Protective Effect Of Polydatin Against Burn-induced Vascular Hyperpermeability And Lung Injury

Burn injury is a common trauma and extensive bums cause a great attention because of high mortality.The most important sign in the acute phase of bums is the markedly edema formation in the locally injured tissue.Visible swelling of the skin,blister formation are among the typical clinical features,due to increased microvascular permeability,vasodilation and increased extravascular osmotic activity.In extensive bum,the injury results in not only local edema but also edema in remote organs such as intact skin,muscle,and internal organs,which indicate that major bum injury induces a systemic hyperpermeability.With the microvascular hyperpermeability induced shifts and losses of fluid from the circulation,hypovolemia will occur after large burns if massive volume resuscitation is not provided,Particularly in the first few hours after injury.However,hypovolemia can be exacerbated by serious burns of limbs which induce lymphatic circulation obstruction.Therefore,adequate fluid resuscitation on the basis of degree of bum is the main clinical treatment on burn junry.Adequate fluid resuscitation may,however,markedly aggravate the edema formation because of the vascular hyperpermeability.Also,edema induces tissueanoxia and infection.Factors effect vascular hyperpermeability in bum injury has not been well described.In current studies:heat injury directly damage blood vessel;enzymes and cytokines such as thrombin,endothelial cell growth factor(ECGF),TNF-?,and so on,cause cytoskeleton rearrangement and endothelial junctions opened,via cellular signal transduction,lead to vascular hyperpermeability.Recent studies have also demonstrated that activation of the apoptotic signaling cascade is involved in endothelial dysfunction,which may result in hyperpermeability.Burn injury results inintrinsic apoptotic signaling activation,which mediated via mitochondria release of mitochondrial cytochrome c and second mitochondria-derived activator of caspases(Smac)into the cytoplasm,and activation of caspase-3.Caspase-35 the key executioner of apoptosis,is either partially or totally responsible for the proteolytic cleavage of many key proteins involved in endothelial cell morphological homeostasis.One such adherens-junctional protein is ?-catenin.?-catenin is a key component of adherens junction in exchange vessels,which connects the transmembrane adhesion protein vascular endothelial cadherin(VE-cadherin)to actin cytoskeleton.?-catenin cleavage cause adherens junction damage and hyperpermeability.Extensive burn injury cause remote organs injuried,and lung are some of the most vulnerable distant organs that are affected by burns.The pathophysiology of ALI involves highly complicated mechanisms,and recent studies have shown that systemic inflammatory response,apoptosis and endothelial cell dysfunction are key to this process.The role of inflammatory mediators in ALI is well described.Many inflammatory factors enter circulation after local burn injury and cause a severe systemic inflammatory response.An excessive inflammatory response constitutes the earliest stages in the development of lung injury after burn injury.Increased levels of local and systemic inflammatory mediators,such as tumor necrosis factor(TNF)-?and interleukin(IL)-1? and IL-6,and activated leukocytes,lead to systemic inflammatory response syndrome(SIRS).In addition,inflammatory mediators recruit neutrophils into the alveolar-eapillary wall,adhere to the endothelium,release various proteases,and infiltrate into the injury site.These processes may result in damage to the alveolar-capillary membrane and lead to increased permeability of the endothelium in lung injury.Vascular hyperpermeability enhances infiltration of inflammatory cells,notably polymorphonuclear neutrophils(PMNs),and subsequently results in cell apoptosis and interstitial edema.In recent years,increasing evidence has suggested that pulmonary cell apoptosis may also play an important role in the pathophysiology of lung injury.All these processes subsequently lead to pulmonary damage.Polydatin(PD),a monocrystalline drug that can be isolated from the traditional Chinese herb Polygonum euspidatum,is mainly used in the treatment of shock,burn and ischemia-reperfusion injury.The effect of PD on acute severe shock and burn injury has been studied