Protective Effects Of Sodium Butyrate And Its Underpining Mechanism In Oxidative Damage Of Diabetic Arterial Endothelium

BackgroundDiabetes is a serious chronic disease that threat to human health.The incidence of diabetes has increased year by year.It has become more serious public health problems.Diabetic macrovascular complications are the main causes of death and disability Diabetic arterial endothelial dysfunction is the main causes of macrovascular complications.The basic pathology of diabetic vascular disease is the formation of atherosclerotic plaques.The basic pathology of diabetic vascular lesions is the formation of atherosclerotic plaques,which is mainly caused by the endothelial damage caused by long-term exposure to hyperglycemia,which further leads to the occurrence of atherosclerosis.Oxidative stress plays a key role in the development of arterial endothelial dysfunction caused by diabetes.Previous studies have shown that oxidative stress can reduce the effective bioavailability of NO,and then lead to vascular endothelial diastolic dysfunction.The oxidative stress of arterial endothelium caused by diabetes can promote and amplify each other with the inflammatory reaction,forming a positive feedback loopand promoting the infiltration of leukocyte and monocytes into the blood vessel wall,and inducing the occurrence and development of atherosclerosis.Therefore,inhibiting oxidative stress is an effective strategy to reduce diabetic arterial endothelial dysfunction and block the progression of diabetic arterial complications.NRF2 is the main regulator of the antioxidant capacity of cells.NRF2 initiates the transcription of various antioxidant genes and produce antioxidants,which can scavenge the free radicals induced by diabetes.NRF2 is an important protective factor for cardiovascular complications of diabetes mellitus.Our previous study shown that Nrf2 gene knockout(KO)diabetic mice had more serious heart and kidney damage compared with wild-type(WT)diabetic mice.Activation of NRF2 reduced diabetic cardiomyopathy and diabetic nephropathy in WT mice.Therefore,activation of NRF2 may be an effective strategy to reduce arterial endothelial injury in diabetes mellitus.The expression and function of NRF2 are negatively regulated by kelch-like ECH related protein 1(KEAP1).KEAP1 binds to NRF2 and anchors NRF2 in the cytoplasm and prevents it to entering the nucleus.So Nrf2 cannot play its transcriptional factor role.On the other hand,KEAP1 causes NRF2 to be ubiquitinated by proteasome.Therefore,in recent years,specific inhibition of KEAP1 and activation of NRF2 at the post-translational processing level have become research hotspots.In addition,up-regulation of Nrf2 gene transcription is another effective strategy to activate Nrf2.Histone acetylation/deacetylation modification is an important regulatory mechanism of gene expression in cells.In general,histone acetylases(HAT,CBP/P300)acetylate histone proteins,which leads to the opening of chromatin spatial structures.This process facilitates the combination of transcription factors and coactivators in the gene promoter region to activate gene transcription.In contrast,histone deacetylase(HDAC)deacetylates histones,resulting in a constricted chromatin space.This process prevents transcription factors and coactivators from binding in the promoter region and inhibits gene transcription.Sodium butyrate(NaB)is a short-chain fatty acid,which is a metabolite of undigested sugars(oligosaccharides,dietary fiber,etc.)fermented by intestinal probiotics(clostridium caseinate,etc).NaB is a potent inhibitor of HDAC.We previously found that NaB can inhibit the activity of HDAC in diabetic mice kidney and alleviate diabetic nephropathy by activating the expression and function of NRF2.Our further study found that NaB significantly increased the level of Nrf2 mRNA in the kidney of diabetic mice.Therefore,NaB may activate the expression and function of Nrf2 gene at the transcriptional level by inhibiting HDAC.The effect of NaB on arterial endothelial injury in diabetes mellitus has not been reported and the mechanism remains unclear.In our study,C57BL/6 WT(Nrf2+/+),Nrf2 KO(Nrf2-/-)mice,and arterial endothelial cells were used as research subjects.The aim is to observe the effect of NaB on diabetic arterial endothelial injury and explore the mechanism of NaB regulating expression of Nrf2 gene.To explore the new mechanism of arterial endothelial injury in diabetes mellitus,and to provide key and effective intervention targets for the prevention and treatment of diabetic macrovascular complications.ObjectsTo explore the effect and mechanism of NaB on oxidative damage of arterial endothelium caused by diabetes.Methods1.The diabetic model of mice was established by intraperitoneal injection of streptozotocin(STZ)for several times at a small dose.2.Blood glucose levels were measured at 0,28,56,84,112,and 140 days.The area of aortic media hyperplasia was determined by HE staining.The systolic and diastolic responses of aorta in mice were measured by in vitro microvascular tension measurement system.3.mRNA expression of inflammatory related factors,Nrf2 and related antioxidant genes in aortic tissue and cells were detected by qPCR.4.The expression levels of oxidative stress,inflammatory reaction-related proteins and Nrf2 and related antioxidant gene proteins in aortic tissue were detected by Immunohistochemical staining(IHC).5.Nrf2 and other related proteins in cells were detected by Western Blot.6.ROS and MDA levels were detected using ROS/MDA detection kits.7.The nucleus was separated by nuclear separation kit,and t-NRF2,c-NRF2 and n-NRF2 were detected by Western Blot.8.The effect of NaB on HDAC activity was detected by HDAC kit.9.The effect of NaB on promoter of Nrf2 was detected by Chromatin immunoprecipitation(ChIP)kit.Results1.NaB had no significant effect on blood glucose level in mice.2.In vivo,NaB alleviated media hyperplasia of aorta,contraction and diastolic dysfunction of aorta in WT diabetic mice,while NaB had no such effect in Nrf2 KO diabetic mice.3.NaB reduced the expression of iNos and Vcam-1 mRNA in the aortic tissues of wild-type diabetic mice,and inhibited the oxidative stress(3-NT,4-HNE,ROS,MDA)and inflammatory response(VCAM-1,ICAM-1)induced by hyperglycemia.4.In vivo and cell experiments,NaB increased the expression of Nrf2 and its downstream antioxidant genes Nqo1 and Ho-1.However,the effects of NaB disappeared in Nrf2 KO diabetic mice and arterial endothelial cells treated with high glucose + Nrf2-siRNA.5.NaB increased the levels of t-NRF2,n-NRF2 and c-NRF2 in endothelial cells treated with high glucose.However,unlike SFN,NaB failed to increase the proportion of NRF2 entering the nucleus,but significantly increased the level of Nrf2 mRNA.6.NaB could significantly increase the binding of AHR,P300 and H3K9 ac with gene promoter regions of Nrf2 and reverse the inhibitory effect of high glucose on the above phenomenon.7.C646,the specific inhibitor of P300,reversed the regulatory effect of NaB on Nrf2 gene expression in endothelial cells treated with high glucose.Conclusion1.NaB relieved oxidative stress,inflammation and dysfunction of arterial endothelium caused by diabetes through activating NRF2 signaling pathway.2.NaB activated the expression and function of Nrf2 gene at the transcription level,but did not increase NRF2 nuclear translocation.3.NaB inhibited the activity of HDAC and increased the binding of transcription factors and coactivators in the promoter region of Nrf2 gene.4.P300 was the key factor that NaB regulated the transcription process of Nrf2 gene.