Sirt1 Improves High Glucose-induced Endothelial Injury By Inhibiting Mff-based Mitochondrial Fission And F-actin Depolymerization

Obgetive:Sirtuin 1(Sirtl)has been found to be involved in diabetic vasculopathy and high glucose-mediated endothelial injury,the underlying mechanisms remain to be fully elucidated.The aim of our study was to explore the role of Sirtl in HG-induced endothelial injury and its potential mechanism.Methods:1.Animal Experiment:(1)Grouping and modeling:ApoE-/-male mice were randomly divided into four groups(Ctrl group,Diabetic group,Ctrl+SRT1720 group,Diabetic+SRT 1720 group).ApoE-/-mice were fed with a high-fat diet from 4 weeks of age and the mice in the Ctrl group were fed with normal diet to the end of the study.8-week-old apoE-/-mice were intraperitoneally injected with streptozotocin(STZ,50 mg/kg)for five consecutive days.Fasting blood glucose levels>16.7 mmol/L was considered successful model establishment.The diabetic mice(12 weeks old)were treated with SRT1720(15 mg/kg/d,defined as the Diabetic+SRT1720 group)for 12 weeks.The mice were anesthetized by intraperitoneal injection of pentobarbital sodium(60 mg/kg body weight)and were sacrificed by euthanasia method.(2)The expression of Sirtl in the arteries of each group was detected by Western Blot,RT-qPCR and immunohistochemical staining.(3)Measurement of biochemical parameters:The levels of fasting blood glucose,glycated hemoglobin(HbA1c),insulin,glutathione(GSH),superoxide dismutase(SOD)and malondialdehyde(MDA)were determined.(4)The measurement of endothelial relaxation functioning was conducted.(5)The expressions of eNOS and p-eNOS in the arteries were detected by Western Blot.The mRNAs of ICAM-1,VCAM-1,CRP,TNF-a and IL-6 in the arteries were measured by RT-qPCR.(6)The apoptosis of mouse arterial endothelial cells was measured by Annexin V/PI-FITC method.2.Cell Experiment:(1)Human umbilical vein endothelial cells(HUVECs)were treated with 30mmol/1 glucose or 30mmol/1 mannitol for 24 hours and 48 hours respectively.The expression of Sirtl in HUVECs was detected by Western Blot.(2)HUVECs were transfected with the Sirtl adenovirus plasmids(ad-Sirtl)and control adenovirus plasmids(ad-Ctrl).Cellular viability was quantitatively measured by MTT assay.TUNEL staining was used to observe the apoptosis.The expression of cleaved-Caspase3 and PARP was measured by Western Blot.(3)The cellular ROS was measured via dihydroethidium(DHE)staining.The expression levels of Caspase9,Bax,Bad,Bcl-2 and x-IAP were detected by Western Blot.The mitochondrial membrane potential(△Ψm)was measured using the JC-1 kit.(4)To examine whether mitochondrial fission is responsible for endothelial apoptosis,FCCP,a mitochondrial fission activator,was used to reactivate mitochondrial fission in ad-Sirtl cells.The mitochondrial morphology was observed by immunofluorescence staining.The apoptosis of HUVECs was detected by TUNEL staining.(5)Western Blot was used to detect the expression of Mff,Fis1,Mid49,Mid51 and Drp1.(6)To suppress and activate the JNK pathway,SP600125(SP)and anisomycin(Ani)were used respectively.The expression levels of t-JNK,p-JNK and Mff were detected by Western Blot.The mitochondrial morphology was observed by immunofluorescence staining.(7)To inhibit the F-action degradation,Jasplakinolide(Jas)was used.Transwell assay was used to detect the cell migration.Western blot was used to detect the expression of F-actin and G-actin.(8)To examine the mechanism by which Sirtl is downregulated through diabetic vasculopathy,miR-195 was examined by RT-qPCR.HUVECs were transfected with agmir-195 and antagomir.The mRNA of Sirtl was measured by RT-qPCR.The luciferase reporter assay was used to analyze whether miR-195 can interact with target protein directly.The mitochondrial morphology was observed by immunofluorescence staining.The cell scratching test was used to detect the cell migration.Results:1.Compared to Ctrl group,the expression of Sirtl in Diabetic group decreased(P<0.05),body weight,fasting blood glucose,fasting insulin,glycosylated hemoglobin,and systolic blood pressure increased(P<0.05).Compared to Diabetic group,the expression of Sirtl in Diabetic+SRT 1720 group increased(P<0.05),and the above metabolic indicators decreased(P<0.05).SRT1720 reversed the downregulation of Sirtl in diabetic mice,as indicated by immunohistochemical analyses.2.Compared to Ctrl group,diabetes inhibited the endothelial-dependent vasodilation in response to Ach(P<0.05).Compared to Diabetic group,the endothelial-dependent vasodilation of the Diabetic+SRT1720 