Polydatin Protects Against LPS-induced Endothelial Barrier Dysfunction Via SIRT3 Activation

Sepsis is a life-threatening organ dysfunction caused by the host's dysregulated reaction to infection.Despite the rapid development of diagnostic levels and advanced life support technologies,the morbidity and mortality of sepsis remain high.There is currently no specific drug for the treatment of sepsis due to its complex pathogenesis.Therefore,it is important to clarify the pathogenesis of sepsis and find potential therapeutic drugs.Mounting evidence indicates that vascular endothelial barrier dysfunction is the pathophysiological basis for multiple organ dysfunction in sepsis.Recently.it has been confirmed that endothelial barrier protection can significantly reduce the mortality rate of sepsis in several septic mouse models.This results suggest that intervention on the endothelial barrier function may be a promsing approach for the treatment of sepsis.Lipopolysaccharides(LPS)is the main component of the cell wall of Gram-negative bacilli and is an important pathogenic factor in the pothogensis of sepsis.Toll-like receptor 4(TLR4)is a classical receptor for LPS.Later studies found that the receptor for advanced glycation end products(RAGE)can also bind to LPS and promote the development of sepsis.Sirtuins are nicotinamide adenine dinucleotide(NAD+)-dependent type III histone deacetylases.The seven mammalian Sirtuins(SIRT1-SIRT7)show different subcellular localizations.SIRT3 is mainly localized in mitochondria and regulates various mitochondrial functions such as fatty acid oxidation,glucose metabolism,antioxidant response and mitochondrial dynamics.Studies show that SIRT3 has a protective effect on vascular barrier function.In advanced glycation end products(AGEs)-stimulated endothelial progenitor cells,RAGE mediates the downregulation of SIRT3 protein expression.Therefore,we hypothesize that RAGE may also be involved in LPS-regulated alterations of SIRT3 signaling.Polydatin(PD)is an effective component extracted from a traditional Chinese medicine,Polygonum cuspidatum.PD exhibits anti-inflammatory,anti-oxidation,anti-thrombosis and myocardial protective effects.In addition,our previous studies have confirmed that PD can protect against shock,microcirculation dysfuction and multi-organ dysfunction.In animal models of sepsis and hemorrhagic shock,we found that PD attenuates intestinal and renal injury by upregulating the deacetylase activity and protein expression of SIRT3.Therefore,PD might play a protective role in endothelial barrier function in sepsis by activating SIRT3.Manganese superoxide dismutase(SOD2)is a key antioxidant enzyme in mitochondria.SOD2 acetylation contributes to a decrease in its activity and increase in intracellular ROS level.ROS generation promotes the dissociation of VE-cadherin and ?-catenin,leading to endothelial hyperpermeability.SIRT3 deacetylates the lysine sites of SOD2,enhances SOD2 activity and promotes ROS clearance.Cyclophilin D(CypD)is an important component protein of mitochondrial permeability transition pores(mPTP)and regulates the opening of mPTP.Inhibition of CypD significantly attenuates the decrease in mitochondrial membrane potential and endothelial hyperpermeability caused by hemorrhagic shock.We previously demonstrated that SIRT3-mediated CypD deacetylation inhibits mPTP opening and mitochondrial damage during hemorrhagic shock.SIRT3-mediated deacetylation of CypD may also exert protective effects on endothelial barrier function.In the present study,we hypothesize that PD can restore LPS-induced mitochondrial dysfunction and endothelial barrier dysruption by enhancing SIRT3-mediated deacetylation of SOD2 and CypD.Objective:This study aims to investigate whether polydatin protects vascular endothelial barrier function by activating SIRT3 and the underlying mechanisms.The findings will provide new ideas for the pathogenesis of sepsis and provide potential therapeutic drugs and targets for the treatment of sepsis.Methods:At the cellular level,primary human umbilical vein endothelial cells(pHUVECs)treated with LPS were used to establish a cell model of sepsis.SIRT3 activity was detected by SIRT3 activity assay kit and SIRT3 protein expression was detected by immunoblotting.SIRT3 specific inhibitor 3-TYP was used to investigate whether PD-mediated endothelial barrier protection is dependent on the SIRT3 activation.The distribution of endothelial cytoskeleton was observed by F-actin staining.VE-cadherin and ?