MSCs Alleviate Liver Fibrosis Via Regulating Kupffer Cell M1 And M2 Balance

【Background】Cirrhosis is a chronic liver damage caused by various factors and develops to the end-stage liver disease, and the morbidity and mortality are increasing year by year. There are as many as 1.03 million deaths caused by cirrhosis each year worldwide. Cirrhosis has been ranked the 14 th in the most common cause of death, and serious harm to human health.Currently, traditional routine medical treatments were unsatisfactory, so liver transplantation is still the most effective therapy, but due to the lack of donors, high cost and other factors, the clinical application is greatly limited the clinical application. Therefore, it is important to figure out the pathogenesis of liver fibrosis and make the early prevention or reverse liver fibrosis. In recent years, mesenchymal stem cells(MSCs) have been found to have self-renewal and multi-directional differentiation potential, especially its immunoregulation has attracted much attention in various clinical researches. In addition, it is also reported MSCs could regulate the inflammation responses in chronic liver fibrosis, significantly improve liver function and prolong survival of patients with end-stage liver disease, which has brought a new hope and appropriate entry point for tissue repair and cell therapy.Liver has an important kind of non-parenchymal immune cells named kupffer cells(KCs), which protect the body from invading foreign substances and play a key role in maintaining homeostasis. Studies have reported that KCs have two different activation pathways, respectively, to produce two different types of KCs, namely the M1 and M2 type. The balance between these two types of cells is closely related with tumor, infection and tissue injury and other inflammatory diseases.Previously, we have explored the effects of MSCs and KCs. We injeted disodium liposome solution into mice from tail vein(CCl4 liver injury model) and removed KCs, then we found that therapeutic effects of the treatment MSCs group were significantly better than the other groups. Besides, the m RNA expression of CD206, Arg-1, and anti-inflammatory cytokine IL-10 that associated with M2 type KCs were increased significantly, whereas M1 type-related CD68 and pro-inflammatory cytokines TNF-α decreased. These results manifest that KCs could play an important role in the treatment functions of MSCs on liver fibrosis by inducing activation of M2 KCs.In this experimental research, using CCl4 induced liver fibrosis model, then transplanted with homologous MSCs, evaluate therapeutic effects of MSCs transplantation, and investigate the mechanism of MSCs by promoting KC type conversion to treat liver fibrosis. We provide theory support for the application of MSCs in clinical treatment of liver fibrosis. 【Objectives】1. To investigate the differences of M1/M2 in mouse CCl4-induced liver injury group at different times.2. To clarify whether MSCs can regulate the M1/M2 ratio to participate in the repair of liver damage repair. 【Methods】1. Using CCl4 intraperitoneal injection to induce liver injury model for 8 weeks. During the development of liver injury, liver samples and serum of the 2nd, 4th, 6th, 8th week modeling were collected. The automatic biochemical analyzer tested mouse serum AST, ALT and ALB levels. H&E staining and Sirius red staining were to analyze liver injury and the degree of fibrosis. Meanwhile we used the liver tissue serial sections, labeled CD68 and CD206 respectively by immunohistochemical staining and investigated the ratio of two types KCs in different time periods during the process of liver injury.2. We isolated primary non-parenchymal cells in mice at 2nd, 4th, 6th, 8th week by collagenase perfusion. After skin preparation, laparotomy, intubation in situ perfusion, and centrifugation, we got non-parenchymal cells and then stained CD68 and CD206 to detect the percentages of two types of KCs at different times of liver injury by flow cytometry.3. We isolated and cultured MSCs from whole bone marrow. Cell morphology was observed under an inverted microscope and relief microscope, and MSCs surface markers of the third generation were identified by flow cytometry.4. CCl4-induced liver fibrosis male mice were randomly divided into three groups, including olive oil group, untreated group and treatment group. MSCs from the same strain of mice were transplanted intravenously ino mice of treated group(1x106cells/mice). Also serum level(AST, ALT and ALB) was tested to monitor liver function. H&E staining and Sirius red staining were to analyze liver inflammation and fibrosis. Similarly, non-parenchymal cells in mouse liver of treatment MSCs group were isolated with collagenase perfusion, and we observed percentage of two types KCs by flow cytometry.5. The m RNA expression of cytokines related M1-type(TNF-α, MCP1, IFN-γ) and M2-type(Arg1, CD163, IL-10) by Real-time PCR in mouse liver injury and MSCs treatment group were tested in different time periods. 【Results】1. After CCl4 treatment for 8 weeks, H&E staining and Sirius red staining displayed infiltration of inflammatory cells and the formation of remarkable fibrosis. Blood biochemistry test results showed transaminases were increased but albumin levels significantly reduced, which suggested that mouse liver fibrosis model was successfully established.2. According to Immunohistochemical staining and flow cytometry, during the pocess of CCl4-induced liver injury, M1-type KCs labeled CD68 marker gradually increased and reached the peak until the eighth week. While M2-type KCs labeled CD206 marker gradually increased from the fourth week to the sixth week, then gradually decreased. The m RNA expression of cytokines related M1-type(TNF-α, MCP1, IFN-γ) and M2-type(Arg1, CD163, IL-10) by Real-time PCR in mouse liver injury are similar to the above results, which suggested that the ratio of M1/M2 KCs is different.3. Compared with control group, the transplantation of homologous MSCs into CCl4-treated mouse significantly ameliorated liver injury. H&E staining and Sirius red staining showed effective improvements of CCl4-induced liver injury in mice, and blood biochemistry test results also displayed that transaminase levels were decreased and albumin levels elevated.4. Immunohistochemical staining and flow cytometry also showed that compared to untreated groups, the ratio of M1-type KCs labeled CD68 marker of the treatment MSCs group was reduced, while M2-type KCs labeled CD206 marker increased. The m RNA expression of cytokines related M1-type(TNF-α, IFN-γ) and M2-type(CD163, IL-10) by Real-time PCR in mouse liver injury are similar to the above results, which suggested that MSCs might change the type of KCs to participation liver damage repair. 【Conclusion】This study suggested that KCs involve in the pathological process of CCl4-induced liver injury in mice.(1) During early inflammation, M1-type KCs is predominant, while M2-type KCs becomes a leading factor in late inflammation development stage. When M1/M2 type KCs is imbalance in the inflammatory process, it will lead to poor recovery of liver injury.(2) MSCs might change the type of KCs that significantly reduced quantity of pro-inflammatory cytokines and increased secretion levels of anti-inflammatory factors, which would play an important role in the recovery of liver injury.