Sirt1 Protects LPS-induced Hyper-permeability In Endothelial Cells

Sepsis is a lethal clinical syndrome,and its feature is the complexity in pathophysiological process,a variety of clinical symptoms,expensive cost for treatment and devastating results.Sepsis does much harm to us,but little is known about its development and treatment.Although sepsis is very complicated,we are trying to notice the important role of the destruction of the micro vascular barrier during sepsis.Critical are the vascular endothelial cells(ECs)in trafficking the nutrients and keeping the vessels in order.Once ECs are disrupted,vascular permeability will be enhanced and fluid leakage will be caused,thus inducing dysfunction in tissue and organs.As is revealed by much evidence,EC disorder is the potent cause of sepsis.Stabilizing the skeleton and connection of ECs could reduce the mortality of sepsis.Sepsis could lead to serious lung injury and the microvascular extravasation,as well as the elevated wet/dry ratio.This pushes us to know more about sepsis from the point of endothelial dysfunction.However,there remains much to be done.As we all know,silent information regulator complex(SIR complex)is composed of four parts,including Sir1 to Sir4,during which Sir2 is highly expressed in most kinds of cells.Sir2 is divided to 4 parts according to its conserved sequence.Part ? includes silent information regulator 2 homolog 1(Sirtl),2 and 3.Part ?includes Sirt4.Part ? includes Sirt5.Part IV includes Sirt6 and 7.To date,Sirtl has been acknowledged extensively.As a nicotinamide adenine dinucleotide(NAD+)-dependent deacetylase,Sirt1 is responsible for modulating aging,apoptosis and stress of ECs.Generally speaking,Sirtl could regulate functions of ECs through targeting its substrates,such as p53 and p66Shc.Moreover,Sirtl is critical in defending lipopolysaccharide(LPS)-sparked lung injury.Resveratrol could defend inflammation,while application of Sirtl small interfering RNA(siRNA)reversed the aforementioned effect.Furthermore,the lack of Sirtl could aggravate the vessel response,morbidity and mortality rate,which could be reversed by Sirtl activator,suggesting Sirtl is paramount in the protection of ECs form sepsis.However,it still remains elusive whether Sirtl is salutary for ECs.The potent mechanism is another question.In this study,we aim to explore the role of Sirtl in barrier dysfunction induced by LPS and the potent mechanism.Furthermore,we hypothesize that Sirtl acts as a node,responding to receptor for advanced glycation end products(RAGE)and toll like receptor 4(TLR4),for one thing,by elevating the manganese superoxide dismutase(SOD2)level,and for another,by attenuating the NADPH oxidase 4(NOX4)level,to diminish the increased reactive oxygen species(ROS)level induced by LPS.In this way,endothelial barrier is protected.RAGE and TLR4 serve as the typical receptor for LPS.In the model of diabetes nephrology,LPS increases the ubiquitin of Sirtl through RAGE,thus downregulating Sirtl protein level.We speculate that LPS could induce Sirtl downregulation through RAGE and TLR4,thus sparking endothelial hyper-permeability.As is well known,SOD2 is famous for cleaning the ROS level and alleviating the increased leakage induced by ROS in ECs.As is reported,interaction between Sirtl and SOD2 is beneficial for the capacity for resisting ROS.Our previous study has confirmed that in the model of ischemic shock,the transition pore permeability of mitochondrial in hepatocyte was increased,accompanied by the mitochondrial swelling,decrease of the SOD2 activity,and the increase of ROS generation.Polydatin inhibited the mitochondrial damage through Sirtl-SOD2 pathways,thus protecting the function of liver.Meanwhile,by inhibiting Sirtl,NOX4 could be elevated to generate more O2·-,thus resulting in EC barrier disruption.Taking all the above into consideration,we speculate that EC barrier function could be maintained via Sirtl pathways to elevate SOD2 and attenuate NOX4 to reduce ROS level.Taking all the above into consideration,our hypothesis is that,LPS induces hyper-permeability in ECs,and Sirtl could abolish the process through enhancing SOD2 level and attenuating NOX4 to decrease ROS level.Object:The study targeted on pulmonary microvascular endothelial cell(PMVECs).human umbilical vein endothelial cell(HUVECs)and C57 mouse to investigate the potent role of Sirtl in