摘要
【目的】观察黄连素调节Hedgehog信号通路对转化生长因子β1(TGF-β1)诱导的肝星状细胞增殖和活化的影响。【方法】培养HSC-T6细胞,分为空白组,TGF-β1组,黄连素低、中、高剂量组及黄连素高剂量+purmorphamine(Hedgehog信号通路激活剂)组。分组处理后,四甲基偶氮唑盐(MTT)法检测HSC-T6细胞增殖能力,流式细胞术检测细胞凋亡,免疫荧光染色法检测细胞Ⅰ型胶原(CollagenⅠ)、α-平滑肌肌动蛋白(α-SMA)表达变化,实时荧光定量聚合酶链式反应(qRT-PCR)法检测细胞增殖细胞核抗原(PCNA)、Bcl-2、Bcl-2相关X蛋白(Bax)mRNA表达水平,蛋白免疫印迹(Western Blot)法检测细胞Hedgehog信号通路相关因子Smo、Gli-1、Shh、Ptch蛋白表达水平。【结果】与空白组比较,TGF-β1组HSC-T6细胞增殖率,CollagenⅠ、α-SMA阳性蛋白表达,PCNA mRNA、Bcl-2 mRNA表达水平及Smo、Gli-1、Shh、Ptch蛋白表达水平显著升高,凋亡率及Bax mRNA表达水平显著降低(均P<0.05);与TGF-β1组比较,黄连素低、中、高剂量组HSC-T6细胞增殖率,CollagenⅠ、α-SMA阳性蛋白表达,PCNA mRNA、Bcl-2 mRNA表达水平及Smo、Gli-1、Shh、Ptch蛋白表达水平显著降低,凋亡率及BaxmRNA表达水平显著升高(均P<0.05),呈剂量依赖性;与黄连素高剂量组比较,黄连素高剂量+purmorphamine组HSC-T6细胞增殖率,CollagenⅠ、α-SMA阳性蛋白表达,PCNA mRNA、Bcl-2 mRNA表达水平及Smo、Gli-1、Shh、Ptch蛋白表达水平显著升高,凋亡率及Bax mRNA表达水平显著降低(均P<0.05)。【结论】黄连素通过调节Hedgehog信号通路促进TGF-β1诱导肝星状细胞凋亡,抑制其增殖和活化。
Objective To observe the effect of berberine on the proliferation and activation of hepatic stellate cells induced by transforming growth factorβ1(TGF-β1)via regulating Hedgehog signaling pathway.Methods HSCT6 cells were cultured and divided into blank group,TGF-β1 group,berberine low-,medium-and high-dose groups,berberine high-dose+purmorphamine(Hedgehog signaling pathway activator)group.After treatment,the proliferation of HSC-T6 cells was detected by methyl thiazolyl tetrazolium(MTT)assay.The apoptosis of HSC-T6 cells was detected by flow cytometry.The expressions of collagen I andα-smooth muscle actin(α-SMA)were detected by immunofluorescence staining.The mRNA expression levels of proliferating cell nuclear antigen(PCNA),Bcl-2 and Bcl-2 associated X protein(Bax)were detected by real-time fluorescence quantitative polymerase chain reaction(qRT-PCR).The protein expression levels of Hedgehog signaling pathway related factors Smo,Gli-1,Shh and Ptch were detected by Western Blot.Results Compared with the blank group,the proliferation rate of HSC-T6 cells,the positive protein expressions of CollagenⅠandα-SMA,the mRNA expression levels of PCNA and Bcl-2,and the protein expression levels of Smo,Gli-1,Shh and Ptch in the TGF-β1 group were significantly increased,while the apoptosis rate and the mRNA expression level of Bax were significantly decreased(all P<0.05).Compared with TGF-β1 group,the proliferation rate of HSC-T6 cells,the expression of Collagen I andα-SMA positive protein,the mRNA expression levels of PCNA and Bcl-2,and the protein expression levels of Smo,Gli-1,Shh and Ptch in berberine low-,medium-and high-dose groups were significantly decreased,while the apoptosis rate and the mRNA expression level of Bax were significantly increased(P<0.05)in a dose-dependent manner.Compared with the high-dose berberine group,the proliferation rate of HSC-T6 cells,the positive proteins expression of CollagenⅠandα-SMA,the mRNA expression levels of PCNA and Bcl-2,and the protein expression levels of Smo,Gli-1,Shh and Ptch in the high-dose berberine+purmorphamine group were significantly increased,while the apoptosis rate and the mRNA expression level of Bax were significantly decreased(all P<0.05).Conclusion Berberine promotes TGF-β1-induced apoptosis of hepatic stellate cells and inhibits their proliferation and activation by regulating Hedgehog signaling pathway.
作者
白璐
张意兰
陈光耀
BAI Lu;ZHANG Yi-Lan;CHEN Guang-Yao(Dept.of Gastroenterology,The Fifth Clinical School of Henan University of Chinese Medicine(Zhengzhou People’s Hospital),Zhengzhou 450000 Henan,China)
出处
《广州中医药大学学报》
CAS
2024年第8期2145-2152,共8页
Journal of Guangzhou University of Traditional Chinese Medicine
基金
河南省医学科技攻关计划联合共建项目(编号:LHGJ20200680)。
