| Background: Acute liver injury(ALI)is a clinical syndrome associated with a sudden impairment of liver function.Although different hepatinica have been used as treatments,morbidity and mortality rates remain high.Mesenchymal stem cells(MSCs)derived from the bone marrow have showed a partial protective effect in ALI.The endometrial regenerative cell(ERC)is a novel type of adult mesenchymal stem cell isolated from menstrual blood.Furthermore,ERCs emerge as an attractive candidate because they present important advantages over other sources,including improved proliferation rates and paracrine response under specific stress conditions.Previous studies also demonstrated that ERCs possess unique immunoregulatory properties in vitro and in vivo,as well as the ability to differentiate into functional hepatocyte-like cells in vitro.For these reasons,the present study was undertaken to explore the inhibitory effects of ERCs on carbon tetrachloride(CCl4)–induced acute liver injury.Objective:The acute liver injury model of mice was established by intraperitoneal injection of carbon tetrachloride,and the inhibitory effect and mechanism of endometrial regenerative cells derived from female menstrual blood were studied.Methods: An ALI model in C57BL/6 mice was induced by administration of intraperitoneal injection of CCl4.In brief,18 mice were randomly assigned to the following three groups(n=6).(1)Normal control group: mice first receiving intraperitoneal(i.p.)injection of corn oil were then injected with 200 ul PBS intravenously 30 min later.(2)Untreated group: mice first receiving i.p.injection of a single dose of CCl4 for induction of acute liver injury were injected 200?l PBS intravenously 30 min later.(3)ERC-treated group: mice first receiving i.p.injection of CCl4 were injected intravenously with 1×106 ERCs at passage 4 resuspended in 200?l of PBS 30 min later.Biochemical,pathological and immunohistological parameters were evaluated,cell tracking was detected,and the systemic and local levels of pro-and anti-inflammatory cytokines were measured through enzyme-linked immunosorbent assay(ELISA)as well as the response of specific lymphocyte subsets were determined by flow cytometry 24 h after the CCl4 induction.Results: 1.ERCs were separated and obtained by Ficoll-Paque density-gradient centrifugation and had a typical spindle shape Under light microscope.The doubling time of the cultured cells was approximate 24 h.In addition,Clusters formed in the course of primary culturing and vortex-like cells appeared at 90% confluence in subcultures.2.The serum levels of ALT and AST in the untreated group were markedly increased compared with the normal control group(p<0.01).Compared with Untreated group,the levels of serum ALT and AST were significantly lower in the ERC-treated group,(p<0.01).3.Compared with the normal control group,the liver in the untreated group became inflamed,turned yellowish-white,and increased in volume at 24 hours after CCl4 injection.H&E staining showed a large number of inflammatory cells infiltration,sinusoid congestion and hemorrhage,and extensive hepatocyte necrosis.Very few residual hepatocytes were found and the hepatocytes present had a swollen cytoplasm.In contrast,liver in the ERC-treated group presented a significantly reduced edema,dark red,and loss of volume.H&E staining appeared a small amount of inflammatory cells accumulation,congestion and hemorrhage of the sinusoids were significantly lightened,and the area of necrosis of hepatocytes and parenchymal cells edema was reduced.4.Immunohistochemical analysis showed that the number of PCNA positive staining-cells in liver of ERC-treated group increased significantly(p<0.01),compared with the normal control group.In addition,compared with Normal control group,the number of Ly6 G positive cells in the liver of Untreated group was dramatically increased,(p<0.01),Ly6 G positive cells decreased significantly in ERC-treated group compared with Untreated group.5.Enzyme-linked immunosorbent assay detection shows that the untreated group exhibited significantly higher levels of pro-inflammatory cytokines(IL-1?,IL-6 and TNF-a)and lower level of anti-inflammatory cytokine IL-10 in both the liver(p<0.01) and the serum(p<0.01),the difference was statistically significant compared with the normal control group.In contrast,these pro-inflammatory cytokine levels were markedly reduced in ERC-treated group(p<0.01).On the other hand,the level of IL-10 was notably elevated after ERC treatment,and the difference was statistically significant(p<0.01).6.The results of cell tracking test showed that the PHK26-positive ERCs were detected by fluorescence microscopy in the liver(injured tissue)and the spleen(lymphoid organ)of ERC-treated mice.Moreover,the labeled ERCs were aslo mainly found in the lung,but not in other normal organs,such as the kidney.7.Flow cytometry results showed that the frequency of CD11 c MHCII positive DCs in the spleen was significantly ascending in Untreated mice compared to normal control mice(p<0.01),but the percentage of CD11c+ MHCII+ DCs was notably descending after ERC injection.8.The Proportion of T-cell subsets in the untreated group were markedly increased compared with the normal control group(p<0.01).the percentages of Splenic CD3+CD4+ and CD3+CD8+ T cells in the ERC-treated mice were dramatically decreased as compared with those of untreated mice(p<0.01).9.The proportion of Tregs in the untreated group was down-regulation than that of the normal control group(p<0.01).In contrast,the percentage of CD4+CD25+Foxp3+Treg population was significantlyup-regulation by ERC treatment in ALI mice(p<0.01).Conclusions: 1.The animal model of acute liver injury in mice was successfully used to verify that ERCs can effectively reduce the levels of AST and ALT,improve liver function and lessen the hepatic pathological damage.2.ERCs can protect the liver from acute injury in mice by increasing the number of PCNA positive cells and promoting the proliferation of hepatocytes,as well as restrain the damaged liver tissue by decreasing the infiltration of Ly6G-positive neutrophils.3.ERCs regulate inflammation and participate in hepatic damage and repairation through reducing the levels of pro-inflammatory cytokines(IL-1?,IL-6 and TNF-a) and improve the level of anti-inflammatory cytokine IL-10 in vivo.4.In vivo tracking experiments showed that ERCs could migrate,distribute and get into the damaged liver,and it can also accumulate in the lung.Besides,more fluorescent labeled ERCs were found in the spleen.5.ERCs transplantation inhibit liver injury by decreasing the percentage of CD11c+MHCII+ cells in the spleen.6.ERCs administration relieve hepatic damage is associatied with down-regulation of the proportion of splenic CD3+ CD4+ and CD3+ CD8+T cells.7.ERCs injection can inhibit and accelerate the restoration of liver injury by promoting the production of spleen Tregs cells,and increase its percentage. |
PDF Full Text Request