Protectiveeffectand Molecular Mechanism Of HDACi Of Research On Inflammatory Cytokines Induced INS-1 Cell Apoptosis

Type 1 diabetes mellitus(T1DM)which is characterized by islet ? cell damage and absolute insulin deficiency,histone deacetylase inhibitor(HDACi)can improve pancreatic islet ?-cell function,promote the synthesis and release of insulin,and reduce the damage of pancreatic ?-cell in inflammatory reaction.At present,HDACi on the injury of islet cells in inflammatory factors is still lack of perfect information.In this paper,the injury model of islet cells was established by cytokines(IL-1?,TNF-? and IFN-?)induced in vitro,exploring the protective effect of hydroxamic acids HDACis on inflammatory factor-induced apoptosis of pancreatic islet cells,and to study the molecular mechanism.It provides new candidate compounds for further research and development of diabetes drugs,providing a theoretical basis for HDACi in the treatment of diabetes.In the next step,we selected 17 hydroxamic acid HDACs inhibitors in the literatures,and 17 HDACi small molecules were docked with HDAC2,HDAC3,HDAC5,HDAC6,HDAC7 and HDAC8 active cavities by GOLD5.2 molecular docking software.TSA,SAHA,PXD101,LBH589 and PCI-24781 were selected for in vitro experiments.Screening of cytokine-induced INS-1 cells was induced by the combination of ligand and protein binding and scoring.Death has a protective effect of HDACi.In order to study the proinflammatory response of cytokines,we used inflammatory cytokines to stimulate INS-1 cells to construct inflammatory response models in vitro.Firstly,the cells were treated with IL-1?,TNF-?,IFN-? and cytokines in different time points,and cell viability was measured by MTT assay.The concentration and time of cytokine action in the subsequent experiments were determined.The number of apoptotic bodies after cytokine exposure was significantly increased by DAPI staining,indicating that cytokines promoted apoptosis of INS-1 cells.Meanwhile,we also detected the effect of cytokines on apoptosis and cycle of INS-1 cells by flow cytometry,and the block period was measured as S phase.Next we screened HDACi that protects INS-1 cells from apoptosis.Five broad-spectrum Hydroxamic acids HDACis(TSA,SAHA,PXD101,LBH589 and PCI-24781)were selected,set up a series of concentration gradients were applied to INS-1 cells.MTT was used to measure the absorbance.The IC10 values for each inhibitor were used to determine the concentration of HDACi used in subsequent experiments.Then the cells were pretreated with non-toxic HDACi and then treated with cytokines.Cell viability was determined by MTT assay,TSA,SAHA,PXD101 and PCI-24781 inhibited the apoptosis of INS-1 cells induced by inflammatory factors.Finally,the mechanism of PXD101 protection was investigated by Western-blot.PXD101 can inhibit the phosphorylation of I?B and the incorporation of p65 into the nucleus,indicating that HDACi can reduce the apoptosis of INS-1 cells by inhibiting the activation of NF-?B signaling pathway mediated by cytokines.In summary,cytokines can promote INS-1 cell apoptosis,Hydroxamic acids HDACi can protect INS-1 cells inhibit the occurrence of apoptosis.The protective effect of PXD101 is achieved by modulating the NF-?B signaling pathway.Therefore,HDACi is hoping to be a potential type 1 diabetes treatment for HDACs as a therapeutic target.