共同病理基础与必经途径，是“慢性肝炎-肝纤维化-肝癌”的中心环节，并且具有可逆性[1-2]。肝星状细胞(hepatic stellate cell，HSC)的活化是导致肝纤维化发生的主要发病机制，肝损伤后导致HSC大量活化增殖，从而合成分泌大量的细胞外基质(extracellular matrix，ECM)，ECM的大量沉积导致肝纤维化的发生[3]。因此促进活化的HSC凋亡可能成为治疗肝纤维化的有效方法。
Wnt/β-catenin信号通路是促肝纤维化细胞凋亡的主要转导通路之一，通路活化后，β-catenin移位至细胞核，与核内T细胞因子/淋巴增强因子(TCF/LEF)结合诱导靶基因转录，促进肝纤维化的发生[4-5]。micoRNA(miRNA)是一类细胞内源性小片段 RNA分子，由20～25个核苷酸组成[6]。可以在转录后水平通过调控靶基因的表达而影响细胞的增殖、分化、凋亡及个体的生长发育等生命过程[7-8]。近年来研究发现，miRNA参与了多种疾病的病理过程，是重要的疾病候选诊断标志物及潜在治疗靶点[9]。本课题组发现miR-154在大鼠HSC中表达具有较大程度的上调，抑制miR-154的表达可有效地促进细胞凋亡。 (张英 张洪 彭锐 魏丹芸)
[关键词] 青蒿琥酯；肝星状细胞；miR-154；Wnt/β-catenin信号通路；肝纤维化
[中图分类号] R914.1 [文献标识码] A [文章编号] 1673-7210(2016)01(a)-0035-04
Mechanism research on Artesunate in the treatment of liver fibrosis by inhibiting miR-154/β-catenin in hepatic stellate cell
ZHANG Ying ZHANG Hong PENG Rui WEI Danyun
Department of Pharmacy， Renmin Hospital of Wuhan University， Hubei Province， Wuhan 430060， China
[Abstract] Objective To investigate the mechanism of Artesunate in the treatment of liver fibrosis by inhibiting miR-154/β-catenin in hepatic stellate cell. Methods HSC-T6 cells were cultured in vitro with different concentrations (0， 5， 10， 20， 30， 40 μg/mL) of Artesunate on rat hepatic stellate cells. Western blot was used to detect the expression of β-catenin protein after drug administration. RT-PCR was used to detect the expression of β-catenin mRNA and microRNA-154 after drug administration. Results After treatment with artesunate for 24 h， the results of Western blot showed that the expression of β-catenin protein significantly decreased in a dose-dependent manner (P < 0.05). The results of RT-PCR showed that the expression of β-catenin mRNA and microRNA significantly decreased in a dose-dependent manner (P < 0.05). Conclusion Artesunate may suppress the occurrence of liver fibrosis through inhibiting the expression of microRNA-154 on Wnt/β-catenin signaling pathway. There may exist a certain correlation between miR-154 and the wnt/β-catenin signaling pathway.
[Key words] Artesunate； Hepatic stellate cell； miR-154； Wnt/β-catenin signaling pathway； Liver fibrosis
肝纤维化是所有慢性肝病发展成肝硬化的共同病理基础与必经途径，是“慢性肝炎-肝纤维化-肝癌”的中心环节，并且具有可逆性[1-2]。肝星状细胞(hepatic stellate cell，HSC)的活化是导致肝纤维化发生的主要发病机制，肝损伤后导致HSC大量活化增殖，从而合成分泌大量的细胞外基质(extracellular matrix，ECM)，ECM的大量沉积导致肝纤维化的发生[3]。因此促进活化的HSC凋亡可能成为治疗肝纤维化的有效方法。
Wnt/β-catenin信号通路是促肝纤维化细胞凋亡的主要转导通路之一，通路活化后，β-catenin移位至细胞核，与核内T细胞因子/淋巴增强因子(TCF/LEF)结合诱导靶基因转录，促进肝纤维化的发生[4-5]。micoRNA(miRNA)是一类细胞内源性小片段 RNA分子，由20～25个核苷酸组成[6]。可以在转录后水平通过调控靶基因的表达而影响细胞的增殖、分化、凋亡及个体的生长发育等生命过程[7-8]。近年来研究发现，miRNA参与了多种疾病的病理过程，是重要的疾病候选诊断标志物及潜在治疗靶点[9]。本课题组发现miR-154在大鼠HSC中表达具有较大程度的上调，抑制miR-154的表达可有效地促进细胞凋亡。 (张英 张洪 彭锐 魏丹芸)