| BackgroundAcute kidney injury(AKI)is one of the common complications in hospitalized patients.Mortality and fatality rate of patients with AKI were significantly increased;renal ischemic injury caused by acute renal injury is a common cause.A significant pathological feature of hemorrhagic shock is decreased perfusion in systemic and local tissue;fluid resuscitation after hemorrhagic shock is one of the important measures in critically ill patient's treatment.Multiple organ failure is a common prognosis after hemorrhagic shock and resuscitation,and the kidney is the major organ involved.It was reported that,although some patients with AKI was successfully survived but renal function appeared irreversible damage and finally get chronic renal failure,leading to decreased survival rate.According to statistics,the mortality of patients with acute renal failure is up to 40-60%.A characteristic of AKI is a sharp decline in glomerular filtration rate.People have clearly realized that,AKI is an important factor to promote the end-stage renal disease process to patients with chronic kidney disease.Previous studies showed that cell apoptosis,oxidant and iron mediated cell injury,endothelial cells change,regeneration and repair,and inflammatory reaction all play a key role in the pathogenesis of AKI in.However,a growing body of research indicates that mitochondrial dysfunction is a major cause of AKI.Mitochondrial dysfunction is a factor in the pathogenesis of many diseases.Since 1962,it was found to be a high metabolic disease as nonthyroid source disease.After that,mitochondrial dysfunction has been gradually concerned.In different cells,different tissues and organs,different energy demand,and the number of mitochondria varies greatly,from 16/cells in human germ cells to 100000/cell in oocytes.The kidney is a very dynamic organ and is rich in mitochondria.Therefore,mitochondrial dysfunction plays a crucial role in the pathogenesis of renal disease.At present,it have been identified that mitochondrial dysfunction and apoptosis mechanism of AKI mainly involving ischemia and hypoxia,oxidative stress,microcirculation disorder,cell apoptosis-mediated by mitochondria and mitochondrial dynamics destruction.To date,few studies targeting at mitochondria in therapeutic of kidney disease were reported.If mitochondrial dysfunction was caused by genetic defects,such therapy as correcting mitochondrial mutations or targeted RNA treatment according to etiology was tried,however,this therapy is still at the laboratory stage.In clinic,allogeneic hematopoietic stem cell transplantation has been confirmed in the treatment of mitochondrial encephalomyopathy(a mitochondrial cell disease in digestive tract),but it is only eliminating toxic substances targeting at mitochondrial gene induced mutation and difficult to apply in other mitochondrial cytopathy.In view of the present condition,the clinician should be aware of nephropathy especially in diagnosis and differential diagnosis of diseases caused by mitochondria,avoid unnecessary and ineffective treatment,especially use harmful drug to patients with mitochondrial cell disease(barbiturates,biguanides,glucocorticoid treatment).At the same time,in addition to the treatment of kidney disease outside of therapy should also consider them for adverse effects in patients with renal cell mitochondrial disease,because they are particularly susceptible to damage of renal function.Other methods include gene therapy,microRNA therapy,antioxidant therapy,thiazolidine two ketones etc.It have been widely reported that calorie restriction can delay aging,prolong life,however,because of many problems in application,it is difficult to therapeutic a disease in a short period in clinical critical patients.Therefore,the sirtuin family which can promote chromatin silencing and transcriptional repression has become a kind of simulation of calorie restriction alternatives and plays an important role in the pathogenesis of renal disease.Silent information regulator 2 related enzyme 1(Sirtuin 1,SIRT1)is a kind of nicotinamide adenine dinucleotide(NAD+)-dependent protein deacetylase,it is one of the 7 homologous chromosomes of mammalian Sir2 and have the longest total sequence,and is most widely researched.Initially,SIRT1 was found to be able to regulate longevity,apoptosis and DNA repair.In addition to deacetylated histone H1,H3 and H4,SIRT1 also can deacetylated many other non-histone proteins includes p53,FOXO,Ku70,P300,Rb,E2F1,NF-kappa B,p73 and PGC-1 alpha.Due to the effect of SIRT1 on the above important protein,it is involved in the regulation a variety of life process in normal