Protective Effects Of Acarbose Against Vascular Endothelial Dysfunction Through Inhibiting Nox4/nlrp3 Inflammasome Pathway In Diabetic Rats

BackgroundCardiovascular disease（CVD）is the main cause of death and disability in patients with type 2diabetes mellitus（T2DM）.Given the prevalence of CVD in this population,a complete understanding of the cardiovascular safety and efficacy of glucose-lowering drugs is needed^[15].Acarbose is anα-glucosidase inhibitor that suppresses postprandial hyperglycemia by delaying the absorption of carbohydrates from the small intestine.Study to Prevent Non-Insulin-Dependent Diabetes Mellitus（STOP-NIDDM）trial first provided some support that Acarbose held particular promise in the prevention of CVD events,especially patients with impaired glucose tolerance^[16].However,the Acarbose Cardiovascular Evaluation（ACE）large-scale cardiovascular outcome trial recently concluded that Acarbose failed to decrease the risk of major adverse cardiovascular events,although it did delay the emergence of diabetes^[17].Thus,the cardiovascular protective effects of Acarbose has still remained controversial.Here,we investigated the cardiovascular protection of Acarbose in the light of its regulation on vascular endothelial dysfunction caused by T2DM.It was reported that Acarbose can pleiotropically inhibit rabbit atherosclerosis by reducing vascular smooth muscle cell proliferation/migration which was associated with the decrease of IL-6and TNF-α^[18].Recent study found that Acarbose effectively inhibited the proliferation and migration of Ras^G12V A7r5 cells by blocking small G proteins and PI3K/Akt signaling^[19].In addition,Acarbose accelerated wound healing and stimulated angiogenesis in T2DM,accompanied by directly improved endothelial progenitor cell functions in vitro^[20].These studies above suggested that Acarbose exerted beneficial effects on vascular cells beyond glycemic control.Endothelial dysfunction was considered as early critical event in atherogenesis involved in the development and progression of vascular complications in patients with T2DM^[21].As demonstrated by clinical trials,Acarbose improved postprandial endothelial dysfunction in newly diagnosed T2DM patients^[22].However,there was not information about the link between Acarbose and vascular endothelial cells,and the direct effects of Acarbose on vascular endothelial function impaired by hyperglycemia in vitro.Nucleotide-binding domain-like receptor 3（NLRP3）inflammasome as an important sensor is involved in the vascular endothelial pathological process,which assembly activates Caspase-1leading to the processing of bio-active IL-1βsecretion in diabetes^[23,24].So far,it is well documented that inflammasome exerts its effects through the production of IL-1βthat instigates inflammatory cells（monocytes and T lymphocytes）leading to local tissue sterile inflammation.These canonical cytokine and inflammatory cell-mediated inflammatory responses can cause or exacerbate the vascular inflammation contributing to the development of cardiovascular diseases such as atherosclerosis.In this respective,we recently reported that the inactivation of NLRP3 inflammasome was associated with the recovery of diabetes-induced endothelial junction disruption to induce vascular permeability^[25],which represented one of the initial pathologic processes leading to endothelial dysfunction.It was also supported by some studies that IL-1βreduced the adhesion strength of tight junctions and adhesion junctions between vascular endothelial cells which leading to increased vascular permeability^[26].Acarbose was demonstrated to suppress inflammatory cytokine production in patients with T2DM.For example,Acarbose suppressed the levels of IP-10,MCP-1,MDC and TNF-αand downregulated phosphorylation of NF-κB-p65 in LPS-stimulated THP-1cells^[27].Acarbose reduced the peaks of the various inflammatory Markers including IL-6 and Hs-CRP in diabetic patients^[28].Additional study found that Acarbose add-on insulin therapy was associated with greater improvements in inflammation and oxidative stress in patients with