LPS-induced EC hyper-permeability,and to unveil the underlying mechanism.This will provide the new theoretical evidence and approach for treatment.Methods:The object of the study focused on PMVECs,HUVECs and C57 mouse.Measures were adopted including isolation,identification and cultivation of cells,Western blot(WB),immunofluorescence,flow cytometer,TER measurement,flux coefficient of FITC-dextran,and extravasation from mesentery microvasculature.To study the receptor of LPS,the protein and activity level of Sirt1 and the downstream of ROS,and morphological change were assessed.The Sirtl specific agonist SRT1720 and antagonist ex527 were pretreated.Sirtl siRNA was used to inhibit Sirt1 expression.WB was used to detect expression of Sirtl,SOD2 and NOX4.Flow cytometer was used to detect ROS level.Immunofluorescence was used to detect the morphological change of VE-cadherin and F-actin,and ROS level.Reagent kit was used to detect MDA and SOD level.The resistance of the monolayer was detected by the resistance instrument.Another method was measuring the flux coefficient of fluorescein isothiocyanate(FITC)-labelled dextran.These two methods reflect the EC barrier function.C57 mouse was also cited through detecting the extravasation of dextran from mesentery venules.All data were showed as mean±SE,and the significance was determined by One-way Anova.P<0.05 was considered as statistical significance.Results:1.LPS induced hyper-permeability in HUVECs1.1 LPS induced EC hyper-permeability in a time-dependent manner500 ng/mL LPS was applied to stimulate ECs for 3,6,9,12,24 h,then FITC-dextran was detected to measure the EC permeability.The result showed the time-dependent increase in EC permeability.There is significance between 24 h and 0 h(P<0.05).In the follow-up,24 h was adopted as the stimulating time to explore the concentration gradient effect upon LPS.1.2 LPS induced EC hyper-permeability in a dose-dependent mannerThe result showed LPS induced EC hyper-permeability in a dose-dependent manner.The flux level was increased from 100 ng/mL,and the level in 200 ng/mL and 500 ng/mL was statistical higher than that in Control group(P<0.05).In the follow-up experiment,500 ng/mL LPS for 24 h was adopted to observe the change in permeability.2.Sirtl inhibited LPS-induced EC hyper-permeability2.1 LPS induced ubiquitination and downregulation of Sirtl protein expression500 ng/mL LPS stimulated ECs for 1,2,4,8,12,24 h,with 0 h as Control.The result showed that there was statistical significance between 0 h and any other time for stimulation(P<0.05).Sirt1 was decreased quickly after 1 h,and the low level was kept until 24 h.The ubiquitination of Sirt1 was observed,indicating the relation between Sirt1 protein drop and ubiquitination.2.2 Sirt1 activator SRT1720 upregulated activity of Sirt1Western blot showed that 5 ?M SRT1720 for 24 h upregulated the activity of Sirt1,compared with the Control group(P<0.05),indicating the high efficiency of activating Sirt1.2.3 Sirt1 inhibitor ex527 downregulated activity of Sirt1Western blot showed that 20 ?M ex527 for 1 h downregulated the activity of Sirt1,compared with the Control group(P<0.05),indicating the efficiency of inactivating Sirt1.2.4 Sirt1 siRNA downregulated the protein expression of Sirt1Western blot showed that transfection of Sirt1 siRNA inhibited the protein expression of Sirt1 compared with the Control siRNA group(P<0.05),indicating the efficiency of inhibiting Sirt1 expression.2.5 SRT1720 inhibited LPS-induced EC hyper-permeabilityThe transendothelial electrical resistance(TER)level and the coefficient of FITC-labelled dextran revealed that both pretreatment and simultaneous treatment of SRT1720(5?M,24 h)could reversed the increase in permeability induced by LPS in HUVECs(P<0.05),hinting the protective and remedial role of Sirt1 in EC barrier.2.6 ex527 aggravated LPS-induced EC hyper-permeabilityPretreatment of ex527(20 ?M,1 h)further sparked EC hyper-permeability induced by LPS(P<0.05),strengthening the possibility of the protective role of Sirt1 in ECs.2.7 Sirt1 siRNA aggravated LPS-induced EC hyper-permeabilityCompared with Control siRNA group,Sirtl siRNA itself induced EC hyper-permeability and also aggravated LPS-induced barrier disruption(P<0.05),2.8 SRT1720 inhibited LPS-evoked morphology change in F-actin and VE-cadherinCompared with Control group,LPS caused the redistribution of F-action