Mfn2Inhibits Rat Chronic Rejection Of The Abdominal Aorta And Liver Fibrosis By Regulating TGF-β

BackgroundChronic rejection belongs to delayed allergy, generally occurs and develops progressively in six months or a few years. Its etiology and mechanism is complex and not very clear, mainly caused by repeated acute rejection. The pathological feature of chronic rejection is degeneration and disruption, characterized by proliferation of vascular intimal smooth muscle cells and interstitial fibrous tissue and mononuclear macrophage infiltration.Chronic rejection has a common histologic appearance which is allograft arteriosclerosis, the early allograft atherosclerosis has obvious VSMC apoptosis, and VSMC apoptosis associated with vascular intimal thickening and allograft atherosclerosis, it is characterized by foam cell occlusive arterial disease, such as artery intimal thickening, infiltration of mononuclear cells and macrophage in intima, interstitial fibrosis.Mfn2, also known as the proliferation inhibition gene (HSG), its expression product inhibits the proliferation of VSMC and induces VSMC convert from the proliferative dedifferentiation to differentiation,participates in the process of VSMC differentiation, and high expression of HSG/Mfn2gene can promote the apoptosis of rat vascular smooth muscle cells (VSMC). But the functions and changes of Mfn2in chronic graft rejection has not been reported. Liver fibrosis is the accumulation of fibrous tissue in the liver caused by disequilibrium of formation and degradation of extracellular matrix, is an extensive damage of the structure of the liver caused by many regenerative nodules which are surrounded by fibrous tissues. Liver fibrosis is the excessive deposition of liver fiber connective tissue, is the result of the unbalanced fiber hyperplasia and decomposition. Mfn2as a new type of proliferation inhibiting factor has been proved that it can inhibit the proliferation of vascular smooth muscle cells, and reduce neointimal elastic fibrous tissue. At the same time, it has been reported that in diabetic nephropathy model, high expression of Mfn2can inhibit renal interstitial fibrosis, decrease the expression of collagen4. But how Mfn2effects on liver fibrosis has not been reported.AimsThis study aimed to find the role and the possible mechanism of Mfn2in the process of fibrosis by studying the influence of Mfn2on chronic rejection of rat abdominal aorta transplantation and changes in the process of liver fibrosis, to provide certain help to the treatment of liver cirrhosis and reduce chronic rejection.Methods1. Construct Mfn2-GFP lentivirus, and transfect the transplanting abdominal aorta in donor Lewis rat.2. Transplant the Mfn2-GFP-transfected donor Lewis rat abdominal aorta to the corresponding position of the abdominal aorta in receptor Bn rat, and make negative control group which transfected with GFP by the same way.3. Take the transplanted abdominal aorta in day30,60,90, and observe vascular intimal thickness with HE staining and masson staining. Determine the relationship of Mfn2and chronic rejection related genes TGF-betal、TGF-beta R2by Immunohistochemistry.4. Inject Mfn2-GFP lentivirus through portal vein to infect rat liver, and construct rat liver cirrhosis model by ccl4.5. Take rat liver after8weeks, and observe liver morphology by HE and masson staining, use western blot, Immunohistochemistry to detect the relationship of expression of Mfn2and fibrosis associated factors TGF-β1、TGF-βR2, Smad3, Smad4, Smad7,collagen IV,a-SMA.6. Transfect rat hepatic stellate cell with Mfn2-GFP lentivirus, detect the relationship of Mfn2and fibrosis associated factors TGF-β1、TGF-βR2, Smad3, Smad4, Smad7.collagen IV by WB.7. Use CCK8,cell apoptosis,migration assay to detect proliferation of Mfn2-transfected hepatic stellate cell.Results1. Use masson staining and imagepro-plus6.0software to measure the intimal thickness of transplanted rat abdominal aorta, the result shows that the intimal thickness of Mfn2-GFP-transfected abdominal aorta is lower than the one transfected with GFP(p<0.001)2. The immunohistochemical results confirmed that the expression of TGF-β1、 TGF-β R2in intima of Mfn2-GFP-transfected abdominal aorta is lower than the one transfectd with GFP in day60,90after transplantation (p<0.01). 3. In construction of rat liver cirrhosis model with ccl4, we observed with masson staining that:At2weeks began to appear obvious fat vacuoles, but the structure of liver is normal; At4weeks began to appear degeneration and necrosis within the lobule,, and disorder of fiber interval with lobular structure;6weeks appear larger range of bridging necrosis, involving multiple lobules with structure disorder, fiber septae is less than0.1mm, but fiber nodule diameter is larger than2mm;8weeks appear larger range of bridging necrosis, involving multiple lobules with structure disorder, fiber septae is less than0.1mm, but fiber nodule diameter is less than1mm. Masson staining also hints that, in construction of rat liver cirrhosis model with ccl4, the fibrosis degree of rat liver injected with Mfn2-GFP lentivirus through portal vein in8weeks has no obvious difference with the one in6weeks(p>0.05), but less than the one in8weeks (P<0.05).4. WB and immunohistochemistry results suggest that the expression of Mfn2in liver cirrhosis group is less than control negative group and Mfn2-virus-transfected group (p<0.05), at the same time the expression of TGF-β1, TGF-(3R2, Smad3, Smad4and collagen IV is greater than the latter two (p<0.05), which suggest that Mfn2may inhibit liver fibrosis by participating in the TGF(3-Smad pathway.Conclusions1. The intimal thickness of Mfn2-GFP-transfected abdominal aorta is lower than the one transfected with GFP, and the immunohistochemical results also confirmed that the expression of TGF-β1,TGF-β R2is lower than the one transfectd with GFP, which suggest that Mfn2may reduce graft chronic rejection by regulating TGF -β1.2. Mfn2has the effect of inhibiting the process of liver fibrosis, and it may participate in the TGFβ-Smad pathway to reduce liver fibrosis.