| Objective:To investigate the role and mechanism of I-BET151 in murine chronic graft-versus-host disease(cGVHD)after allogeneic hematopoietic stem cell transplantation(allo-HSCT).Methods:1.We establishd a mouse model of chronic graft-versus-host disease:C57BL/6(H-2b)?BALB/c(H-2d).2.Mice were randomly assigned to two groups at day 21,and the experimental group was administered I-BET151(10 mg/kg)every three days while the control group was administered only vehicle.Clinical manifestations of cGVHD,survival and weight changes were monitored and recorded after allo-HSCT.3.The lung,kidney,small intestine,skin and liver of mice were collected for histopathological examination after transplantation at day 30,45 and 60.4.The proportions of lymphocytes in spleens were measured by flow cytometry at day 30 and 60,and the proportions of lymphocytes in the mesenteric lymph nodes were detected by flow cytometry at day 30.In addition,the counting of thymocytes in the thymi were measured at day 45,60 and the percentages of CD3+T,CD4+T,CD8+T cells in the thymi were measured by flow cytometry day 60.5.The spleens of both control group and I-BET151 group at day 60 were detected by RNA-sequencing(RNA-seq)to explore the possible target genes of I-BET151,and the accuracy of RNA-Seq results were verified by real-time qPCR.Results:1.I-BET151 significantly alleviated cGVHD compared with recipients that received only vehicle.Compared with the control group,mice in the experimental group significantly increased weight and the cGVHD score was significantly reduced.2.I-BET151 significantly reduced histopathological damage in lung,kidney,small intestine,skin and liver.3.At day 30,I-BET151 treatment significantly decreased donor-derived Th1 cells in spleens compared with control group and the percentages of CD3+T,CD8+T cells and dendritic cell(DC)were significantly decreased in mesenteric lymph nodes compared with control group.In addition,in the I-BET151 group the numbers of total thymocytes in the thymi were significantly increased at day 45 and 60,and the percent of CD3+T and numbers of CD3+T?CD4+T and CD8+T cells in the thymi were significantly increased at day 60.However,at day 30,CD3+T,CD4+T,regulatory T cells(Treg),T follicular helper(Tfh)cells,PD-1(programmed cell death-1)+CD4+T,T follicular regulatory(Tfr)cells,PD-1+Treg,B cell,CD24hiCD38hi regulatory B cells(Breg)in spleen were similar between 2 groups.4.Through RNA-seq technology 711 DEGs were identified in the I-BET151 group,311 of which were up-regulated and 397 were down-regulated.In the enrichment analysis of GO(Gene Ontology),most of the DEGs were involved in immune system process,immune response and regulation of immune system process.In the enrichment analysis of KEGG(Kyoto Encyclopedia of Genes and Genomes),most of the DEGs were enriched in hematopoietic cell lineage,immune system and cytokine-cytokine receptor interaction.Through consulting relative literatures and verification by qPCR we screened 2 genes related to immune system process(Ctnnbipl and AnxA1)for future study.Conclusion:Our study demonstrated that I-BET151 could alleviate the development of cGVHD.The main mechanism was to inhibit the proliferation of donor-drived T cells and improve thymic immune reconstruction in cGVHD mice in murine HSCT models compared with vehicle.The results of RNA-seq indicated that DEGs were mainly enriched in immune system process which was consistent with previous results.These 2 DEGs Ctnnbipl and AnxA1 could be the possible targets of I-BET151 treatment for cGVHD. |
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