at our laboratory for many years.The Sino Food and Drug Administration(SFDA)has approved of clinical trial on the effect of PD on hypotension after hemorrhagic shock and the trial has entered stage ? now.In addition,the effect of PD on the intrinsic apoptotic pathway inhibition via the mitochondria protective effect,which evidenced by scavenging oxygenfree radicals and inhibiting mitochondrial permeability transition pore(mPTP)opening,has been demonstrated in our recent researches.Since inflammatory response and apoptosis play key roles in the pathogenesis of burn-induced vascular hyperpermeability and lung injury,and PD has potential anti-inflammatory and anti-apoptosis effects,we hypothesized that PD may provide protection against burn-induced vaseular hyperpermeability and lung injury.Method Part one:Polydatin improve burn-induced vascular hyperpermeabilityIn the preliminary dose-response experiments,rats were randomly divided into eleven groups(n=10 for each group):(1)sham burn,(2)bum+normal saline(NS),(3)bum+Low-dose Cyclosporin A(CsA),(4)burn+Medium-dose Cyclosporin A(CsA),(5)bum+High-dose Cyclosporin A(CsA),(6)bum+Low-dose Resveratrol(Res),(7)bum+Medium-dose Resveratrol(Res),(8)bum+High-dose Resveratrol(Res),(9)bum+Low-dose Polydatin(PD),(10)burn+Medium-dose Polydatin(PD),(11)bum+High-dose Polydatin(PD).Low,medium and high dose CsA respectively is 3 mg/kg,6 mg/kg and 9 mg/kg.Low,medium and high dose Res respectively is 15 mg/kg,22.5 mg/kg and 30 mg/kg.Low,medium and high dose PD respectively is 30 mg/kg,45 mg/kg and 60 mg/kg.Rats were received normal saline(NS)or homologous drug after burn or sham bum,and the survival time were recorded.Burn injury was induced in rats on the dorsal skin covering about 30%total body surface area by 98? water for 30 s.Animals in the sham group were immersed in water at room temperature instead of water at 98?.In the vivo experiment,bum injury was induced in rats on the dorsal skin.Briefly,a dorsal area that equals 30%of the total body surface area was shaved.The rat was placed in a mold with an adjustable opening to expose the shaved area to 98? water for 30 s.Animals in the sham group were immersed in water at room temperature instead of water at 98?.5 min after bum injury,homologous volume of drug was given via the jugular vein cannula followed by a 6-h period constantly infusion of Ringer's lactate solution(4 mL/kg/%TBSA)according to Parkland formula to mimic clinical condition of fluid resuscitation.The dose was based on our previous preliminary dose-response experiments(CsA6 mg/kg,Res 22.5mg/k,PD 45 mg/kg).The vascular permeability measurement has been performed 5h postbum.The rats were placed on a Plexiglas platform mounted to an intravital upright microscope and a midline laparotomy was performed,then the permeability of mesentery venules was measured.The transvascular flux of plasma proteins was assessed in vivo by measuring the relative changes over time in fluorescence intensity of FITC-BSA in the intravascular versus extravascular space.The following formula was used for calculating change in integrated optical intensity:?I=1-(Ii-Io)/Ii,where ?I is the change in light intensity,Ii is the light intensity inside the vessel,and Io is the light intensity outside the vessel.Lipid peroxides content in serum and mesenteric vasculatrure were detected by LPO assay kit;cytosolic cytochrome c and smac by western blot;caspase-3 activity by caspase-3 fluorometric assay kit;?-catenin shift by immunofluorescence staining.In the vitro,burn injury was performed at skin of abdomen which induced by 80? water.Animals were randomly divided into three groups:(1)sham burn,(2)NS+burn,(3)PD+burn.PD(45mg/kg weight)and the same volume NS were administrated via the femoral vein 30min before the burn injury.A venule of 60-100?m in diameter was dissected from stripped abdoruen skin and cannulated with a micropipette on each end.The permeability of the vessel was quantified by measuring the ratio of transvascular flux to transmural concentration difference of albumin.The apparent solute permeability coefficient of albumin(Pa)was calculated to reflect the vascular permeability(Pa?