Pharmacological Effects Of The Specific RNA Polymerase I Inhibitor CX-5461 On Acute Rejection Folling Skin Transplantation In Mice

BackgroundThe skin is the largest organ in the human body[1].The skin is wrapped on the surface of the human body and directly in contact with the external environment.It is the first line of defense of the human immune system.It not only protects the human from being attacked by external microorganisms,but also It has the functions of excreting sweat,regulating body temperature and feeling the stimulation of the external environment[2].Once a major accident occurs,or in other cases,a large-scale skin defect occurs,it cannot heal itself.In order to protect the wound surface and prevent infection,clinically usually Skin transplantation is required to promote wound healing.When allogeneic organisms undergo skin transplantation,the graft acts as a foreign antigen,which stimulates the recipient to produce an immune rejection reaction[3].Studies have shown that the antibodies against grafts and MHC molecules in the recipient serum play an important role in immune rejection[6].Because T lymphocytes express abundant MHC-I molecules,lymphocytes in immune rejection reactions The role is crucial[7].After the graft enters the animal body,the MHC antigen of the graft contacts and sensitizes the recipient immune cells,prompting the activated T lymphocytes to produce multiple cytokines[8-10]to recognize the graft cells and cause immune rejection.If an inhibitor that inhibits immune rejection in the body cannot be applied at the same time during allograft transplantation,it will cause the transplanted skin to be attacked by the recipient’s immune cells and cause necrosis.Traditional immunosuppressive agents,such as cyclosporine A,cyclophosphamide,tacrolimus,mycophenolate mofetil,etc.,mainly suppress the growth and differentiation of lymphocytes,so as to achieve the purpose of suppressing the occurrence of immune rejection[4,5],Long-term application will cause many adverse reactions,such as liver and kidney toxicity,and other tumors caused by the weakening of the body’s immune system and other diseases.Therefore,finding a new mechanism of action and a highly effective and low-toxic immunosuppressant may provide new ideas for the clinical treatment of rejection of allogeneic skin transplantation.CX-5461 is a novel drug that specifically inhibits RNA polymerase I(POL I)[6].Previous studies have confirmed that CX-5461 can be used as an anti-tumor drug,which can specifically induce autophagy and tumor cells such as certain lymphatic system leukemia,human colorectal cancer,melanoma and pancreatic cancer through the p53-independent pathway.Aging has a good therapeutic effect on these diseases.According to the previous study of the research group,CX-5461 has a good therapeutic effect on psoriasis,a common chronic immunoinflammatory proliferative skin disease.CX-5461 can significantly inhibit the intima of arteries caused by balloon injury Hyperplasia[7],which also inhibits the progression of macrophage-mediated angiogenesis intimal hyperplasia and vascular inflammation after vascular transplantation,and we continue to study the effect of CX-5461 on myocardial fibroblasts and found this drug It can inhibit the activation and phenotypic transformation of myocardial fibroblasts[8,9].However,it has not been investigated whether there is a transplantation effect on the immune rejection caused by skin transplantation.Objectives1.Explore whether the specific inhibitor of RNA polymerase I(POL I)CX-5461 can inhibit Jurkat T lymphocyte proliferation and the expression and secretion ofinflammatory factors after Jurkat T lymphocyte activation.2.To explore whether the specific inhibitor of RNA polymerase I(POL I)CX-5461 can inhibit the immune rejection caused by allogeneic skin transplantation in mice.Methods1.In vivo(animal)experiment40 SPF grade BALB/c mice and 30 C57BL/6 mice were adaptively fed for one week.In order to rule out the artificial influence caused by the operation in this trial,10 BALB/c mice were randomly selected as autologous skin transplantation models and used as the Sham group.The remaining BALB/c mice and C57BL/6 mice were randomly divided into three groups for heterologous skin transplantation models:(1)Control group,(2)CX-5461 group,and(3)CMC-Na group.The mice in the Control group were treated with saline.Mice in the CX-5461 group were given 2 mL of CX-5461 solvent at a concentration of 9.025mg/Kg by intragastric administration.CX-5461 was dissolved in CMC-Na(sodium carboxymethyl cellulose).Mice in the CMC-Na group were treated with the same dose of CMC-Na.Treat the mice according to the experimental requirements.After 12 days,first observe the weight of the mice and the damage degree of the grafts,and then take the materials for histopathological staining to observe the pathological changes of the skin.2.In vitro(cell)experimentThe Jurkat T lymphocyte line was cultured.Divide Jurkat T lymphocytes into four groups:1.Control group of cells2.CX-5461 group:CX-5461(2uM)treated cells3.PI group:Jurkat T lymphocytes were stimulated with phorbol ester ionmycin mixture(50ng/ml+0.5uM)for activation.4.CX-5461+ PI group:cells treated with CX-5461(2uM)and phorbol ester ionomycin mixture(50ng/ml+0.5uM)Cell proliferation was tested with Cell Counting Kit-8(CCK-8),and the expression of tumor necrosis factor-β,interleukin and gene expression of interferon were detected by real-time quantitative PCR.The expression of TNF-β,IL-2,IFN-γ and β-actain protein was measured by Western blot and the extracellular secretion of IL-2 was detected by ELISA.Results1.In the mouse skin transplantation test model,we found that CX-5461 can significantly improve skin shrinkage,blackening and shedding caused by skin transplantation.2.HE staining results showed that compared with the Control group,CX-5461 treatment significantly inhibited the proliferation and aggregation of inflammatory cells caused by immune rejection.In addition,we found that CX-5461 can reduce the inflammation in the lesion.Compared with the control group,the number of skin inflammatory cell infiltration in the CX-5461 treatment group was significantly reduced.3.In the cell experiment,the results of Cell Counting Kit-8(CCK-8)showed that the number of cells in the CX-5461 treatment group was significantly reduced.4.Real-time quantitative PCR results showed that the expression of IFN-γ,TNF-βand IL-2 in CX-5461 treatment group decreased significantly.5.The expression of TNF-β,IL-2 and IFN-γ protein measured by Western blot also decreased.6.Enzyme-linked immunosorbent assay(ELISA)detected that in the supernatant secreted by the cells,the IL-2 secretion of the CX-5461 treatment group was significantly reduced.Conclusions1.CX-5461 can inhibit T lymphocyte proliferation and the expression and secretion of inflammatory factors after T lymphocyte activation.2.CX-5461 can inhibit the immune rejection caused by heterologous skin transplantation in mice and reduce the inflammatory cell infiltration of grafted skin.