T2DM compared with insulin alone therapy^[29].Therefore,it was hypothesized that the inhibition of NLRP3inflammasome and consequent improvement of vascular barrier dysfunction were important determinants of vascular endothelial protection by Acarbose in diabetic rats.Given the postprandial surge of plasma glucose could lead to severe endothelial dysfunction in diabetic patients^[30],30m M glucose was currently used to impair vascular endothelial barrier dysfunction in RAECs.In vivo study,vascular endothelial permeability was induced in the T2DM rats that were fed with high fat diet（HFD）combined with low dose streptozotocin（STZ）administration.Next,we further detected the upstream mechanism of NLRP3 inflammasome activation in RAECs.In the endothelial cells,nicotinamide adenine dinucleotide phosphate（NADPH）oxidase,rather than cytochrome p450,xanthine oxidase,etc.,has been implicated as the major source of superoxide(O^2.-)production in high glucose stress^[31],and NADPH oxidase-mediated redox signaling triggered the dissociation of thioredoxin-interacting protein（TXNIP）from thioredoxin,which allowed TXNIP bind to the NLRP3 protein leading to formation and activation NLRP3inflammasome^[32].It was reported that Acarbose was helpful to prevent the increase in NADPH oxidase in aorta,heart,and kidney and vascular dysfunction in obese Zucker rats^[33].Thus,NADPH oxidase,especially its subtype Nox4-dependent O^2.-was expected to mediate the inhibition NLRP3inflammasome responding to Acarbose in RAECs.AimsThe cardiovascular efficacy of glucose-lowering drugs is needed due to the cardiovascular complication in type 2 diabetes mellitus（T2DM）.Acarbose is an alpha-glucosidase inhibitor that suppresses postprandial hyperglycemia,however,the cardiovascular protection of Acarbose remains controversial.NLRP3 inflammasome activation mediated tight junction disruption,a hallmark event of endothelial barrier dysfunction leading to endothelial hyperpermeability in diabetes.Given the anti-inflammatory property of Acarbose,it was investigated that Acarbose protected against vascular endothelial barrier dysfunction through inhibiting NLRP3 inflammasome in vascular endothelial cells in T2DM.Methods1.RAECs were cultured in vitro with high glucose（HG,30mm）for 24,48 and 72 hours.Meanwhile,the normal cultured RAECs were treated with different concentrations（1,3 and 9μM）of Acarbose,MTT was used to detect the effects of high glucose and Acarbose on cell proliferation and survival rate;ELISA was used to select the optimal time and concentration of HG and Acarbose treated cells;Western blot,ELISA and confocal fluorescence microscopy were used to observe the effects of NLRP3 inflammasome activation.2.RAECs were treated with HG,Acarbose,NADPH oxidase inhibitors apocynin（Apo 10μM）and Nox4 si RNA,and the production of superoxide anion in cells was detected by fluorescence probe DHE fluorescence spectrometry;the protein expression levels of Nox1,Nox2 and Nox4 were detected by Western blot;RT-PCR was used to detect the level of Nox4 m RNA after transfection of Nox4 si RNA.3.RAECs were treated with HG and Nox4 si RNA.The protein expression of NLRP3 and P20 was analyzed by Western blot.The activity of Caspase-1 and the production of IL-1βwere detected by ELISA kit.The co-localization of NLRP3 and ASC was observed by confocal fluorescence microscopy.4.RAECs were treated with HG,Acarbose,NLRP3 si RNA,NLRP3 inflammasome specific inhibitor MCC950 10μM.Western blot was used to analyze the expression of tight junction protein ZO-1 and adhesion junction protein VE-Cadherin.Flowcytometry detected the expression level of ZO-1 protein in cell membrane;Transwell chamber was used to detect the fluorescence intensity of FITC dextran leakage.5.After treatment with Acarbose and MCC950,the blood glucose of FBG,HOMA-IR and OGTT in the serum of T2DM rats was measured;the content of MDA and IL-1βin the serum of T2DM rats was measured by ELISA.The leakage of Evans blue dye from plasma to interstitium was quantitatively observed by intravenous injection of Evans blue dye.Isometric vascular tone test was used to detect the