Pharmacological Effects Of Novel RNA Polymerase Ⅰ Inhibitor CX-5461 On Acute Transplant Rejection

Background:Organ transplantation is an effective treatment for end-stage organ failure.However,due to the individual differences of major histocompatibility antigens,innate and adaptive immune responses are initiated,resulting in acute rejection in the early stage.CD4+Th cells play a key role in mediating allogeneic tissue injury and subsequent inflammatory response.Therefore,it is very important to use immunosuppressants to inhibit the excessive proliferation and activation of recipient T cells after transplantation.CX-5461 is a specific RNA polymerase I(Pol I)inhibitor,which affects ribosomal biosynthesis by inhibiting the transcription of ribosomal DNA.It is now in phase II clinical trials as an anti-tumor drug and effectively inhibits a variety of proliferative vascular diseases.More importantly,we unexpectedly found that CX-5461 had significant anti-inflammatory activities.Based on the above results,in this study,we used mouse allogeneic skin transplantation model and human primary T cells cultured in vitro to explore the potential immunosuppressive effects of CX5461 and to clarify whether it had inhibitory effects on acute rejection after organ transplantation.Objective:To explore the pharmacological effects of CX-5461 on T cell function and acute allograft rejectionResearch contents:1.To detect the effects of CX-5461 on the function of human primary T cells in vitro2.To explore whether CX-5461 can effectively inhibit acute allograft rejection in vivoMethods:1.Human primary T cells were treated with different concentrations of CX-5461.The effects of CX-5461 on T cell proliferation was detected by EDU incorporation test and flow cytometry;The effects of CX-5461 on T cell activation was detected by qRT-PCR.2.The mice models of full-thickness back skin transplantation were established.The recipient mice were randomly divided into control group,CX-5461 treatment group and FK506(positive control)treatment group.From the day of operation to day 14 after operation,photos were taken to record the survival area of grafts in acute rejection stage;Extended the observation window to day 30 after operation to monitor the long-term protective effects of CX-5461;On the day 9 and 14 after operation,the grafts and recipient secondary lymphoid organs were taken for histopathological examinations:histopathological changes were detected by H&E staining,and the level of immune cell infiltration was detected by immunohistochemistry;Safety evaluation of drugs:the body weight changes of mice were recorded from the day of operation to day 14.The peripheral blood was taken for blood routine tests on the 9th and 14th day after operation.3.On the 9th and 14th day after operation,the effects of CX-5461 treatment on the proliferation of CD3+,CD4+and CD8+T cells in the recipient’s secondary lymphoid organs were detected by flow cytometry.qRT-PCR was used to detect the expression of related inflammatory cytokines in grafts and secondary lymphoid organs.4.On the 9th and 14th day after operation,the effect of CX-5461 treatment on the proportion of CD3+CD4+CD25+Foxp3+Treg cells in the recipient’s secondary lymphoid organs was detected by flow cytometry;TGF-β expression level-the marker of Treg cells was detected by qRT-PCR.In vitro,the effects of CX-5461 on expressions of cytokines IL-10 and TGF-β were detected by qRT-PCR in human primary T cells.Results:1.CX-5461 inhibited the proliferation and activation of human primary T cells in a concentration dependent manner in vitro.2.During the acute rejection period,CX-5461 treatment significantly improved the survival rate of grafts.In the long-term observation experiment,CX-5461 played protective effects on the recipient mouse graft.During the acute rejection period,CX-5461 reduced the inflammatory level of graft and spleen,but it did not cause non-specific bone marrow suppression,and the recipient mice had a good tolerance to CX-5461.3.CX-5461 inhibited the proliferation of T cells in spleens and lymph nodes and downregulated the mRNA expression of pro-inflammatory cytokines in skin grafts and secondary lymphoid organs in vivo.4.On day 14,the abundance of CD3+CD4+CD25+Foxp3+Treg cells in spleens and lymph nodes in CX-5461 treatment group was significantly increased.CX-5461 treatment upregulated the expression level of TGF-β in skin grafts and spleens.In addition,CX-5461 treatment in human primary T cells also significantly increased IL-10 and TGF-β expression levels.Summary:In vitro,CX-5461 can significantly inhibit the proliferation and activation of T cells,and can induces differentiation of Treg cells.In vivo,CX-5461 can significantly inhibit acute rejection of skin allografts,T cell proliferation,and the expression of proinflammatory cytokines.CX-5461 can also promote Treg differentiation in vivo.Conclusion:CX-5461 may be used as a novel immunosuppressant to prevent acute rejection after organ transplantation.