Autologous Mesenchymal Stem Cells Transplantation Protect Against Experimental Liver Fibrosis Rats During Inhibition Of Hepatic Stellate Cell Activation

Background:The hepatic fibrosis is the abnormal hyperplasia of the connective tissue caused by various kinds of pathogenic factors, which lead to pathological process of excessive matrix deposition. Many chronic liver disease were eventually develop into liver fibrosis.In the course of hepatic fibrosis, activation of hepatic stellate cells is the key event. Extracellular matrix such as collagen fibers is generated by fibroblasts which from the activated hepatic stellate cells. There are four states of hepatic stellate cells: the primary quiescent hepatic stellate cells, the activated hepatic stellate cells, the quiescent fibroblasts, and the apoptotic hepatic stellate cells. Some scholars proposed that the main strategies of anti hepatic fibrosis include apoptosis of muscle fibroblasts, maintaining the static phenotype of cells or inhibiting the activation of cells. To date, there is no effective inhibition drugs of hepatic stellate cells for anti fibrosis and there is still no effective treatment for hepatic fibrosis.Mesenchymal stem cells(MSC) is an adult multipotent stem cell from mesoderm.In addition to have the basic characteristics of stem cells which inlude: self-renew and multi lineage differentiation general biological, it also has its own characteristics. MSC have biological characteristics of immune regulation, homing, targeted anti-inflammatory, actively adapt to micro environment and transform it which can be used for repairment of various tissue. Researches show that MSC has the effect on cardiac fibrosis caused by ischemic myocardial infarction, renal fibrosis caused by autoimmune diseases and idiopathic pulmonary fibrosis. Recent studies have found that MSC has a certain effect on the anti fibrosis of liver. The studies inclued: I-II clinical stage research of using MSC to treat of liver cirrhosis patients inducted by Kharaziha P et al and using MSC to treat end-stage hepatitis C liver disease patient inducted by Salama H et al. It suggests that MSC transplantation has broad application in the treatment of hepatic fibrosis. However, the outcomes of clinical studies are not the same, the treatment mechanism is not clear yet, and all above need to be further studied and clinical research. Objective:To investigate the effects of mesenchymal stem cells(MSC) on hepatic fibrosis and activation degree of hepatic stellate cells(HSC) after transplantation and the possible internal mechanism. Methods:The methods of Ficoll- Hypaque density gradient centrifugation and adherent culture were used to separate and purify Bone marrow mesenchymal stem cells(BM-MSC) respectively. Liver fibrosis model of SD rats were induced by long-term intraperitoneal injection of low-dose CCl4 for 8 weeks. 18 rats were used to make model, since 4 weeks after the modeling process, 9 of them was taken out to receive treatment of 6×106 MSCs for 4 weeks by tail vein injection, the remains were injected the same volume of saline without MSCs. Normal control group containing 9 rats were only given the same volume of saline injection. ALT, AST, TBIL, ALB level of serum were determined by automatic biochemical analyzer every week since the research start and, to the end of the research, it has tested 8 times on total.The localization and expression of alpha-smooth muscle actin(α-SMA), transforming growth factor beta 1(TGF-β1),collagen type I(Col-I) in liver tissues was researched by Immunohistochemical method. Density gradient centrifugation after situ perfusion and 326 nm ultraviolet excitation together with α-SMA immunofluorescence staining were used to isolate and identify HSC respectively. The mRNA and protein expression lever of α-SMA, TGF-β1, COLⅠ, UCP2 and UCP3 in HSC were detected by qRT-PCR and Western Blot respectively. All above were done on the end of the experiment at 8 weeks. Results:1.The determination results of automatic biochemistry analyzer showed that: compared with the model group, serum ALT, AST, TBIL levels of the treatment group gradually decreased after MSC transplantation, there was significant difference by statistical analysis;2.The liver pathology study shows: liver fibrosis and inflammatory cell infiltration of the treatment group was significantly improved compared with the model group, the expression levels of liver tissue α-SMA, alpha TGF- beta 1 and COLⅠwere significantly reduced as compared with the model group, there were significant differences by statistical analysis;3.Founds in qRT-PCR and Western Blot test: compared with the model group,α-SMA, alpha TGF- beta 1, COLⅠgene and protein expression level of treatment group in HSC were decreased and the gene and protein expression level of UCP2, UCP3 were increased, there are significant differences by statistical analysis. Conclusion: 1. Small dose long-term intraperitoneal injection of CCl4 can induced the typical model of liver fibrosis, intervention of BM-MSC through tail vein injection on modeling process can reduce the inflammation of the liver, inhibit the formation of liver fibrosis, promote the recovery of liver function; 2. The effect of BM-MSC on anti hepatic fibrosis may be related to the anti inflammation effect of MSC and the inhibition of HSC activation. BM-MSC can inhibit the activation of HSC by up regulating UCP2 expression; 3. BM-MSC transplantation can be used to treat hepatic fibrosis.