Triptolide Regulates P300-mediated H3K27 Acetylation To Reshape The Inflammatory ’Brake’ Mechanism Of RA-FLS

Objective:The purpose of the study was to explore the mechanisms of the persistence of rheumatoid arthritis(RA)synovial inflammation and the persistent expression of inflammatory gene in fibroblast-like synoviocytes(FLS).Then,from the perspective of histone acetylation modification,the mechanism of triptolide(TP)blocking the sustained expression of RA-FLS inflammatory genes and restoring the ’brake’ of RA synovial inflammation was explored.Finally,the target of TP regulating histone H3K27 acetylation was confirmed.Methods:1.RNA-seq sequencing of the synovial membrane was performed to detect the persistence of synovial inflammation in CIA rats,which was further verified by synovial H&E staining and immunohistochemistry.The progressive destruction of j oint bone in rats was observed by safranin fast green staining and tartrate-resistant acid phosphatase staining.Then,RT-qPCR,ELISA,Western blot and IF were used to detect the expression of TNF-α,IL-1β and IL-6 in FLS and macrophages induced by TNF-α.The expression of nuclear factor kappa B(NF-κB)and the acetylation of histone H3K27 in FLS were detected by Western blot and IF.2.The effects of 9.31 μg/(kg*d)and 18.62μg/(kg*d)TP on liver and kidney function and reproductive toxicity in CIA rats were detected by kit and H&E staining.The effect of TP on bone destruction was detected by safranin green staining and tartrate-resistant acid phosphatase staining.The effect of TP on synovial inflammation was detected by RNA-seq sequencing,ELISA and immunohistochemistry.The effect of TP on histone H3K27 acetylation was detected by Western blot.The effect of TP on the proliferation activity of FLS was detected by CCK8.The effect of TP on FLS apoptosis was detected by flow cytometry.The effects of TP on the migration and invasion of FLS were detected by cell migration assay and invasion assay.Western blot was used to detect the expression of TNFα,IL-1β,IL-6,NF-κB and H3K27 acetylation in FLS after treatment with TP.Combined RNAseq,CUT&Tag,and ATAC-seq techniques to detect the effect of TP on H3K27 acetylation and chromatin accessibility at inflammation-related genes in FLS.3.The binding ability of TP to histone acetylase(HAT)and histone deacetylase(HDAC)was analyzed by reverse molecular docking,and the protein targets with less binding energy were screened according to the level of free binding energy.Western blot and immunofluorescence were used to detect the expression of p300 and HDAC3/6/7/8 in FLS at different time points after TNF-α intervention.The effects of TP intervention on the expression of p300 in FLS were detected by Western blot and immunofluorescence.The effects of TP on the expression of p300 in synovium of CIA rats were detected by Western blot.The p300 competitive inhibitor A485 was used to inhibit the expression of p300,and the histone deacetylase inhibitor TSA was used to promote the expression of p300.The effects of TP on the expression of p300,and H3K27 acetylation in FLS were observed.TP was labeled by biotin,and the protein target of TP was pulled down by similar Pull-down method.Then mass spectrometry and Western blot were used to identify the protein target of TP.Results:1.The lung tissue and synovial membrane of CIA rats showed obvious inflammatory cell infiltration.And lung inflammation will disappear within a month,while synovial inflammation persists.The expression of inflammatory factor TNF-α in serum and synovium of CIA rats continued to increase,and synovial inflammation-related genes continued to express.And the cartilage and bone tissue of CIA rats showed progressive damage with the prolongation of modeling time.Compared with macrophages,FLS and its cell line MH7A showed persistent expression of inflammatory genes and increased expression of inflammatory factors TNFα,IL-6 and IL-1β.The expression of transcription factor NF-κB and histone H3K27 acetylation levels in FLS were consistent with the continuous expression trend of inflammatory genes in FLS.2.TP at 9.31μg/(kg*d)and 18.62μg/(kg*d)had no significant effect on the expression of serum alanine aminotransferase,aspartate aminotransferase,blood urea nitrogen,creatinine and acid phosphatase in CIA rats.TP can significantly alleviate joint swelling and improve arthritis score in rats,and its effect is not weaker than methotrexate(1.05 mg/kg/w).TP can alleviate the progressive destruction of articular cartilage and bone tissue,inhibit the expression of synovial inflammatory factor TNF-α and transcription factor NF-κB,and inhibit the transcription of synovial inflammatory genes and matrix metalloproteinase genes.TP can inhibit the proliferation of FLS in a time-dependent and dose-dependent manner.TP at 20 nM and 40 nM can inhibit FLS inflammation-related invasive phenotypes,such as apoptosis,migration and invasion.TP can inhibit the expression of inflammatory factors TNF-α,IL-1β,IL-6 and transcription factor NF-κB in FLS.TP can reduce the acetylation level of H3K27 in FLS.TP can inhibit the H3K27ac and chromatin accessibility of inflammatory genes(such as TNFSF15,TNFRSF18,NFKB)in FLS.3.Reverse molecular docking confirmed that TP had high affinity with p300 and HDAC3/6/7/8.The expression of p300 in FLS increased with the prolongation of TNF-αintervention time,which was consistent with the expression trend of inflammatory factors.The expression of HDAC3/6/7/8 did not change significantly with the prolongation of TNFα intervention time.In vitro experiments confirmed that TP can inhibit the expression of p300 in FLS.In vivo experiments confirmed that TP can inhibit the expression of histone acetylase p300 in synovium of CIA rats.The combined intervention of A485 and TSA confirmed that TP may inhibit H3K27 acetylation by inhibiting the expression of p300 in FLS.Pull-down experiments confirmed that transcription factor NF-κB and histone acetylase p300 may be potential targets of TP.Conclusions:The persistent expression of inflammatory genes in FLS may be a key factor for the persistent synovitis and progressive destruction of articular cartilage and bone tissue in CIA rats.The high acetylation of H3K27 is an important mechanism for the continuous expression of inflammatory genes in FLS.TP can block the continuous expression of inflammatory genes in FLS by regulating the expression of transcription factor NF-κB and the acetylation level of H3K27,and restore its inflammatory "brake" mechanism,ultimately inhibiting persistent synovitis.The regulation of H3K27 acetylation by TP may be achieved by regulating the expression of p300.