青蒿琥酯通过抑制ERK1/2蛋白活化负性调控HSC-T6细胞cyclin D1的表达和AP-1活性

目的: 通过观察青蒿琥酯(artesunate,Art)对肝星状细胞(hepatic stellate cells,HSCs)细胞外信号调节蛋白激酶1/2(extracellular signal-regulated kinase 1/2,ERK1/2)、激活蛋白1(activator protein 1,AP-1)和细胞周期蛋白D1(cyclin D1)的影响,探讨Art抗肝纤维化的分子机制。方法: 用血小板源性生长因子BB(platelet-derived growth factor BB, PDGF-BB)诱导HSC-T6细胞的增殖。实验分为对照组、PDGF-BB组、PDGF-BB+Art组(Art终浓度6.25 mg/L、25 mg/L、50 mg/L)和PDGF-BB+ERK抑制剂PD98059组。ELISA法测定细胞上清液中Ⅰ型胶原的含量,RT-PCR检测各组ERK1/2和cyclin D1 mRNA水平变化,Western blotting法检测各组p-ERK1/2和cyclin D1蛋白表达水平,凝胶电泳迁移率实验(EMSA)检测AP-1的活性。结果: (1)PDGF-BB 可明显促进HSC-T6细胞的增殖,PDGF-BB+Art各剂量组和PDGF-BB+PD98059均可抑制HSC-T6细胞上清液中Ⅰ型胶原的含量(P<0.05,P<0.01)。(2)PDGF-BB+Art组(50 mg/L)ERK1/2 mRNA的表达低于PDGF-BB组(P<0.05),其中PDGF-BB+PD98059组ERK1/2 mRNA的表达最低(P<0.01);PDGF-BB+Art组(25 mg/L、50 mg/L)和PDGF-BB+PD98059组cyclin D1 mRNA的表达明显低于PDGF-BB组(P<0.05,P<0.01)。(3)p-ERK1/2、cyclin D1蛋白表达水平在PDGF-BB组最高,而PDGF-BB+Art各剂量组和PDGF-BB+PD98059组降低(P<0.05,P<0.01)。(4)Art可使AP-1与DNA结合活性下降。结论: Art可抑制HSC-T6细胞增殖,其机制可能与其抑制ERK1/2蛋白活化从而降低AP-1的活性和下调cyclin D1的表达有关。

Artesunate negatively controls expression of cyclin D1 and activity of AP-1 by inhibiting the activation of ERK1/2 protein in HSC-T6 cells

AIM: To investigate the effects of artesunate(Art) on the expression of ERK1/2,AP-1 and cyclin D1 in rat hepatic stellate cells(HSCs),and to elucidate the molecular mechanism of Art against hepatic fibrosis.METHODS: HSC-T6 cells were treated with platelet-derived growth factor BB(PDGF-BB) to induce cell proliferation.The cells were divided into control group,PDGF-BB group,PDGF-BB+Art groups(with 6.25 mg·L-1,25 mg·L-1or 50 mg·L-1 of Art) and PDGF-BB+PD98059 group.The level of collagen type I in the supernatant was detected by enzyme-linked immunosorbent assay(ELISA).The mRNA expression levels of ERK1/2 and cyclin D1 were measured by RT-PCR.The protein levels of p-ERK1/2 and cyclin D1 in HSC-T6 cells were detected by Western blotting.The activity of AP-1 was analyzed by electrophoretic mobility shift assay.RESULTS: The concentration of collagen type I was significantly higher in PDGF-BB group than that in control group(P<0.05),and decreased in PDGF-BB+Art group and PDGF-BB+PD98059 group in comparison with that in PDGF-BB group(P<0.05,P<0.01).The protein level of ERK1/2 in PDGF-BB+Art group(50 mg·L-1) was lower than that in PDGF-BB group(P<0.05),and was even lower in PDGF-BB+PD98059 group(P<0.01).The mRNA expression of cyclin D1 in PDGF-BB+Art groups(25 mg·L-1and 50 mg·L-1) and PDGF-BB+PD98059 group were significantly lower than that in PDGF-BB group(P<0.05).The protein levels of p-ERK1/2 and cyclinD1 were the highest in PDGF-BB group,and significantly lower in PDGF-BB+Art groups(6.25 mg·L-1,25 mg·L-1 and 50 mg·L-1) and PDGF-BB+PD98059 group(P<0.05,P<0.01).The AP-1 binding activity in HSC-T6 cells was down-regulated by Art.CONCLUSION: Artesunate inhibits the proliferation of HSC-T6 cells in vitro by inhibiting the activation of ERK1/2,thus down-regulating the activity of AP-1 and expression of cyclin D1.