-catenin complex were detected by immunoprecipitation.TER and FITC-dextran leakage were detected to evaluate endothelial monolayer permeability.SIRT3 overexpressing adenovirus and SIRT3 siRNA were used to further confirm the role of SIRT3 in endothelial barrier protection.SOD2 acetylation level was detected by immunoprecipitation.DCFH-DA was used to detect cellular ROS levels.MDA assay kit was used to examine MDA levels.SOD2 siRNA and mito-TEMPO were used to verify the role of SIRT3-SOD2-ROS pathway in PD-mediated barrier protection.Mitochondrial membrane potential and mPTP opening were detected by JC-1 and calcein-AM,respectively.CypD siRNA was used to verify the role of the SIRT3-CypD axis in PD-mediated barrier protection.RAGE antibody was used to demonstrate the role of RAGE in LPS-mediated SIRT3 signaling alterations.At the animal level,an animal model of septic mice was established by LPS injection,and Evans blue dye was used to detect vascular leakage in multiple organs of septic mice.Results:1.Effect of LPS on SIRT3 activity and protein expression in endothelial cells1.1 LPS induces a decrease in SIRT3 activity in endothelial cells in a time-dependent mannerThe primary endothelial cells were stimulated with 1 ?g/ml LPS for 0,3,6,12,and 24 h,respectively,and the activity of SIRT3 was measured.Results showed that LPS induced a significant decrease in SIRT3 activity at 3 h,and reached the lowest value at 12 h,and this effect was still significant at 24 h(F=17.023,P=0.000).1.2 LPS induces a decrease in SIRT3 protein expression in endothelial cells in a time-dependent mannerThe primary endothelial cells were stimulated with 1 ?g/ml LPS for 0,3,6,12,and 24 h,respectively,and the expression levels of SIRT3 protein were detected.The results showed that SIRT3 protein expression was decreased from 12 h to 24 h in response to LPS(F=3.952,P=0.035).2.PD protects against LPS-induced endothelial barrier dysfunction dependent on SIRT3 activation2.1 Effects of PD and 3-TYP on SIRT3 activityPrimary endothelial cells were treated with PD(50 ?M)or 3-TYP(50 ?M)for 2 h,and the activity of SIRT3 was detected.The results showed that PD significantly enhanced SIRT3 activity,while 3-TYP significantly inhibited SIRT3 activity(F=623.534,P=0.000).2.2 Effect of 3-TYP on SIRT1 activityPrimary endothelial cells were treated with 3-TYP(50 ?M)for 2 h to detect SIRT1 activity.The results showed that 3-TYP had no effect on SIRT1 activity in primary endothelial cells(F=4.506,P=0.101).The results confirmed the specificity of 3-TYP on SIRT3 inhibition.2.3 PD alleviates LPS-induced decrease in SIRT3 protein expression in endothelial cellsThe experiment was divided into 5 groups:Control group;LPS group was stimulated with 1 ?g/ml LPS for 12 h;PD group was stimulated with 50 ?M PD for 2 h;PD+LPS group was pretreated with 50 ?M PD for 2 h,then stimulated with 1?g/ml LPS for 12 h;PD+LPS+3-TYP group was pretreated with PD(50 ?M)and 3-TYP(50 ?M)for 2 h,and then stimulated with 1 ?g/ml LPS for 12 h.The expression levels of SIRT3 protein were then detected.The results showed that PD effectively inhibited LPS-induced decrease in SIRT3 protein expression,but this effect of PD was inhibited by 3-TYP(F=8.891,P=0.002).These results suggest that PD may exert an endothelial protective effect by upregulating SIRT3 protein expression.2.4 PD reverses LPS-induced endothelial cell F-actin stress fiber formation and dissociation of cadherin-catenin complex by activating SIRT3It was found that PD pretreatment significantly inhibited the formation of stress fibers caused by LPS,while 3-TYP abolished the protective effect of PD.Immunoprecipitation experiments showed that PD inhibited LPS-induced dissociation of VE-cadherin and ?