| Backgrounds:In the early 2000s,Th17 cells,a unique subgroup of CD4~+T cells,were found to be different from other CD4~+T cell subsets,such as Th1 cells,Th2 cells and Treg cells,in their function and function,and their functions and functions were different from those of other CD4~+T cell subsets,such as Th1 cells,Th2 cells and treg cells.Th17 cells play an important role in autoimmune diseases and defense responses,including rheumatoid arthritis,multiple sclerosis,encephalomyelitis,colitis,psoriasis and so on.When Th1cells and Th2 cells respond effectively to intracellular bacterial infections and parasitic pathogens,Th17 cells provide protection against extracellular bacterial and fungal infections,and Th17 cells secrete IL-17a,IL-17f,IL-6,And tumor necrosis factor-?(tumor necrosis factora,TNF-?(TNF-?),which effectively mediates tissue inflammation by recruiting neutrophils.When this protective effect is excessive,it may lead to the occurrence of autoimmunity.Th17 cells are inflammatory,characterized by the ability to express a specific transcription factor,retinoic acid-associated orphan nuclear receptor(retinoid-related orphan receptor gammat,ROR?t).IL-17 is the main effector of Th17cells,which has strong proinflammatory cytokine properties of neutrophil recruitment,and can promote the release of inflammatory factors by many kinds of cells.IL-17 is a key cytokine of interleukin 17(IL-17A),which is a key cytokine in Th17 cells.Retinoic acid associated orphan nuclear receptor(retinoid-related orphan receptor gammat,ROR?t)is a specific nuclear transcription factor of Th17 cells.It can promote the differentiation of Th17 cells together with other transcription factors.Overexpression of ROR?t can induce the differentiation of th0 into Th17 cells.It can induce the expression of genes encoding IL-17 and IL-17f cytokines,and directly activate the transcription of IL-17 and other transcription factors together with other transcription factors to promote the differentiation of Th17 cells by binding to the IL-17 promoter.p300 protein is a member of the histone acetyltransferase hats family,which has the functions of ubiquitin,acetylation,phosphorylation and so on.As far as acetylation is concerned,it can acetylate the histone,and it can modify the histone by acetylation.Some transcription factors can also be acetylated,in which the modification of transcription factors can affect protein stability,subcellular localization and DNA binding ability,participate in cell differentiation,transcription,immune response and other regulation.In this study,the immunoprecipitation method was used to prove that p300 can interact with ROR?t and acetylation of ROR?t in both over-expressed HET293T cells and human Th17cells.According to the results of previous studies,p300 is acetylation of ROR?t by binding to lysine residue at position 81 of ROR?t.We found that in Th17 cells,p300 was induced by this acetylation of ROR?t,and that p300 could bind to the lysine residue at position 81 of ROR?t to acetylate ROR?t.It promotes the binding of ROR?t with IL-17promoter to up-regulate the transcription of IL-17 mediated by Th17 cell differentiation.JQ1 is a acetyltransferase inhibitor,which can inhibit the acetylation of ROR?t induced by p300.Thus,the secretion of IL-17 and the differentiation of Th17 cells were inhibited.After JQ1 was used,the symptoms of hepatic granuloma fibrosis were alleviated and the expression of IL-17 was down-regulated in the granuloma model of Schistosoma japonicum infected with Schistosoma japonicum.In addition,we determined that the inhibitory effect of JQ1 on the acetylation of p300 was determined by detecting the activity of acetyltransferase,which was due to the competitive binding of JQ1 to the brd domain of p300,and thus to the down-regulation of the acetylation of p300 to ROR?t.Research contents:1.In HEK293T cells,the protein interaction and protein acetylation were detected by immunoprecipitation,immunoprecipitation and Westernblot in the presence or absence of JQ1.2.In human Th17 cells,the results of Westernblot showed that the acetylation of ROR?t in JQ1 treated group was down-regulated compared with that in DMSO solvent control group.3.The results of qPCR showed that IL-17A,IL-17F expression of Th17 cells in JQ1treated group was lower than that in DMSO solvent control group.4.ELISA results showed that compared with the DMSO solvent control group,JQ1treatment group inhibited the expression of IL-17.5.The results of qPCR of mouse liver granuloma cells showed that the gene levels of IL-17 were significantly down-regulated compared with those of JQ1 group treated with DMSO solvent.6.Flow cytometry was used to detect liver granuloma cells in mice.Compared with DMSO solvent control group,the expression of IL-17 and ROR?t in JQ1 treated group was significantly down-regulated.7.Up to now,no studies have shown which part of the p300 domain interacts with JQ1.In order to determine the structure region,p300 protein is expressed partly in bromine domain and p300 protein is absent in bromine domain.In the presence or absence of JQ1,the acetylation activity of these proteins was detected by the kit.Research results:1.JQ1 inhibited the acetylation of ROR?t by inhibiting the acetylase activity of p300.2.JQ1 can inhibit the expression of cytokines such as IL-17A,IL-17F,IL-21,IL-22,IL23R and GM-CSF.3.JQ1 can competitively bind to the BRD domain of p300,thus inhibit the acetyltransferase activity of p300 and the acetylation of ROR?t by p300.4.JQ1 inhibited the expression of IL-17 and ROR?t in liver granuloma cells of mice infected with Schistosoma japonicum.5.JQ1 may inhibit the production of IL-17 by inhibiting the acetylation of ROR?t by p300.JQ1 may contribute to the treatment of granulomatous inflammation and fibrosis caused by Schistosoma japonicum eggs.Conclusions:1.JQ1 inhibited the acetylation of ROR?t by inhibiting the acetylase activity of p300.2.JQ1 can inhibit the expression of cytokines such as IL-17A,IL-17F,IL-21,IL-22,IL-23R and GM-CSF.3.JQ1 can competitively bind to the BRD domain of p300,thus inhibit the acetyltransferase activity of p300 and the acetylation of ROR?t by p300.4.JQ1 inhibited the expression of IL-17 and ROR?t in liver granuloma cells of mice infected with Schistosoma japonicum.5.JQ1 may inhibit the production of IL-17 by inhibiting the acetylation of ROR?t by p300.JQ1 may contribute to the treatment of granulomatous inflammation and fibrosis caused by Schistosoma japonicum eggs. |
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