和血柔肝方含药血清通过鞘氨醇激酶-1对肝星状细胞增殖与凋亡的作用  

Effect of medicated serum of Hexuerougan formula on regulates the proliferation and apoptosis of hepatic stellate cells through sphingosine kinase-1

作　　者：王燕[1] 肖雄 马丽 蒲诗云 WANG Yan;XIOA Xiong;MA Li(Department of Traditional Chinese Medicine,The Fifth People′s Hospital Affiliated to Chengdu University of Traditional Chinese Medicine,Chengdu Sichuan,611130,China;不详)

机构地区：[1]成都中医药大学附属第五人民医院,四川成都611130 [2]中国科学院新疆理化技术研究所 [3]中国科学院大学

出　　处：《中西医结合肝病杂志》2024年第10期903-908,共6页Chinese Journal of Integrated Traditional and Western Medicine on Liver Diseases

基　　金：国家自然科学基金项目(No.81860808)。

摘　　要：目的:和血柔肝方(HXRGF)含药血清调节鞘氨醇激酶-1(SphK1)对肝星状细胞(HSC)增殖与凋亡的作用。方法:采用Resistin处理HSC-T6细胞,建立纤维化HSC模型,采用LV-rSphK1-OE、LV-rSphk1-shRNA慢病毒方法构建SphK1过表达、低表达稳转细胞株,运用实时定量PCR、WB检测TGF-β、α-SMA、Col I、FN、Smad、SphK1mRNA及蛋白的表达、CCK8实验检测细胞活力、AnnexinV-APC/7-AAD流式细胞学检测细胞凋亡情况。结果:与对照组比较,MF-HSC-T6细胞中可见TGF-β、α-SMA、Col I、FN、SphK1mRNA及蛋白表达水平升高,Smad mRNA水平降低(P<0.01);HSC-T6 SphK1过表达细胞株中SphK1及相关纤维化标志物的表达水平升高(P<0.01);HSC-T6 SphK1低表达细胞株中TGF-β、α-SMA、Col I、FN mRNA等纤维化相关标志物降低,Smad mRNA水平升高(P<0.01)。与模型组比较,中药复方HXRGF含药血清干预后,可见MF-HSC T6 SphK1mRNA及蛋白表达水平降低,并同时伴有α-SMA、Col I、FN表达水平的下降(P<0.01),HSC细胞增殖率下降(110.85%);shRNA-SphK1转染组细胞增殖率下降,凋亡率(12.14%)升高(P<0.01);HSC-T6 SphK1低表达组细胞凋亡率(16.48%)升高(P<0.01)。结论:SphK1可作为肝纤维化潜在治疗靶点。HXRGF干预通过抑制SphK1表达,下调TGF-β、α-SMA等促纤维化因子的表达水平,抑制活化的HSC增殖,诱导HSC凋亡,缓解肝纤维化。Objective:Investigation of the effect of serum containing Hepatic Soften and Nourish Formula(HXRGF) on the regulation of sphingosine kinase-1(SphK1) in the proliferation and apoptosis of hepatic stellate cells(HSC).Methods:Resistin was applied to treat HSC-T6 cells with Resistin to establish a fibrotic HSC model,and LV-rSphK1-OE and LV-rSphK1-shRNA lentivirus methods to construct stable cell lines with overexpression or low expression of SphK1.The mrna and protein expressions of TGF-β,α-SMA,Col I,FN,Smad and SphK1 were detected by real-time quantitative PCR and Western blot.The cell viability was detected by CCK8 assay and apoptosis was detected by flow cytometry.Results:(1)Compared with the control group,TGF-β,α-SMA,Col I,FN,SphK1mRNA and protein expression levels were increased in MF-HSC-T6 cells,whil Smade mRNA levels were decreased(all P<0.01).The expression levels of SphK1 and related fibrosis markers were increased in HSC-T6 SphK1 overexpression cell lines(P<0.01).Fibrosis related markers such as TGF-β,α-SMA,Col I and FN mRNA were decreased in HSC-T6 SphK1 low expression cell lines,while Smad mRNA levels were increased(P<0.01).(2)Compared with model group,the mrna and protein expression levels of MF-HSC T6 SphK1 were decreased after the intervention of TCM compound HXRGF containing serum,along with the expression levels of α-SMA,Col I and FN(P<0.01),and the proliferation rate of HSC cells was decreased(110.85%).Cell proliferation rate decreased and apoptosis rate increased(12.14%) in shRNA-SphK1 transfection group(P<0.01).The apoptosis rate of low expression group of HSC-T6 SphK1 was increased(16.48%,P<0.01).Conclusion:SphK1 can be a potential therapeutic target for hepatic fibrosis.By inhibiting the expression of SphK1,HXRGF can down-regulate the expression level of TGF-β,α-SMA and other pro-fibrosis factors,inhibit the proliferation of activated HSC,induce the apoptosis of HSC,and alleviate liver fibrosis.

关 键 词：肝星状细胞 鞘氨醇激酶-1 和血柔肝方 

分 类 号：R575[医药卫生—消化系统]