(1/?If)(dIf/dt)o(r/2),where Alf is the initial stepincrease in fluorescence intensity,(dIf/dt)o is the initial rate of gradual increase in intensity as solutes diffuse out the vessel,and r is the venule radius).Part two:Protective effect of Polydatin against burn-induced lung injuryRats were randomly divided into three groups:(1)sham burn,(2)burn+normal saline(NS),and(3)burn+PD.Rats were subjected to 30%total body surface area burn injury followed by resuscitation.The treatment group received 45 mg/kg of PD and the bum group received the same amount of normal saline.Microvascular permeability,interstitial edema,neutrophil recruitment and histopathologic changes were detected by measuring Evans blue concentration,wet-to-dry weight ratio(W/D),myeloperoxidase(MPO)activity,and hematoxylin-eosin staining respectively.To investigate the mechanism of action of PD,ELISA,cell counting,TUNEL staining,fluorometric assay and western blot were used for assessing inflammatory cytokines(TNF-?,IL-1?,IL-6),total cells and polymorphonuclear neutrophils(PMNs)in bronchoalveolar fluid,the number of apoptotic cells,caspase-3 activity,and apoptosis-related proteins including Bax and Bcl-xl respectively.Results:Part one:Polydatin improve burn-induced vascular hyperpermeabilityDose experiment:rat survival time in sham burn group is more than 72 h;in NS group 1s 13.8±2.9 h;in Low-dose CsA,Medium-dose CsA,High-dose CsA group respectively is 10.7±4.0h,14.8±3.0 h,10.6±2.9 h;in Low-dose Res,Medium-dose Res,High-dose Res group respectively is 14.42±4.5 h,17.3±5.5 h,15.1±3.1 h;in Low-dose PD,Medium-dose PD,High-dose PD group respectively is 14.1±2.8 h,28.3±12.1 h,26.9±10.3 h.Burn-injuried rats treated with middle dose of CsA,Res and PD had a longer survival time.Therefore,the intermediate doses of three drugs were chosen in the main experiments.PD inhibits burn-induced local vascular hyperpermeabilityIn the vitro experiment,the permeability coefficient to albumin(Pa)was measured in venules of similar diameters.At T30,the value of Pa was increased from 4.3±1.8 in sham burned skin venules to 7.9±2.6 in burned skin venules(P<0.01),and reduced to 5.2±1.2 in PD pre-treated skin venules(P<0.05).Research data demonstrate that PD treatment attenuates burn-induced local vascular hyperpermeability.PD prevents burn-induced mesenteric vascular hyperpermeabilityIn the vivo experiment,burn-induced hyperpermeability was evidenced by a marked increase in extravasation of FITC-albumin into the extravascular space versus the sham burn group(P?0.01),which were respectively reduced at different level by the three treatments,and the protective effect of PD and Res were obviously.At T60,the ?I increased from 0.25±0.06 in sham burn group to 0.55±0.07 in burn+NS group(P<0.01),then decreased to 0.47±0.07,0.43±0.06(p<0.05),0.35±0.07(P<0.01)respectively in CsA,Res and PD treatment group.PD prevents burn-induced oxidative stressThe serum LPO level were increased from 0.21±0.02?mol/L in sham bum group to 0.46±0.06?mol/L in burn+NS group(P<0.01),then decreased to 0.41 ±0.05?mol/L?0.33±0.04?mol/L(p<0.05),0.28±0.04?mol/L(P<0.01)respectively in CsA,Res and PD treatment group.PD prevents burn-induced Cytochrome c and Smac ReleaseIn rat mesenteric tissue collected from sham burn animals,cytosolic cytochrome c and smac levels were both low.6 hours post bum injury,the cytosolic cytochrome c and snac dramatic elevated in the burn+NS group(3.20±0.42 of sham burn group values,for cytochrome c;4.32±1.01 for smac).The data indicated that the cytosolic cytochrome c and smac levels were decreased at different degree compared with bum+NS group after CsA,Res or PD treatment(3.03±0.33 of sham bum group values in CsA treatment group,2.02±0.40 in Res treatment group,1.71±0.25 in PD treatment group,respectively for cytochrome c;3.99±0.71 of sham burn group values in CsA treatment group,2.86±0.59 in Res treatment group,2.24 ± 0.37 in PD treatment group,respectively for smac).PD decreases burn-induced Caspase-3 activationThe burn+NS group showed a significant increase in caspase-3 activity compared with the sham bum group(2.37±0.66 of sham bum group values).Experimental data demonstrated that the burn-induced high caspase-3 activity was decreased at different degree after CsA,Res or PD treatment(2.23±0.75 of sham bum group values in CsA treatment group,1.78±0.26 of sham burn group values in Res treatment group,1.43±0.48 of sham burn group values in PD treatment group).PD prevent burn-induced remarkable morphological changes of ?-cateninMassive staining for ?-catenin along with the border of vessels,exiguous and orderly inner vessels was seen in mesentery of sham burned rats.Burn injury caused a noticeable alteration of ?