Therapeutic Effect And Mechanism Of Human Endometrial Mesenchymal Stem Cells On Acute-on-Chronic Liver Failure In Mice

Objective:To investigate the therapeutic effect of human endometrial mesenchymal stem cells on mice with acute-on-chronic liver failure and explore the mechanism.Methods:1.Chronic liver injury was induced in female C57BL/6J aged 10-12 weeks by intraderitoneal injection of 20%CCl4,0.2ml/CCl4,about 20-25g,twice a week for 8-10weeks,and 6ml/kg,50%CCl4 was injected 3 days after the end of chronic liver injury to induce ACLF model.2.After the induction of chronic liver injury,mice were randomly divided into 4 groups,A Oil group:Oil group was used as blank control group and received equal volume of olive oil injection.B PBS group:After 4h intraperitoneal injection of 50%CCL4,mice were injected with 200μl PBS through the tail vein as the negative control group.C BMSCs group:2×10~6 h BMSCs were injected into the tail vein after 4h intraperitoneal injection of 50%CCL4.D EMSCs group:2×10~6 hEMSCs were injected into the tail vein after 4h intraperitoneal injection of 50%CCL4.After caudal vein injection,mice in each group were randomly divided into two groups.The blood samples of group X were collected continuously on day 1,day 2,day 3 and day 7.The liver tissues of group Y were collected on day 1,day 2,day 3 and day 7,respectively.3.Survival rates were recorded at day 1,day 2,day 3 and day 7 after transplantation of mesenchymal stem cells.4.Ocμlar venous blood was collected 4h after 50%CCl4injection and 1,2,3,and 7 days after transplantation of mesenchymal stem cells.Serum levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),total bilirubin(TBIL),and prothrombin(PT)of mice were detected to reflect the liver function.5.Liver tissue sections were collected at different times for HE staining to observe the necrosis of liver cells,MASSON staining to observe the degree of fibrosis and immunofluorescence staining labeled F4/80,CD163,Ki67 reaction immune cell infiltration level and liver cell proliferation.The levels of inflammatory cytokines TNF-α,IL-1β,IL-10 and TGF-β2 were detected by Western Blot.The therapeutic effect of hEMSCs on ACLF was demonstrated by observing the general condition,survival rate,serological changes,liver histopathological changes,and collagen fiber deposition.Macrophage surface marker F4/80 and M2-type macrophage surface marker CD163 were used to reflect the infiltration level of M2-type macrophages,and cell proliferation marker Ki67 was used to reflect the proliferation level of hepatocytes.The levels of inflammatory cytokines TNF-α,IL-1β,IL-10 and TGF-β2 were detected by Western Blot.These methods were designed to explore the possible mechanism of hEMSCs in the treatment of ACLF mice.Results:1.Chronic liver injury was induced by intraderitoneal injection of 20%CCl4,0.2ml/piece,twice a week for 8-10 weeks,and ACLF was successfμlly induced by injection of6ml/kg,50%CCl4 after induction of chronic liver injury.Compared with the Oil group,the survival rate of mice in the CCl4 group decreased significantly,and the survival rate dropped to 62.5%one day after 50%CCl4 injection.The survival rate of mice in Oil group had no change,and the difference was statistically significant(P<0.01).The serum ALT,AST and TBIL levels of mice in CCl4 group were significantly increased,and the difference was statistically significant(P<0.05).PT decreased significantly in CCl4 group.The difference was statistically significant(P<0.05).2.One day after transplantation of mesenchymal stem cells,mice in PBS group,BMSCs group and EMSCs group began to die,and the survival rate decreased.The 2-day survival rate after transplantation of mesenchymal stem cells was stable(74%in PBS group,80%in BMSCs group and 85%in EMSCs group).Transplantation of hEMSCs can significantly improve the survival level of ACLF mice.Compared with Oil group,the serum ALT and AST levels in PBS group were significantly increased,while the serum ALT and AST levels in BMSCs group and EMSCs group were significantly decreased,with statistical significance(P<0.0001).TBIL levels in the BMSCs and EMSCs groups were lower than those in the PBS group.PT level in PBS group showed a downward trend,while BMSCs group and EMSCs group showed an upward trend compared.Due to small sample size and large standard deviation,the difference was not statistically significant.Transplantation of hEMSCs significantly improved the liver function of ACLF mice.3.HE staining resμlts showed that compared with Oil group,the degree of liver damage in PBS group was significantly increased,while that in BMSCs group and EMSCs group was significantly decreased,the difference being statistically significant(P<0.05).Transplantation of hEMSCs coμld reduce the degree of liver cell necrosis of ACLF mice and alleviate liver damage.4.One day after transplantation of mesenchymal stem cells,the degree of liver fibrosis in PBS group was significantly increased,the degree of liver fibrosis in BMSCs group and EMSCs group was significantly improved compared with PBS group,and the degree of liver fibrosis in EMSCs group was lower than that in BMSCs group.Transplantation of hEMSCs can significantly improve the liver fibrosis level of ACLF mice.5.One day after transplantation of mesenchymal stem cells,macrophage surface marker F4/80 increased significantly in the BMSCs and EMSCs groups,and the level of F4/80 in the EMSCs group was higher than that in the BMSCs group.The resμlts of M2-type macrophage surface marker CD163 were consistent with those of F4/80(P<0.01).Transplantation of hEMSCs significantly increased the level of macrophage infiltration,and M2-type macrophage infiltration was dominant.6.Two day after transplantation of mesenchymal stem cells,Ki67 levels in Oil group and PBS group did not change,but significantly increased in BMSCs group and EMSCs group(P<0.01),indicating that hEMSCs transplantation promoted hepatocyte proliferation in ACLF mice.7.One day after transplantation of mesenchymal stem cells,he levels of pro-inflammatory cytokines IL-1βand TNF-αwere increased significantly in PBS group,but decreased significantly in BMSCs and EMSCs groups,the difference was statistically significant(P<0.05).The levels of anti-inflammatory cytokines IL-10 and TGF-βwere significantly decreased in PBS,but significantly increased in BMSCs and EMSCs groups,the difference was statistically significant(P<0.05).In conclusion,transplantation of hEMSCs decreased the levels of pro-inflammatory cytokines IL-1βand TNF-α,and increased the levels of anti-inflammatory cytokines IL-10 and TGF-β.Conclusion:1.Transplantation of hEMSCs can improve the survival rate of ACLF mice and reduce liver injury after ACLF induced by CCl4.2.Transplantation of hEMSCs may play a therapeutic role by regμlating immune response,improving the level of M2-type macrophages,promoting hepatocyte proliferation,and alleviating local liver inflammation.