Functional Role Of High-mobility Group Box1(HMGB1) Expression In Diabetic Retinopathy Through Inflammation And Redox

Background and Objectives : Diabetic retinopathy(DR)is one of the main microvascular complications of diabetes,and is a major cause of adult blindness.Because the pathogenesis of DR is very complex and has not been fully elucidated,it is necessary to understand its pathogenesis and search for new therapeutic targets.The main pathological changes of DR include retinal cell apoptosis and angiogenesis.Previous studies have shown that the development of DR includes oxidative stress,mitochondrial dysfunction and inflammation.Recent studies have shown that diabetes can cause many epigenetic changes,including histone modification and DNA methylation,which is present in DR.High-mobility group box B1(HMGB1)is a highly conserved non-histone DNA binding protein,which plays an important role in many biological behaviors.As an advanced inflammatory factor,HMGB1 can be actively or passively released into the extracellular fluid after stress.In the cytoplasm,HMGB1 is involved in the imbalance between autophagy and apoptosis.When released,HMGB1 regulates angiogenesis,cell metastasis,and the release of inflammatory cytokines.Recent studies have shown that HMGB1 is associated with diabetic peripheral neuropathy and is involved in the occurrence and development of DR,but its mechanism is still unclear.As a multi-functional nuclear transcription factor,NF-kB has a wide range of biological activities and is involved in the expression and regulation of many genes.It is closely related to the major pathological and physiological processes such as inflammatory response,immune response,cell proliferation,transcription and apoptosis.In vivo and in vitro experiments have confirmed that the activation of NF-kB will lead to the increased expression of inflammatory factors such as IL-1,IL-6 and TNF,which will lead to an obvious inflammatory response.In recent years,NF-kB has become a research hotspot in various medical fields.Superoxide dismutase(SOD)is a kind of metal enzyme that catalyzes superoxide dismutase reaction.There are three types of SOD in humans and mammals: Cu in cells,zn-sod(SOD1),mn-sod(SOD2)in mitochondria,and ec-sod(SOD3)in extracellular fluid containing Cu and Zn.SOD2 mainly exists in the mitochondrial matrix of prokaryotes and eukaryotes,and is a key enzyme inmitochondria to remove superoxide anion from oxidative stress,so it is considered as a key barrier to resist mitochondrial damage.It has been reported that SOD activity in vivo decreases during inflammation,and exogenous SOD can stop inflammation.In macrophages stimulated by LPS,the blocking of NF-kB signaling pathway can improve SOD activity.Other studies have shown that the gene polymorphism of SOD2 is significantly correlated with the generation of diabetic neuropathy.In animal models of diabetic neuropathy and in vitro studies,overexpressed SOD2 also shows protective effects.In diabetic state,the activity of SOD2 decreased.Chen feng et al.observed that the protein synthesis of SOD2 may be inhibited to some extent under high glucose.Epigenetic mechanisms include DNA methylation,histone modification and miRNAs expression regulation,etc.,that is,genetic changes can be made to gene expression without changing DNA sequence.In the pathological process of diabetes and its complications,a series of pathological and physiological changes caused by hyperglycemia as the initiating factor can all originate from and lead to a variety of abnormal epigenetic regulation.Epigenetic regulation of SOD2 involves the development of diabetic retinopathy and metabolic memory.H3K4me1 and me2 of SOD2 promoter decrease and LSD1 of histone methylation-related enzymes increase in diabetic environment.LSD1 gene silencing can reduce H3K4 methylation of SOD2 promoter and block the down-regulation of SOD2 gene by high glucose.These studies indicate that SOD2 has a wide range of histone modification regulation in DR,and the manipulation of related enzymes can regulate SOD2 expression,thus playing an epitherapeutic role in DR.Apoptosis is a process of programmed cell death regulated by genes and plays a key role in many diseases.The early pathological changes of diabetic retinopathy are capillary endothelial cells and pericyte apoptosis.The number of apoptotic cells increased gradually,and some capillary wall permeability changed,resulting in no perfusion area and capillary necrosis and occlusion.In order to clarify the trends of HMGB1,NF-kB,SOD2 and LSD1 in diabetic retinopathy,the relationship between HMGB1 and cell proliferation and apoptosis was studied in this study.Methods:Part I: Expression of HMGB1,NF-KB,SOD2 and LSD1 in retinal cells undernormal and high glucose conditions.Diabetes mellitus model was established by intraperitoneal injection of streptozotocin into rats,and retinal vascular endothelial cells(HREC)were treated with high concentration glucose medium in vitro.Then the