-catenin spreading the inner vessel,blurred and disordered distribution.These changes can be obviously improved by treatment of Res and PD,but CsA.Part two:Protective effect of Polydatin against burn-induced lung injuryPD treatment attenuated burn-induced lung injuryLung tissue of rats in the bur+NS group showed accumulation of a large number of neutrophils in the intra-and interalveolar space,a thickened alveolar wall,less alveolar space,interstitial congestion and edema.PD treatment was found to markedly attenuate these signs of bum-induced lung injury.PD treatment inhibited burn-induced pulmonary microvascular hyperpermeabilityEB concentration in the lung was markedly increased in postburn rats(11.84±1.88 in sham group vs.24.00±5.22 in bum+NS group,p?0.01).Treatment of PD significantly reduced the concentration of EB in the lung(17.66±2.99 in bum+PD group vs.burn+NS group,P?0.01).PD treatment reduced pulmonary edema induced by burn injuryThe W/D in the bum injury animals was signifieantly inereased compared with that in the sham controls(3.62±0.33 in sham group vs.4.83±0.38 in burn±NS group,p<0.01).PD markedly decreased the W/D as compared with that in burn+NS rats(4.00±0.25 in burn+PD group vs.bum+NS group,p?0.01).PD treatment attenuated local and systemic levels of inflammatory mediators induced by burn injuryTNF-? and IL-1?,IL-6 in the lungs obtained from burn animals were dramatically higher than those from sham animals(TNF-? 23.92±7.46,IL-lp 13.42±4.17,IL-6 30.87±11.3 in sham group TNF-? 331.98±68.5,IL-1?172.4±73.78,IL-6 188±65.18 in bum+NS group,P<0.01),and these levels were significantly reduced in PD treatment group(TNF-? 95.177±22.55,IL-1? 84.87±25.39,IL-6 120.43±27.03 in burn+PD group vs.bun+NS group,P<0.05 or 0.01).In line with the measurements of lung tissues,TNF-?,IL-1p and IL-6 in the peripheral blood of bum animals were significantly higher than those in sham animals(TNF-?18.62±12.32,IL-1? 33.08±20.8,IL-6 25.82±12.51 in sham group vs.TNF-?432.38±122.7,IL-1? 310.03±102.8,IL-6 229.68±88.5 in burn+NS group,P<0.01).The increased levels of TNF-?,IL-1? and IL-6 were markedly attenuated by PD treatment(TNF-? 228.626±66.81,IL-1? 193.7±60.77,IL-6 150.22±48.68 in burn+PD group vs.burn+NS group,P<0.05 or 0.01).PD treatment reduced the total cells and PMNs in the BALFMarkedly increased total cells and PMNs in the BALF has been detected in bum-injured rat(total cells 0.49±0.2,PMNs 0.24±0.16 in sham group vs.total cells 9.53±2.11,PMNs 6.42±2.31 in bum+NS group,P<0.01),which were markedly reduced by PD treatment(total cells 5.9±1.97,PMNs 4.29±1.73 in burn+PD vs.bum+NS group,P<0.01).PD treatment decreased neutrophils recruitment in the lungLung MPO activity dramatically increased(sham group vs.bumH+NS group,P<0.01),and it was inhibited by PD treatment(burn+NS group vs.burn+PD group,P?0.008)PD treatment prevented lung cell apoptosis in burn injury ratsBum-injured animals showed a significant increase in apoptotic cells in comparison with the sham burn group(1.7±1.2 in sham group vs.39.5±12.7 in burn+NS group,P<0.01),which was significantly reduced by PD treatment(18.7±10.6 in bum+PD group vs.burn+NS group,P<0.01).Burn-injury resulted in down-regulation of Bcl-xl protein and PD prevented this decrease.The pro-apoptotic protein,Bax,can be up-regulated by burn-injury and inhibited by PD treatment.The caspase-3 activity was significantly increased in the lungs of animals postbum,which was prevented by PD treatment(sham group vs.burn+NS group,P<0.01,burn+NS group vs.burn+PD group,P<0.05).PD improves the survival time of the burn-injured ratsThe average survival time extended from 30.2±11.6 h in the burn+NS group to 50.7±17.0 h in the bum+PD group(sham group bum+NS group,P<0.01,burn+NS group vs.bum+PD group,P<0.05).Conclusion1.PD attenuates burn-induced systemic vascular hyperpermeability,which may be caused by its inhibitory effects on oxidative stress and intrinsic apoptotic signaling pathway.2.PD inhibits burn-induced locally hyperpermeability,further studies may be required to investigate the impossible mechanism.3.PD-induced significant attenuation of lung injury induced by extensive bum and improvement survival time in a rat model.4.The potential mechanism of this action that PD attenuates lung injury induced by extensive burn is through amelioration of inflammation and apoptosis.