Mesenchymal Stem Cells-conditioned Medium Reduces CCL₄-induced Liver Fibrosis By Regulating Hepatic Stellate Cells

Background:Mesenchymal stem cell(MSC)therapy is a relative new approach for the treatment of liver fibrosis.Our previous study has showed that bone marrow derived MSC(BM-MSC)may reduce liver fibrosis in CCL4-induced cirrhotic Sprague-Dawley(SD)rats models.However,the few numbers of BM-MSCs that migrated into the liver and their low differential rate into functional hepatocyte like cells may not responsible for the direct effect on the cirrhotic liver.So we hypothesized that BM-MSCs may play a role primarily due to their trophic secretic molecules by the paracrine effect.In this study,we investigated whether the MSC-conditioned medium(MSC-CM)can protect injured liver and reduce liver fibrosis and its potential mechanisms.Methods:Six-week-old SD rats were allocated into three groups(each group n=8)as follows:model group(CCL4-control);treatment group(CCL4+MSC-CM);normal group(normal control).The liver fibrosis model was established by intraperitoneal injection of low dose(1.5ml/kg)of CCL4 twice a week for eight weeks.BM-MSCs were grown for 3-5 generation for the preparation of MSC-CM which was concentrated 25-fold using ultrafiltration units with 3-kDa molecular weight cut-off.From weeks 5 to 8,MSC-CM was injected every day with a dose 2mg/kg by tail vein in treatment group;at the same time,the other two groups injected with the same dose of L-DMEM.At the end of experiment,the hepatic stellate cells(HSCs)were isolated for further analysis.The blood samples were taken once a week from the rats during the experiment to detect liver function.The collagenous fiber was detected by Masson staining,while the expression of alpha-smooth muscle actin(a-SMA)in liver tissues was measured by immunohistochemical staining.These data were analyzed by Image Pro Plus software.In HSCs study,the gene and protein expression of a-SMA,transforming growth factor beta 1(TGF-(31),collagen type I(Col-I),uncoupling protein 2(UCP2),Matrix metalloproteinases-2(MMP-2)and tissue inhibitor of metalloproteinases-2(TIMP-2)were evaluated by quantitative real-time PCR and western blot.Results:1.The level of serum ALT,AST and TBIL in treatment group was lower than that in model group,especially TBIL at the fifth week significantly lower than model group.2.In liver tissues,there was an improvement of the inflammatory cells infiltration in treatment group(G2S1)compared with model group(G3S4).The expression of a-SMA and the level of collagenous fiber were significantly lower in treatment group(CCL4-MSC-CM)than in model group(CCL4-control group)(P<0.05).3.In HSC study,the mRNA expression of a-SMA,TGF-?1,collagen I in treatment group were significantly decreased than that in model group(P<0.05);and their protein expressions were also lower in treatment group(P<0.05);and the expression of gene UCP2 was increased in treatment group at the level of mRNA and protein;suggesting the suppression of HSC activation after the MSC-CM therapy.The level of MMP-2 mRNA in HSCs was significantly higher in the treatment group than that in the model group,indicating that MSC-CM promoted collagen degradation.Conclusions:1.MSC-CM can reduced the level of liver fibrosis induced by CCL4,suppressed the infiltration of liver inflammation.2.Our results showed that MSC-CM have a therapeutic effect on liver fibrosis.And this process may be related to the inhibition of HSCs activation through down-regulating the expression of TGF-?1 and up-regulating the expression of UCP2;MSC-CM promotes collagen degradation through up-regulated MMP-2 level in HSC.