柴胡含药血清通过Smad3/Rheb轴调节HFL1细胞凋亡和肌成纤维细胞转化  

Medicated Serum of Bupleuri Radix Regulates HFL1 Apoptosis and Fibroblast-myofibroblast Transition via Smad3/Rheb Axis

作　　者：李妲[1] 沈枭 吴趋荟 郑蕾 赵四林[1] 金朝晖[1] 肖雪飞 范伏元[1] LI Da;SHEN Xiao;WU Quhui;ZHENG Lei;ZHAO Silin;JIN Zhaohui;XIAO Xuefei;FAN Fuyuan(The First Hospital of Hunan University of Chinese Medicine,Changsha 410007,China;Hunan University of Chinese Medicine,Changsha 410208,China)

机构地区：[1]湖南中医药大学第一附属医院,长沙410007 [2]湖南中医药大学,长沙410208

出　　处：《中国实验方剂学杂志》2023年第11期89-96,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：湖南省卫生健康委员会科研计划课题项目(202203023299);湖南省教育厅科学研究项目(20B442);湖南省中医药科研计划项目(2021165);长沙市科技局重点研发计划项目(Kq1901096)。

摘　　要：目的:利用转化生长因子-β_(1)(TGF-β_(1))刺激人胚肺成纤维细胞(HFL1)模拟特发性肺纤维化(IPF)的病理过程,探讨柴胡含药血清对IPF的作用和机制。方法:采用10μg·L^(-1)TGF-β_(1)刺激HFL1细胞,给予不同体积分数柴胡含药血清(5%、10%、15%、20%)干预24 h后,利用细胞增殖与活性检测试剂盒-8(CCK-8)检测细胞增殖率。随后细胞分为空白血清组(20%空白血清)、TGF-β_(1)组(20%空白血清和10μg·L^(-1)TGF-β_(1))、TGF-β_(1)+柴胡含药血清组(5%空白血清、15%含药血清和10μg·L^(-1)TGF-β_(1))、TGF-β_(1)+SIS3组(3μmol·L^(-1)SIS3、20%空白血清和10μg·L^(-1)TGF-β_(1))。采用原位末端标记法(TUNEL)检测细胞凋亡率;通过实时荧光定量聚合酶链式反应(Real-time PCR)检测凋亡相关蛋白B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)和肌成纤维细胞标志物α-平滑肌肌动蛋白(α-SMA)mRNA表达情况;通过免疫荧光法检测α-SMA、脑Ras同源蛋白(Rheb)、磷酸化(p)-Smad3蛋白表达;蛋白免疫印迹法(Western blot)检测Rheb、p-Smad3、Smad3蛋白表达。结果:与空白血清组比较,TGF-β_(1)组细胞增殖率明显上升(P<0.05)。与TGF-β_(1)组比较,TGF-β_(1)+15%柴胡含药血清组和TGF-β_(1)+20%柴胡含药血清组细胞增殖率显著降低(P<0.01)。与空白血清组比较,TGF-β_(1)组细胞凋亡率显著下降,Bcl-2、α-SMA mRNA表达增加,Bax mRNA表达减少,α-SMA、Rheb蛋白表达增加,Smad3磷酸化水平明显上升(P<0.05);与TGF-β_(1)组比较,TGF-β_(1)+柴胡含药血清组和TGF-β_(1)+SIS3组细胞凋亡率显著上升,Bcl-2、α-SMA mRNA表达减少,Bax mRNA表达增加,α-SMA、Rheb蛋白表达减少,Smad3磷酸化水平下降(P<0.05)。结论:柴胡能够抑制TGF-β_(1)诱导的HFL1细胞增殖和肌成纤维细胞转化,促进成纤维细胞凋亡,该作用可能与Smad3/Rheb轴有关。Objective:Transforming growth factor-β_(1)(TGF-β_(1))was used to stimulate human fetal lung fibroblast 1(HFL1)for simulating the pathological process of idiopathic pulmonary fibrosis(IPF)and thereby the effects and mechanism of medicated serum of Bupleuri Radix against IPF were investigated.Method:TGF-β_(1)(10μg·L^(-1))was employed to stimulate HFL1,and cells were treated with medicated serum of Bupleuri Radix(5%,10%,15%,20%)for 24 h.Then cell proliferation rate was determined with cell counting kit-8(CCK-8).Subsequently,cells were classified into the control group(20%blank serum),TGF-β_(1) group(20%blank serum and 10μg·L^(-1) TGF-β_(1)),TGF-β_(1)+medicated serum of Bupleuri Radix group(5%blank serum,15%medicated serum,and 10μg·L^(-1) TGF-β_(1)),and TGF-β_(1)+SIS3 group(3μmol·L^(-1) SIS3,20%blank serum,10μg·L^(-1) TGF-β_(1)).Based on in situ end labeling(TUNEL)staining,the apoptosis rate was examined,and mRNA expression of apoptosis-related proteins B-cell lymphoma 2(Bcl-2),Bcl-2 associated X protein(Bax)and myofibroblast markerα-smooth muscle actin(α-SMA)was detected by Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR).The protein expression ofα-SMA,Ras homolog enriched in brain(Rheb),and phosphorylated(p)-Smad3 was determined by immunofluorescence.Expression of Rheb,p-Smad3,and Smad3 was examined by Western blot.Result:The cell proliferation rate of TGF-β_(1) group increased compared with that of the control group(P<0.05).The cell proliferation rate of TGF+15%medicated serum of Bupleuri Radix group and TGF+20%medicated serum of Bupleuri Radix group decreased compared with that of the TGF-β_(1) group(P<0.01).Compared with the control group,TGF-β_(1) group showed decrease in apoptosis rate,increase in mRNA expression of Bcl-2 andα-SMA,reduction in Bax mRNA expression,and rise ofα-SMA and Rheb protein expression and p-Smad3 level(P<0.05).Compared with TGF-β_(1) group,TGF-β_(1)+medicated serum of Bupleuri Radix group and TGF-β_(1)+SIS3 group demonstrated high

关 键 词：特发性肺纤维化 柴胡 成纤维细胞 SMAD3 脑Ras同源蛋白 成纤维细胞向肌成纤维细胞转化 

分 类 号：R2-0[医药卫生—中医学] R33R289R256.1