with increased fiber stress.Disruption of VE-cadherin was also observed.SRT1720 inhibited the aforementioned changes by LPS,indicating the protective role of Sirtl in skeleton and adherence protein.2.9 SRT1720 inhibited LPS-induced increased permeability in mesentery venulesCompared with Control group,pre-injection of LPS(15 mg/kg)for 6 h induced the increase in extravasation of FITC-labelled dextran from the mesentery venules.Pretreatment of SRT1720(10 mg/kg,2 h)could inhibit the leakage of dextran.That indicated the pivotal role of SRT1720 in the EC barrier from the perspective of animal.3.RAGE and TLR4 participated in LPS-induced downregulation of Sirtl3.1 RAGE soluble antibody and TLR4 inhibitor TAK242 inhibited LPS-induced Sirtl activity downregulation.Compared with LPS group,pretreatment of 10 ?g/mL RAGE antibody for 1 h or 3 u M TAK242 for 1 h remarkably inhibited the downregulation of Sirtl in activity level(P<0.05).In view of the Sirtl protein level,RAGE antibody inhibited LPS-sparked Sirtl activity downregulation(P<0.05).3.2 RAGE antibody inhibited LPS-induced Sirtl ubiquitinationCompared with LPS group,RAGE antibody inhibited Sirtl ubiquitination induced by LPS,indicating the critical role of RAGE in LPS-induced Sirtl decrease.3.3 RAGE-/-inhibited LPS-induced Sirtl protein decrease in PMVECsLPS evoked the remarkable downregulation of Sirtl protein in Wild type mouse,while the decrease was reversed in RAGE-/-mouse.The difference was statistical significant(P<0.05).It meant that RAGE responded to the LPS stimulation to trigger Sirtl decrease.3.4 RAGE-/-inhibited LPS-induced increase in permeability of mesentery microvasculatureLPS could spark Sirtl decreases in PMVECs of Wild type mouse,while this phenomenon was not observed in RAGE-/-mouse,strengthening the potent role of RAGE in hyper-permeability of mesentery microvasculature.4.Sirtl inhibited increase in ROS and MDA induced by LPS in HUVECs4.1 Sirtl inhibited LPS-induced ROS increaseThe flow cytometer with probe H2DCF-DA showed that 500 ng/mL LPS for 6 h induced the remarkable increase in ROS level,while SRT1720 reversed it.The difference was statistical significant(P<0.05).4.2 Sirtl inhibited LPS-induced MDA increaseThe MDA reagent kit showed that 500 ng/mL LPS for 1 h increased MDA level,while SRT1720 prevented the MDA increase,the difference was statistical significant(P<0.05).5.Sirtl inhibited SOD2 decrease induced by LPS in HUVECs5.1 LPS induced SOD2 decrease in a time-dependent mannerTreatment of 500 ng/mL LPS for different time(0,1,2,4,8 h)showed that protein level of SOD2 began to decrease after 1 h,and reached the bottom at 4 h,rebounding at 8 h,implying the time-dependent manner after LPS treatment(P<0.05).In the following experiment,500 ng/mL LPS for 1 h was adopted to detect the protein level.5.2 SRT1720 inhibited LPS-induced SOD2 decrease,while ex527 and SirtlsiRNA aggravated the SOD2 decreasePretreatment of 5 ?M SRT1720 for 24 h alleviated SOD2 decrease in protein level induced by LPS(P<0.05).ex527 itself induced SOD2 decrease,and ex527 could also aggravate LPS-induced SOD2 decrease,compared with LPS group(P<0.05).Moreover,compared with Control siRNA,Sirtl siRNA aggravated LPS-induced SOD2 decrease(P<0.05).All these indicated the paramount role of Sirtl in SOD2 increase.5.3 SRT1720 inhibited LPS-induced decease in SOD2 activitySOD reagent kit showed that SRT1720 inhibited LPS-induced SOD activity decrease,and the difference was statistical significant(P<0.05).5.4 ex527 aggravated LPS-induced decease in SOD activityPretreatment of ex527 for 1 h aggravated LPS-induced SOD activity decrease,the difference was statistical significant(P<0.05).5.5 Apocynin inhibited LPS-induced ROS generationPretreatment of 300 ?M apocynin for 1 h and subsequent application of 500 ng/mL LPS for 6 h showed that LPS led to ROS increase while apocynin reversed the increase(P<0.05).5.6 SRT1720 inhibited LPS-induced NOX4 upregulationCompared with Control,LPS induced NOX4 upregulation significantly.Pretreatment of 5 ?M SRT1720 for 24 h alleviated NOX4 upregulation in protein level induced by LPS(P<0.05).Conclusion:1.Sirtl protects LPS-induced vascular endothelial hyper-permeability.2.RAGE and TLR4 mediated LPS-induced decrease in Sirtl activity and protein.3.Sirtl protects LPS-induced barrier dysfunction through upregulating SOD2 and downregulating NOX4,to decrease ROS level.