Effect And Mechanism Of Heme Oxygenase-1 Modified Bone Marrow Mesenchymal Stem Cells On Liver Fibrosis In Rats

Objective:To investigate the effect and mechanism of heme oxygenase-1（HO-1）modified bone marrow mesenchymal stem cells（BMMSCs）on hepatic fibrosis in rats.Methods:1.BMMSCs were isolated from femur and tibia of SD rats by adherent method,and identified by observing cell morphology,detecting cell surface specific markers and adipogenic and osteogenic differentiation potential;2.BMMSCs overexpressing HO-1 gene（HO-1/BMMSCs）were prepared by adenovirus transfection,and the overexpression of HO-1 gene was verified;3.The rat liver fibrosis model was established by intraperitoneal injection of CCl₄,and the fibrosis grade was identified by Ishak scoring system;4.HO-1/BMMSCs were infused into liver fibrosis rats via dorsal penile vein;5.The distribution of BMMSCs in rats after transplantation was detected by in vivo imaging system;6.According to different treatment methods,the rats were divided into four groups:control group,model group,BMMSCs group and HO-1/BMMSCs group.After different interventions,the rats were sacrificed,and the blood,liver tissue and fecal samples were taken.Biochemical analyzer,ELISA,HE staining,Sirius red staining,immunohistochemistry,Western blot,RT-PCR,Flow cytometry and 16S r DNA high-throughput sequencing were used to detect the changes of liver function,liver fibrosis index,liver histopathology,EMT marker,T lymphocyte subgroup ratio and intestinal flora ratio in each group.7.SPSS 23.0 was used for statistical analysis,and Graph Pad Prism 5.0 was used to make pictures.The measurement data are expressed by mean±standard deviation（x±s）.The data between the two groups were compared by independent sample t-test,and the data between the three groups and above were compared by one-way ANOVA.When P<0.05,the difference was considered statistically significant.Results:1.BMMSCs can be isolated by adherent method.Through the identification of cell morphology,surface characteristic molecular markers and adipogenic and osteogenic differentiation potential,it is confirmed that our isolated BMMSCs meet the international standards.HO-1/BMMSCs that can overexpress HO-1 gene can be successfully prepared by transfecting BMMSCs with adenovirus vector;2.Liver fibrosis model of rat can be successfully established by intraperitoneal injection of CCl₄,and the transplanted BMMSCs can be colonized in rat liver tissue;3.The long-term survival rate of BMMSCs group and HO-1/BMMSCs group were significantly higher than that of the model group（P<0.05）,the Ishak score and stage of liver histopathology were significantly lower than those of the model group（P<0.05）,and the liver function and liver fibrosis indexes were significantly better than those of the model group（P<0.05）.Further,HO-1/BMMSCs group was superior to BMMSCs group（P<0.05）;4.Comparing with the model group and BMMSCs group,the expression of E-cadherin in the liver tissue of HO-1/BMMSCs group was significantly higher（P<0.05）,while the expression of vimentin was significantly lower（P<0.05）;5.Compared with the model group and BMMSCs group,the ratio of CD4⁺T/CD8⁺T in peripheral blood and spleen of the rats in HO-1/BMMSCs group was significantly higher（P<0.05）,and the ratio of Th17/Treg was significantly lower（P<0.05）;6.Compared with the model group,the abundance of desulfovibrionaceae and desulfovibrio in HO-1/BMMSCs group increased,while clostridiaceae and clostridium decreased.Conclusions:1.HO-1/BMMSCs can effectively improve the degree of liver fibrosis in rats,which is better than BMMSCs alone.2.The protective mechanism of HO-1/BMMSCs on liver fibrosis may be related to the inhibition of EMT process,immunomodulatory effect and regulation of intestinal flora.