Sirt1 And HMGB1 Regulate The AGE-Induced Pro-Inflammatory Cytokines In Human Retinal Cells

Diabetic retinopathy (DR) is one of the most common microvascular disorders of diabetes and leads blindness worldwide among adults? Diabetes occurrence and development from the retinal microvascular disease and microvascular leakage, retinal ischemia and hypoxia, cause the formation of a large number of non perfusion area, and finally the formation of retinal neovascularization value-added lesions, resulting in severe vision loss. Dr pathogenesis is complex, including increased polyol pathway flux, activation of protein kinase C, hexosamine pathway flux increasing way, and advanced glycation end products increase, advanced glycation end products (ages) ages accumulation in diabetic retinopathy and vascular cells, neurons and glial cells in Dr pathogenesis.Part 1 The expression of HMBG1 and SIRT1 in the AGE-BSA-treated ARPE-19 cellsObjective:To investigate he expression of HMGB1 and SIRT1 in the AGE-BSA-treated with AGEsMethods:AGEs and ARPE-19 cells were incubated together, at 4,8, and 12 hours later, using PCR to detect TNF-a, IL-1,IL-6, MCP-1,RANTES and IP-10 and HMGB1, SIRT1 mRNA level,24h after Western blot analysis of HMGB1, SIRT1 protein leve?Results:Compared with the control group, experimental group il-lmrna up-regulated, compare the differences between the two groups have statistical significance, and with the increase of the dose, more significant differences between the two groups; and that of the control group of HMGB1 mRNA and protein expression was up-regulated, between the two groups HBMGlmRNA differentially expressed was statistically significant; and compared to the control group, the down-regulation of SIRT1 mRNA and protein levels and activity, and between the two groups in the expression difference was statistically significantConclusion:The results indicate that AGEs induces the expression of TNF-, IL-1, IL-6, MCP-1, RANTES, and IP-10, with a dose-dependence. AEGs treatment also significantly promoted the expression of HMGB1 in mRNA and protein levels, which was dose dependent, and decreased the activity and protein expression of SIRT1.Part 2 Knockdown of HMBG1 affects AGE induced inflammatory cytokinesObjective:To investigate the influences of down-regulation of HMGB1 on TNF-, IL-1, IL-6, MCP-1, RANTES and IP-10Methods.ShRNA was designed and synthesized, and transfected into ARPE-19 cells under different conditions?The expression of TNF-, IL-1, IL-6, MCP-1, RANTES and IP-10 were examined by RT-real time PCR after knockdown of AEG-1.Results:Compared with the control, exogenous HMGB1 could significantly increase the expression of IL-1. Exogenous HMGB1 group, HMGB1 knock does not affect IL-I. In the AGEs group, the knockdown of HMGB1 significantly reduced the expression of IL-1. The same results were obtained for the detection of interleukin 6, tumor necrosis factor and chemokines.Conclusion:Downregulation of HMGB1 can induce the expression of AGE induced inflammatory cells and chemokinesPart 3 Effects of SIRTl activator and inhibitor on HMGB1Objective:To investigate the association of SIRT1 and HMGB1 in the inflammatory response induced by AGE in ARPE-19.Methods:The activity of SIRT1 was regulated by SIRT1 activators and inhibitors. RSA (SIRT1 activator) and Sirtinol (SIRT1 inhibitor) were used to intervene in the AGE-BS A-treated ARPE-19 cells and then do Western blot analysisResults:The Sirt 1 inhibitor, Sirtinol aggravated the promotion to cytosolic HMGB1 but inhibited the promotion to nuclear HMGB1 by AGEs. On the other hand, the Sirt 1 activator, RSV inhibited the promotion to cytosolic HMGB1 but aggravated the promotion to nuclear HMGB1. Moreover, the AGE-induced IL-l? and IL-6 were aggravated by Sirtinol but inhibited by RSV.Conclusion:Sirt 1 was confirmed to regulate the AGE-induced pro-inflammatory cytokines and chemokines in retinal ARPE-19 cells.