group improved(P<0.05).There was no significant difference in the endothelial-independent vasodilation levels among the four groups.Compared to Ctrl group,diabetes decreased the protein levels of p-eNOS(P<0.05)and increased the mRNA levels of ICAM1,VCAM1,TNF-a,IL-6 and CRP(P<0.05).In the Diabetic+SRT1720 group,the expression of p-eNOS increased(P<0.05)and the mRNA levels of ICAM1,VCNAM1,TNF-a,IL-6 and CRP decreased(P<0.05)compared to Diabetic group.In addition,diabetic mice generated more MDA(P<0.05)and consumed more antioxidant factors,including GSH/SOD(P<0.05),and this effect was reversed by SRT1720(P<0.05).Relative to the Ctrl group,the Diabetic group exhibited clearly elevated ratios of apoptotic cells(P<0.05),and this effect was reduced by SRT1720(P<0.05).3.The expression of Sirtl was downregulated in high glucose(HG)group(P<0.05),with the lowest expression levels of Sirtl following 48h of HG treatment.The overexpression of Sirtl had no influence on cellular viability.However,compared to HG+ad-Ctrl group,cellular viability improved(P<0.05)and the TUNNEL staining positive cells decreased(P<0.05)as indicated by the expression of cleaved-Caspase 3 and PRAP decreased(P<0.05)in HG+ad-Sirtl group.4.Compared to the Ctrl roup,HG increased the cellular ROS content levels,which were reduced in HG+ad-Sirtl group(P<0.05).HG also induced higher expression levels of Caspase9,Bax and Bad but lower expression levels of Bcl-2 and x-IAP,which were reversed in HG+ad-Sirtl group(P<0.05).HG also impaired △Ψm,with evidence of decreased levels of red fluorescence and increased levels of green fluorescence(P<0.05).However,the overexpression of Sirtl reversed the stability of △Ψm(P<0.05).5.Compared to the ad-Ctrl group,HG led to a markedly larger volume of fragmented mitochondria and apoptotic cells in the HG+ad-Ctrl group(P<0.05).The HG+ad-Sirtl group exhibited lower levels of fragmented mitochondria and apoptotic cells compared to the HG+ad-Ctrl group(P<0.05).To examine whether mitochondria are responsible for endothelial apoptosis,FCCP was used to reactivate mitochondrial fission in HG+ad-Sirtl cells.HG+ad-Sirtl+FCCP group showed lower mitochondrial length(P<0.05)and increased apoptotic cells(P<0.05)than HG+ad-Sirtl group.6.HG enhanced the expression of Mff and Drp1 in HG+ad-Ctrl group(P<0.05)and the expression of Mff and Drpl decreased in HG+ad-Sirtl goup(P<0.05).Relative to the control group,JNK was activated in HG+ad-ctrl group,as indicated by an increase observed in the expression of p-JNK(P<0.05).This tendency was reversed in HG+ad-Sirt1 group(P<0.05).The JNK pathway was triggered in HG+ad-Ctrl group and HG+ad-Sirt1+Ani group(P<0.05),but was inhibited in HG+ad-Sirtl group and HG+ad-ctrl+SP group(P<0.05).The expression of Mff and mitochondrial fission were enhanced in HG+ad-Ctrl group,but decreased to normal levels in HG+ad-Sirtl group and HG+ad-Ctrl+SP group(P<0.05).The HG+ad-Sirtl+Ani group eliminated the inhibition of Mff activation and the protective effects on mitochondrial fission induced by the overexpression of Sirtl(P<0.05).7.HG treatment reduced the migration of HUVECs,whereas the overexpression of Sirtl prevented this change from occurring in HG+ad-Sirtl group(P<0.05).HG treatment clearly reduced the expression of F-actin and enhanced the expression of G-actin,which was reversed in HG+ad-Sirtl group and HG+ad-Ctrl+Jasp group(P<0.05).Jasplakinolide also promoted cellular migration in HG+ad-Ctrl+Jasp group(P<0.05).8.The expression of miR-195 was significantly enhanced in HG group(P<0.05).Agmir-195 reduced the expression of Sirtl,echoing the results derived following HG treatment(P<0.05).However,inhibitors of miR-195 reversed the downregulated level of Sirtl under treatment with HG(P<0.05).Compared with that of the MUT type,luciferase activity was downregulated in cells co-transfected with miR-195 mimics and the WT Sirtl-3’UTR(P<0.05),suggesting that Sirtl is a target gene of miR-195.Agmir-195 promoted mitochondrial fission and inhibited cellular migration(P<0.05).However,the inhibition of miR-195 inhibited the HG-induced mitochondrial fission and inhibited migration(P<0.05).Conclusion:1.In HUVECs,high glucose triggers the downregulation of Sirtl by activating miR-195.2.Reduced expression of Sirtl contributes to glucose metabolic abnormalities,aortic endothelial dysfunction and EC apoptosis.3.Downregulated Sirtl triggers mitochondrial fission by evoking the JNK/Mff pathway,promoting endothelial cell apoptosis.4.Sirtl deficiency limits EC migration through affecting F-actin dyshomeostasis.