-catenin,while 3-TYP abolished the protective effect of PD.These results indicate that the protective effect of PD on endothelial cytockeleton and AJs is dependent on the activation of SIRT32.5 PD reverses LPS-induced endothelial hyperpermeability by activating SIRT3The results showed that PD significantly attenuated LPS-induced decrease in endothelial monlayer TER(F=78.571,P=0.000)and increase in FITC-dextran leakage(F=16.549,P=0.000).Inhibition of SIRT3 significantly reversed the protective effect of PD,These results suggest that the protective effect of PD on LPS-induced endothelial permeability is dependent on the activation of SIRT3.2.6 PD alleviates vascular leakage in multiple organs of septic micMice in the LPS group were intraperitoneally injected with 15 mg/kg LPS,and the control group was injected with an equal volume of normal saline.In the PD+LPS group,30 mg/kg of PD was injected through the tail vein,and LPS was intraperitoneally injected 2 hours later.In the PD+LPS+3-TYP group,30 mg/kg of PD and 5 mg/kg of 3-TYP were injected through the tail vein.LPS was given intraperitoneally 2 h later.Results showed that LPS resulted in an increased vascular leakage in lung(F=5.233,P=0.010),kidney(F=4.636,P=0.016),liver(F=16.726,P=0.000),and small intestine(F=15.991,P=0.000).PD pretreatment significantly relieved vascular leakage in multiple organs,while inhibition of SIRT3 abrogated the protective effect of PD on vascular barrier.In vivo experiments showed that PD effectively improved the vascular barrier function of multiple organs in septic mice,and this protection depends on the activation of SIRT3.3.Protective effect of SIRT3 on LPS-induced endothelial barrier dysfunction3.1 SIRT3 overexpressing adenovirus upregulates SIRT3 protein expressionExperiments were performed using Flag and GFP-tagged SIRT3 overexpressing adenovirus.The results showed that green fluorescence was detected after transfection of primary endothelial cells with Ad-GFP and Ad-SIRT3.Ad-SIRT3 significantly upregulated the expression of SIRT3 protein,but there was no significant change in the Control group and the Ad-GFP group.The label protein Flag was only detectable in the Ad-SIRT3 group(F=10.516,P=0.011).The results demonstrate that SIRT3 overexpressing adenovirus can effectively upregulate the protein expression of SIRT3.3.2 Overexpression of SIRT3 inhibits LPS-induced endothelial F-actin stress fiber formation and cadherin-catenin complex dissociationThe results showed that LPS significantly caused the formation of stress fiber in endothelial cells,while overexpression of SIRT3 effectively attenuated the formation of stress fiber.Co-immunoprecipitation experiments also confirmed that overexpression of SIRT3 effectively blocked the dissociation of VE-cadherin and?-catenin complex induced by LPS3.3 Overexpression of SIRT3 attenuates LPS-induced endothelial barrier disruptionOverexpression of SIRT3 significantly attenuated LPS-induced decrease in TER value(F=7.020,P=0.012)and increase in FITC-dextran leakage(F=4.284,P=0.044).The results suggest that SIRT3 overexpression can effectively alleviate LPS-induced endothelial hyperpermeability.3.4 SIRT3 siRNA downregulates SIRT3 expression in endothelial cellsSIRT3 siRNA significantly downregulated the expression of SIRT3 protein in primary endothelial cells(t=6.020,P=0.004).3.5 SIRT3 deletion exacerbates LPS-induced stress fiber formation and dissociation of cadherin-catenin complexSIRT3 deletion significantly aggravated LPS-induced F-actin stress fiber formation and cadherin-catenin complex dissociation.The results further confirm the protective effect of SIRT3 on endothelial barrier function.3.6 SIRT3 deletion exacerbates LPS-induced endothelial hyperpermeabilitySIRT3 deletion aggravated LPS-induced decrease in TER value(F=15.043,P=0.001)and increase in FITC-labeled dextran leakage(F=33.627,P=0.000).The results further confirm the barrier protective effects of SIRT3.4.PD protects endothelial barrier function through SIRT3-SOD2-ROS pathway4.1 PD activates SIRT3 and influences the acetylation and activity level of SOD2LPS caused a significant increase in the acetylation of SOD2.PD pretreatment inhibited the effect of LPS,while 3-TYP relieved the protective effect of PD.Correspondingly,the SOD2 activity assay showed a trend opposite to the acetylation level(F=55.429,P=0.000).4.2 PD attenuates LPS-induced ROS increase via SIRT3 activationPD pretreatment significantly attenuated LPS-induced ROS production,whilr 3-TYP counteracted the effect of PD(F=14.699,P=0.000).The results