骨髓间充质干细胞来源外泌体通过铁蛋白自噬抑制肝星状细胞活化的研究  

Inhibition of hepatic stellate cell activation by bone marrow mesenchymal stem cell-derived exosomes via ferritin autophagy

作　　者：周慧媛 王玉鑫 张新如 左怀文 皮艺林 邓腊梅 宋红丽[3] Zhou Huiyuan;Wang Yuxin;Zhang Xinru;Zuo Huaiwen;Pi Yilin;Deng Lamei;Song Hongli(Clinical College of First Centre,Tianjin Medical University,Tianjin 300192,China;Medical College of Nankai University,Tianjin 300074,China;Department of Liver Transplantation,Tianjin First Central Hospital,Tianjin Key Laboratory of Organ Transplantation,Tianjin Clinical Research Centre for Organ Transplantation,Tianjin 300192,China)

机构地区：[1]天津医科大学一中心临床学院,天津300192 [2]南开大学医学院,天津300074 [3]天津市第一中心医院肝移植科,天津市器官移植重点实验室,天津市器官移植临床医学研究中心,天津300192

出　　处：《中华实验外科杂志》2024年第6期1194-1199,共6页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金(82070639)。

摘　　要：目的探讨骨髓间充质干细胞来源的外泌体(BMMSC-exos)对活化肝星状细胞(HSC)的作用及其机制。方法应用脂多糖(LPS)诱导HSC形成激活体外模型。按照实验要求分为对照组(Con)、活化组(LPS)、LPS+BMMSCs(L+B)组、LPS+BMMSC-exo(L+B-exo)组、L+B-exo+小干扰RNA阴性对照组(si-NC)及L+B-exo+敲降NCOA4组(si-NCOA4)。通过细胞增殖测定细胞活力;实时定量聚合酶链反应(RT-qPCR)检测平滑肌激动蛋白(α-SMA)、核受体共激活因子4(NCOA4)和铁蛋白(FTH1)mRNA;蛋白质免疫印迹法检测NCOA4、FTH1、波形蛋白(Vimentin)、α-SMA、谷胱甘肽过氧化物酶4(GPX4)、p62、环氧合酶-2(COX-2)、苄氯素1(Beclin 1)及微管相关蛋白(LC3)蛋白表达水平;免疫荧光化学检测NCOA4、FTH1及Vimentin表达;检测丙二醛(MDA)、还原型谷胱甘肽(GSH)及脂质活性氧(Lipid ROS)含量变化,多组间的数据比较采用单因素方差分析。结果BMMSC-exos对活化的HSC-T6细胞的影响:L+B-exo组纤维化标志物α-SMA和Vimentin表达量(0.58±0.04、0.25±0.05)低于LPS组(1.26±0.04、1.06±0.03)和L+B组(0.76±0.04、0.75±0.15,F=50.607、35.916,P<0.05)。BMMSC-exos促进活化的HSC-T6细胞铁死亡和自噬:L+B-exo组MDA与Lipid ROS的表达量[(4.27±1.06)nmol/(mg·prot)、8950.49±114.34]高于LPS组[(2.27±0.73)nmol/(mg·prot)、5492.73±25.69]和L+B组[(3.27±0.90)nmol/(mg·prot)、7198.05±116.63,F=1.263、1372.009,P<0.05];L+B-exo组GSH水平[(10.47±0.31)μg/10^(6)cells]低于LPS组[(15.94±0.11)μg/10^(6)cells]和L+B组[(12.60±0.15)μg/10^(6)cells,F=646.842,P<0.05];L+B-exo组NCOA4蛋白表达量[(1.19±0.06)]高于LPS组[(0.73±0.12)]和L+B组[(0.95±0.13),F=44.456,P<0.05];L+B-exo组FTH1蛋白表达量(0.25±0.05)低于LPS组(1.06±0.03)和L+B组(0.75±0.15,F=35.916,P<0.05)。NCOA4介导的铁蛋白自噬参与BMMSC-exos对HSC-T6细胞的作用:si-NCOA4组的MDA[(0.83±0.11)nmol/(mg·prot)]低于si-NC组[(1.17±0.09)nmol/(mg·prot),F=17.217,P<0.05]。结论BMMSC-exos通过NCOA4介导的铁蛋白自噬Objective To investigate the effects of bone marrow mesenchymal stem cell-derived exosomes(BMMSC-exos)on the activation of hepatic stellate cells(HSC)and its mechanism.Methods Lipopolysaccharide(LPS)was used to induce the formation of an in vitro model of HSC activation.According to the experimental requirements,they were divided into control(Con),LPS,LPS+BMMSCs(L+B),LPS+BMMSC-exo(L+B-exo),L+B-exo+small interfering RNA-negative control(si-NC),and L+B-exo+knockdown of NCOA4(si-NCOA4)groups.Cell viability was determined by cell proliferation.The mRNA expression levels of smooth muscle agonist protein(α-SMA),nuclear receptor coactivator 4(NCOA4),and ferritin(FTH1)were detected by real-time quantitative polymerase chain reaction(RT-qPCR).The protein immunoblotting was performed for the detection of NCOA4,FTH1,Vimentin,α-SMA,glutathione peroxidase NCOA4,FTH1,Vimentin,α-SMA,glutathione peroxidase 4(GPX4),p62,benzyl chloride 1(Beclin 1),and microtubule-associated protein(LC3).The immunofluorescence chemistry was used to detect the expression of NCOA4,FTH1 and Vimentin.The changes in the content of malondialdehyde(MDA),reduced glutathione(GSH),and lipid ROS were detected.One-way analysis of variance(ANOVA)was used to compare the data among multiple groups.Results Effects of BMMSC-exos on activated HSC-T6 cells:the expression of fibrosis markersα-SMA and Vimentin in the L+B-exo group(0.58±0.04,0.25±0.05)was lower than that in the LPS group(1.26±0.04,1.06±0.03)and the L+B group(0.76±0.04,1.06±0.03,F=50.607,35.916,P<0.05).BMMSC-exos promoted iron death and autophagy in activated HSC-T6 cells:the expression of MDA and Lipid ROS in the L+B-exo group[(4.27±1.06)nmol/(mg·prot),8950.49±114.34]was higher than that in the LPS group[(2.27±0.73)nmol/(mg·prot),5492.73±25.69]and L+B group[(3.27±0.90)nmol/(mg·prot),7198.05±116.63,F=1.263,1372.009,P<0.05];GSH level in the L+B-exo group[(10.47±0.31)μg/10^(6)cells]was lower than that in the LPS group[(15.94±0.11)μg/10^(6)cells]and L+B group[(12.60±0.15)μg/10^(6)ce

关 键 词：铁蛋白 自噬 铁死亡 肝星状细胞 肝纤维化 

分 类 号：R575.2[医药卫生—消化系统]