Inhibition of hepatic stellate cell activation by bone marrow mesenchymal stem cell-derived exosomes via ferritin autophagy
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.Accord-ing 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 prolifera-tion.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 malondial-dehyde(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 activa-ted HSC-T6 cells:the expression of fibrosis markers α-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),8 950.49±114.34]was higher than that in the LPS group[(2.27±0.73)nmol/(mg·prot),5 492.73±25.69]and L+B group[(3.27±0.90)nmol/(mg·prot),7 198.05±116.63,F=1.263,1 372.009,P＜0.05];GSH level in the L+B-exo group[(10.47±0.31)μg/106 cells]was lower than that in the LPS group[(15.94±0.11)μg/106 cells]and L+B group[(12.60±0.15)μg/106 cells,F=646.842,P＜0.05];and the expression of NCOA4 in the L+B-exo group[(1.19±0.06)]was higher than that in the LPS group[(0.73±0.12)]and L+B group[(0.95±0.13),F=44.456,P＜0.05];FTH1 expression in the L+B-exo group(0.25±0.05)was lower than that in the LPS group(1.06±0.03)and the L+B group(0.75±0.15,F=35.916,P＜0.05).NCOA4-mediated ferritin autophagy was involved in the effect of BMMSC-exos on HSC-T6 cells:the MDA in the si-NCOA4 group[(0.83±0.11)nmol/(mg·prot)]was lower than that in the si-NC group[(1.17±0.09)nmol/(mg·prot),F=17.217,P＜0.05].Conclusion BMMSC-exos induced iron death of activated HSC through NCOA4-mediated ferritin autophagy,thereby inhibiting HSC activation and exerting anti-hepatic fibrosis effects.
NETL
NSTL
万方数据