intact thoracic aorta vasodilation of ach(1×10^-9-10^-5M)and SNP(1×10^-9-10^-5M).ZO-1 and VE-Cadherin were detected by immunohistochemistry The fluorescence co-localization of NOX4/VWF,NLRP3/ASC,NLRP3/VWF,Caspase-1/VWF was observed by confocal laser microscopy.Results1.MTT assays showed that cell viability decreased in RAECs incubated with HG（30m M）for 72hours compared to control.It was found that 9μM Acarbose significantly impaired cell viability（P<0.05）.As shown in ELISA result,HG led to a significant increase in IL-1βrelease after the incubation for 24,48 and 72 hours.No statistical significance was found between 24 hours and 48 hours group（P>0.05）,and 3μM Acarbose effectively reduced HG-induced IL-1βrelease（P<0.05）.Therefore,3μM Acarbose was used to treat RAECs incubated with 30m M HG for 24 hours for the following analysis.Western blot analysis as well as the secretion of Caspase-1 and IL-1βin culture supernatants by ELISA were dramatically induced by HG.These changes were significantly reversed by Acarbose treatment（P<0.05）.The co-localization of NLRP3 and ASC were compared with HG,barely yellow dots were detected in RAECs pretreated with Acarbose（P<0.05）.2.RAECs incubated with HG showed a strong red fluorescence produced from DHE oxidization by O^2.-.Acarbose intervention apparently blocked the increase of fluorescent intensity in respond to HG（P<0.05）,which was similar with Apo.Nox4 m RNA and protein dramatically increased in RAECs incubated with HG,which was inhibited by Acarbose（P<0.05）.there was no significant difference in Nox1 or Nox2 protein expression after the treatment with Acarbose（P<0.05）.Nox4si RNA was transfected into RAECs,and the transfection efficacy was significantly compared with control group（P<0.05）.The apparent inhibition of O^2.-production due to Nox4 si RNA transfection compared with HG group（P<0.05）.3.After Nox4 si RNA transfection,the expression of NLRP3 and p20/pro-Caspase-1 in cell lysate,Caspase-1 activity and IL-1βproduction in culture supernatants,as well as the co-localization of NLRP3 and ASC were significantly decreased compared with HG group（P<0.05）.4.HG treatment markedly degraded the protein expression of ZO-1 and VE-Cadherin,which were reversed by Acarbose or MCC950 or NLRP3 si RNA（P<0.05）.Such amelioration of Acarbose in ZO-1 was further confirmed by flowcytometry assay（P<0.05）.FITC-dextran result was observed a significant increase in the relative permeability of endothelial cell monolayers by HG,which was effectively prevented in cells pretreated with Acarbose or MCC950 or NLRP3 si RNA（P<0.05）.5.In vivo,compared with T2DM rats,after 5 weeks of Acarbose intervention,FBG,HOMA-IR and OGTT blood glucose decreased significantly（P<0.05）.ELISA showed that Acarbose and MCC950treatment could significantly reduce the increase of MDA and IL-1βin T2DM rats（P<0.05）.Quantitative observation of Evans blue dye leakage showed that Acarbose and MCC950 could significantly improve the increase of fluorescence absorption in T2DM rats（P<0.05）.Isometric vascular tone test showed that Ach significantly impaired endothelium-dependent vasodilation in T2DM rats,which was significantly improved after treatment with Acarbose and MCC950（P<0.05）.There was no significant difference in vasodilation induced by SNP between these groups（P>0.05）.In vitro,GKT137831 treatment could effectively improve the aortic diastolic function in HG（P<0.05）.There was no significant difference in vasodilation induced by SNP between these groups（P>0.05）.The co-localization efficiency of NOX4/v WF,NLRP3/ASC,NLRP3/v WF and Caspase-1/v WF in aortic endothelium of T2DM rats was significantly increased,and Acarbose could significantly inhibit these effects（P<0.05）.Conclusions1.Acarbose inhibited HG-induced NLRP3 inflammasome activation in RAECs.2.Acarbose inhibited the activation of NLRP3 inflammasome by reducing Nox4 dependent O₂.^-production.3.Acarbose reversed HG-induced endothelial cell barrier dysfunction through inhibiting Nox4/NLRP3 inflammasome pathway.4.Acarbose improves vascular permeability and diastolic function in T2DM rats by inhibiting NLRP3inflammasome.