The Effects Of Mouse Bone Marrow Mesenchymal Stem Cells On Alcoholic Liver Injury And Its Underlying Mechanisms

Objectives:Alcoholic liver disease(ALD)is a common disease that seriously endangers human health.Its prevalence rate increases year by year in the world,and it is a global public health problem.Therefore,it is of great significance to study the pathogenesis of ALD and to explore its intervention strategies.Bone marrow mesenchymal stem cells(BM-MSCs)are pluripotent adult cells derived from the mesoderm.They have high self-renewal and multidifferentiation potentials and are widely used in basic and clinical studies of a variety of diseases.The purpose of this study was to investigate whether BM-MSCs can effectively alleviate alcoholic liver injury in mice and its related molecular mechanisms.Methods:This study consists of two parts:Part I:Isolation,culture,identification and transfection of mouse BM-MSCs1.Stem cells were isolated from the hindlimb marrows of 4-6 week-old male C57BL/6N mice by means of whole bone marrow adherent method.2.Immunomolecular markers on the surface of the isolated stem cells,including CD29,CD45,CD90 and CD 105,were detected by flow cytometry.3.Osteogenic and lipogenic induction and differentiation cultures were conducted on the isolated stem cells to judge their multidifferentiation potential.4.Small interfering RNA(siRNA)was introduced into the isolated BM-MSCs by lentiviral transfection,and reverse transcription quantitative polymerase chain reaction(RT-qPCR)and Western blot(WB)were performed to assess tumor necrosis factor(TNF)α stimulate gene/protein(TSG)6 expression and the preparation of stable BM-MSCs transfection strains.Part II:Effects of mouse BM-MSCs on mouse alcoholic liver injury and its underlying mechanisms1.Experimental groups:70 female C57BL/6N mice were randomly divided into the following seven groups with 10 mice per group:normal control group,model group,MSCs group,sc-MSCs group,siTSG6-MSCs group,rmTSG-6 group and AG490 group.Mice were fed with liquid control diet or liquid ethanol diet.2.Alcoholic liver injury model:In this study,alcoholic liver injury model was established according to the method of the national institute of alcohol abuse and alcoholism(NIAAA),which was to induce alcoholic liver injury in female mice through chronic feeding of alcoholic liquid diet for 10 days combined with three high-dose alcohol gavages.3.Stem cell transplantation:On day 10 of the modeling period,mice in the normal control group,model group,the MSCs group,sc-MSCs,siTSG6-MSCs group,rmTSG-6 group and AG490 group were given intraperitoneal injection of sterile saline(normal control group)and normal BM-MSCs(MSCs),BM-MSCs transfected by control lentivirus(sc-MSCs group),BM-MSCs transfected by TSG-6 interfering lentivirus(siTSG6-MSCs group),recombinant mouse TSG-6(rmTSG-6 group),and normal BM-MSCs plus AG490(AG490 group).4.Sampling:On the 11th day of the modeling period,9 hours following all mice were treated with gavage,their blood samples and liver tissues were collected after inhalation anesthesia.5.Sample measurement:biochemical,histopathological,flow cytometry,RT-qPCR and WB analyses were performed on serum and liver tissue samples of each group.Results:Part Ⅰ:1.The stem cells isolated by the whole bone marrow adherent method were triangular,fusiform and spindle-shaped,with radial or whirlpool growth.The expression rate of CD45,a marker of cell surface immunity,was only 0.65%,while the expression rates of CD29,CD90 and CD 105 were as high as 82.84%,85.27%and 84.17%,respectively.The cells could be induced to differentiate into adipocytes and osteoblasts.After oil red O staining and alizarin red staining,the cells were positive,fulfilling the identification criteria of BM-MSCs.2.After transfection of mouse BM-MSCs with TSG-6-interference lentivirus,the transcription and translation levels of TSG-6 gene were significantly down-regulated,suggesting that the stable TSG-6-downregulated strain of mouse BM-MSCs was successfully established.Part Ⅱ:1.Compared with the normal control group,the liver/body weight ratio,serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST),serum and liver total cholesterol(TC)and triglyceride(TG),and hepatic lipid peroxidation product malondialdehyde(MDA),serum and liver pro-inflammatory mediators interleukin(IL)6 and TNF-α levels were significantly increased;hepatic infiltration by neutrophils and macrophages increased significantly,whereas hepatic antioxidant reduced glutathione(GSH),anti-inflammatory cytokines IL-10 and TSG-6 levels were significantly reduced.All of these results suggest the successful establishment of alcoholic liver injury in mice.2.After treatment with BM-MSCs,mouse liver weight/body weight ratio,serum transaminases(ALT,AST)and serum and liver lipids(TC,TG)and hepatic MDA,serum and liver pro-inflammatory mediators(IL-6,TNF-α)and hepatic infiltration by neutrophils and macrophages were significantly decreased,whereas serum and hepatic GSH and anti-inflammatory mediator(IL-10,TSG-6)levels were increased significantly.These results suggest that BM-MSCs can effectively relieve alcoholic liver injury in mice.3.Only a few BM-MSCs engrafted the liver tissues of mice in the MSCs group,sc-MSCs group and siTSG6-MSCs group.4.Down-regulating TSG-6 gene expression in BM-MSCs after transfection with TSG-6-interference lentivirus led to a significant decrease in the efficacy of BM-MSCs on mouse alcoholic liver injury,while transfection with control lentivirus did not affect the efficacy of BM-MSCs,and the efficacy of exogenous injection of rmTSG-6 was similar to that of normal BM-MSCs,suggesting that TSG-6 molecule produced by BM-MSCs is the key molecule that can effectively alleviate mouse alcoholic liver injury.5.Compared with mice of the normal control group,hepatic phosphorylated signal transducer and activator of transcription 3(p-STAT3)level in mice of the model group was significantly increased,and hepatic p-STAT3 level in mice with alcoholic liver injury was significantly decreased after BM-MSCs treatment.Transfection with TSG-6-interference lentivirus and down-regulation of TSG-6 gene expression resulted in decreased inhibition by BM-MSCs on p-STAT3 level in mouse liver tissues.However,transfection of control lentivirus did not affect the effect of BM-MSCs,and exogenous injection of rmTSG-6 also significantly inhibited the p-STAT3 level in mouse liver tissues.These results suggest that BM-MSCs can inhibit the hepatic activation of STAT3 signaling pathway mice with alcoholic liver injury through the TSG-6 molecule.6.Compared with MSCs group,hepatic p-STAT3 level in AG490 group was markedly decreased.Serum aminotransferase(ALT,AST),serum and liver lipids(TC,TG),and liver MDA level in AG490 group were significantly decreased,while hepatic antioxidant GSH level was prominently increased,suggesting that further inhibition of STAT3 activation could improve the efficacy of BM-MSCs on mice with alcoholic liver injury.Conclusions:1.BM-MSCs can be successfully isolated and extracted from mouse hindlimb bone marrows by whole bone marrow adherent method.2.Stable TSG-6 gene-interfering strain of BM-MSCs can be successfully established by lentiviral transfection.3.Mouse BM-MSCs transplantation can effectively treat alcoholic liver injury.4.Mouse BM-MSCs mainly produce TSG-6 molecule through paracrine mechanism to exert the effect on alcoholic liver injury.5.Mouse BM-MSCs inhibits hepatic activation of STAT3 signaling through TSG-6 molecules,and further inhibition of this pathway can improve the efficacy of BM-MSCs.