expression of HMGB1,NF-kB,SOD2 and LSD1 in retinal cells was detected by Western Blot,immunohistochemistry and PCR.Part II: The mechanisms of changes in NF-KB,SOD2 and LSD1 induced by HMGB1 in retinal cells.The method of constructing diabetes model in vivo and in vitro is the same as the first part.In vivo: HMGB1 was silenced by injecting siRNA into the vitreous cavity of diabetic rats.HMGB1 was overexpressed by injecting HMGB1 into the vitreous cavity of normal rats.In vitro: HMGB1-shRNA was transfected into retinal vascular endothelial cells to overexpress HMGB1.Then the expression of HMGB1,NF-kB,SOD2 and LSD1 in retinal cells was detected by Western Blot,immunohistochemistry and PCR.Chip-seq was used to detect the binding sites of HMGB1 in HREC and Western Blot was used to detect the binding sites.PartIII: The effects of HMGB1 on retinal neovascularization and retinal cell proliferation and apoptosis.In vivo and in vitro modeling methods are the same as the second part.ADP enzyme retinal staining is used to detect retinal neovascularization.Cell proliferation was detected by CCK-8 method,and apoptosis of HREC was detected by flow cytometry.Results:Part I: Expression of HMGB1,NF-KB,SOD2 and LSD1 in retinal cells under normal and high glucose conditions.After the successful establishment of the diabetic rat model,the weight and blood glucose of the control group and the diabetic group were gradually different with the prolongation of the course of disease.The weight of the control group was higher than that of the diabetic group,and the blood glucose of the diabetic group was lower than that of the diabetic group,and there was a statistical difference(p < 0.05).Western blot in vivo confirmed the high expression of HMGB1,NF-KB and LSD1 and the low expression of SOD2 in diabetic model.Immunohistochemical staining showed that the expression of HMGB1 and NF-KB increased in diabetic model(p <0.05).At the same time,the expression of HMGB1,NF-KB,SOD2 and LSD1 was confirmed in vitro(p < 0.05).Human retinal vascular endothelial cells(HREC)were cultured under low and high glucose conditions respectively.It was found that the expression of HMGB1,NF-KB and LSD1 increased in high glucose medium(p <0.05),while the expression of SOD2 decreased in high glucose medium(p < 0.05).This is consistent with the results of in vivo experiments.Part II: The mechanisms of changes in NF-KB,SOD2 and LSD1 induced by HMGB1 in retinal cells.After siRNA was injected into vitreous cavity of diabetic rats,the expression of NF-KB and LSD1 decreased(p < 0.05)and SOD2 increased(p < 0.05)after HMGB1 silencing.After injecting HMGB1 into the vitreous cavity of normal rats,the expression of NF-KB and LSD1 increased(p < 0.05)and the expression of SOD2decreased(p < 0.05)after overexpression of HMGB1.These results suggest that the changes of expression of HMGB1 can induce the changes of expression of NF-KB,SOD2 and LSD1 in vivo,which is consistent with the changes of expression of NF-KB,SOD2 and LSD1 in diabetic model.HMGB1-shRNA was transfected into HREC.RT-PCR results showed that HMGB1-shRNA transfection could cause overexpression of HMGB1.Western Blot and RT-PCR were used to detect the expression of NF-KB,SOD2 and LSD1.After overexpression of HMGB1,the expression of NF-KB and LSD1 increased(p < 0.05),while the expression of SOD2decreased(p < 0.05).This is consistent with the results of animal experiments in vivo.It further demonstrates that HMGB1 induces high expression of NF-KB and LSD1 and inhibits SOD2 expression at the cellular level.From the result of Chip-seq,HMGB1 binding sites were found to be enriched in promoter region and near TSS,which confirmed that HMGB1 might play a role as a transcription factor.In addition,the results of Chip-Seq confirmed that HMGB1 could directly affect the expression of IKB-?,thereby affecting the expression of NF-kB.PartIII: The effects of HMGB1 on retinal neovascularization and retinal cell proliferation and apoptosis.By ADP enzyme staining,we found that retinal neovascularization increased in diabetic rats compared with normal rats.HMGB1 can increase retinal neovascularization in normal rats,while siRNA can decrease retinal neovascularization in diabetic rats.Cell proliferation was detected by CCK-8 method in vitro,and apoptosis of HREC was detected by flow cytometry.HMGB1-shRNA was transfected into HREC cells to overexpress HMGB1.It was found that HMGB1 could reduce cell proliferation and increase cell apoptosis(p < 0.05).Conclusions:The expression of NF-kB and LSD1 in retinal tissue of rats and HREC showed a positive correlation with HMGB1,while the expression of SOD2 showed a negative correlation with HMGB1.HMGB1 may play a role in the development of DR through the interaction of NF-KB and SOD2.HMGB1 promotes retinal neovascularization,inhibits retinal vascular endothelial cell proliferation and promotes retinal vascular endothelial cell apoptosis.The inhibition of HMGB1 may provide a new idea for the treatment of DR.