indicate that PD reduces LPS-induced increase in ROS levels by activating SIRT3.4.3 PD attenuates LPS-induced increase in MDA level via SIRT3 activationPD significantly inhibited LPS-induced increase in MDA levels,while 3-TYP abolished the effect of PD(F=7.963,P=0.004).In summary,PD inhibits LPS-induced increase in ROS generation and MDA level by activating SIRT3,suggesting that PD is likely to exert barrier protection through SIRT3-SOD2-ROS pathway.4.4 SOD2 siRNA inhibits SOD2 protein expressionWestern blot results showed that SOD2 siRNA could effectively downregulate the expression of SOD2 in primary endothelial cells(t=9.353,P=0.001).4.5 SOD2 knockdown abolishes SIRT3 overexpression-mediated barrier protectionSOD2 siRNA significantly inhibited SIRT3 overexpression-mediated increase in TER value(F=7.467,P=0.010)and decrease in FITC-dextran leakage(F=6.620,P=0.015).The results suggest that SIRT3-mediated SOD2 deacetylation exerts endothelial barrier protection.4.6 Mitochondrial ROS detoxification improves LPS-induced endothelial hyperpermeabilityMito-TEMPO significantly improved LPS-induced decrease in TER(F=9.701,P=0.005)and increase in FITC-dextran leakage(F=6.026,P=0.019),while administration of SIRT3 inhibitor 3-TYP eliminated the barrier protective effect of mito-TEMPO.Taken together,PD protects against LPS-induced endothelial barrier dysfunction via SIRT3-SOD2-ROS pathway.5.PD improves LPS-induced endothelial hyperpermeability through SIRT3-CypD pathway5.1 PD inhibits LPS-induced increase in CypD acetylation by SIRT3 activationLPS induces an increase in CypD acetylation and inhibits the direct interaction between SIRT3 and CypD.PD improved the above effects caused by LPS,whereas the protective effect of PD is abolished by 3-TYP.The results indicate that PD enhances the direct interaction between SIRT3 and CypD,thereby promoting CypD deacetylation.5.2 PD inhibits LPS-induced decrease in mitochondrial membrane potential via SIRT3 activationPD pretreatment significantly inhibited LPS-induced decrease of mitochondrial membrane potential in endothelial cells,while inhibition of SIRT3 reversed the effect of PD.The results suggest that PD inhibits LPS-induced decrease in mitochondrial membrane potential by activating SIRT3.5.3 PD inhibits LPS-induced mPTP opening through SIRT3 activationPD pretreatment inhibited LPS-reduced intracellular calcein fluorescence,while 3-TYP significantly inhibited this effect of PD.The results suggest that PD can inhibit LPS-induced mPTP opening by activating SIRT3.5.4 CypD siRNA inhibits CypD protein expression in endothelial cellsCypD siRNA effectively downregulated the expression of CypD in primary endothelial cells(t=16.215,P=0.000).5.5 CypD knockdown relieves LPS-induced endothelial hyperpermeabilityCypD knockdown significantly attenuated LPS-induced decrease in TER(F=4.975,P=0.031)and increase in FITC-dextran leakage(F=8.684,P=0.007).The results suggest that SIRT3 activation enhances CypD deacetylation,thereby attenuating LPS-induced increase in endothelial permeability.6.RAGE is involved in LPS-mediated SIRT3 activity and protein changes6.1 RAGE is involved in LPS-mediated decline in SIRT3 activityLPS group was stimulated by LPS(1 ?g/mL)for 12 h,RAGE antibody group was treated with RAGE neutralizing antibody(10 ?g/mL)for 1 h,and RAGE antibody+LPS group was pretreated with RAGE neutralizing antibody(10 ?g/mL)for 1 h and then stimulated with LPS(1 ?g/mL)for 12 h.The results showed that RAGE neutralizing antibody attenuated LPS-induced decrease in SIRT3 activity(F=6.497,P=0.015).The results suggest that RAGE is involved in LPS-mediated decrease in SIRT3 activity.6.2 RAGE is involved in LPS-mediated decline in SIRT3 protein expressionRAGE neutralizing antibody abolished LPS-mediated decrease in SIRT3 protein expression(F=4.113,P=0.049).In summary,RAGE is involved in LPS-induced decline in SIRT3 activity and protein expression.Conclusions:1.LPS induces a decrease in SIRT3 activity and protein expression.2.PD inhibits LPS-induced vascular endothelial barrier dysfunction by activating SIRT3.3.PD protects endothelial barrier function by enhancing SIRT3-mediated deacetylation of SOD2 and CypD.4.RAGE is involved LPS-elicited alterations of SIRT3 activity and protein expression.