and abnormal cells,such as tumor metabolism,stress response,aging etc.As a tumor suppressor,p5 3 can effectively regulate cell apoptosis.Under normal circumstances,p53 protein ubiquitination and degradation pathway mediated by MDM2 and maintains at a low level.However,when the cells were exposed to stress environment including genotoxic stress,p53 protein can rapid accumulate and its downstream targets was activated,leading to related biological effects.The stability and activity of p53 are in part affected by various posttranslational regulations,including phosphorylation,ubiquitylation and acetylation etc.p53 gene is the first non-histone targets discovered of SIRT1 deacetylation.Even the HDAC inhibitor trichostatin A(TSA)is exist,p53 acetylation is very unstable in cell lysate,which may be due to the deacetylase.Subsequent research has shown that despite the presence of HDAC inhibitor TSA in common conditions,SIRT1 deacetylase activity still cannot be effectively suppressed.Further studies confirm p53 is indeed a target of SIRT1,i.e.SIRT1 regulation p53 function through its acetylation activity.This phenomenon was also verified by Vaziri and Langley et al.respectively.Recently,resveratrol,which is widely found in red grapes and red Wine,was greatly reported as SIRT1 activator.It was reported that resveratrol can improve the mitochondria function and lipid levels,promote and maintain PGC-1 alpha expression,ultimately maintain the integrity of the podocyte.Interestingly,resveratrol was first proved to be a SIRT1 agonist and has been shown to reduce renal ischemia/reperfusion injury.Furthermore,resveratrol has also been confirmed to be able to reduce cisplatin induced mouse proximal tubular epithelial cell injury and doxorubicin induced myocardial cell apoptosis through SIRT1-deacetylase pathway.In addition to resveratrol,other new SIRT1 agonists are also reported.Since coenzyme NAD+ is a key factor of SIRT1 signaling molecules.Dietary supplementation with NAD+ precursor nicotinamide nucleotide or nucleoside can enhance oxidative metabolism.Therefore,the sirtuin family members and their activation agent may be a promising therapeutic target.Polydatin(PD),also known as piceid,PD crystal No.4.It is extracted from the stem and root of traditional Chinese medicine Polygonum cuspidatum with the effective active ingredient.The chemical structure of PD has been identified as 3,4,5-three hydroxy two styrene-3-D-glucoside.As a single crystal of drug,PD has been studied by Professor Zhao Ke-seng(Department of pathophysiology,Southern Medical University,its former name is First Military Medical University)for more than 30 years,PD has been confirmed to have exact effect in several disease models.Over the years,no obvious toxic side was found in PD application.Through the cooperative development by Professor Zhao Ke-seng and Neptunus Co.,a formal clinical trial of PD was approved(approval No.2006L00301)and PD is new drug listed in class I.Currently,clinical hemorrhagic shock and burn ?b test of PD application is studied and is in a good progress.Several studies show that PD exerts therapeutic effect on experimental animal shock caused by various diseases.Our previous work in experimental animal models involving hemorrhagic shock and burn proves that PD can expand capillaries,improve microcirculation,increase cardiac output,reduce systemic peripheral resistance,improve cardiac function and pulse pressure,change hemorheology characteristics,inhibit leukocyte adhesion etc.Secondly,PD can improve the ischemia reperfusion model of multiple organ damage.In our laboratory,Wang and Song etc.found that PD can reduce mitochondrial injury of vascular smooth muscle in severe hemorrhagic shock rat model.Through the rat myocardial ischemia/reperfusion injury experiment model,Zhang et al.summary some mechanism related to PD effects following:1.PD can inhibit mitochondrial KATP and mPTP to protect mitochondria,exerting a cardioprotective effect;2.PD increases Bcl-2 protein,reduces Bax,attenuates ischemia/reperfusion induces myocardial apoptosis;3.PD decreases calcium influx,open KATP,increases potassium efflux,exerts obvious anti myocardial ischemia/reperfusion and arrhythmia effect;4.PD inhibits platelet aggregation,reduces the leukocytes adhesion to the vascular endothelium,improved microcirculation,inhibits lipid peroxidation.Also some scholars presented that PD reduce ischemia/reperfusion induced myocardial damage through its anti-oxidative effect.In pregnant rabbits hemorrhagic shock model,PD may induce HSP-70 expression in lung tissue,inhibit the activation of NF-kB,reduce the production of inflammatory cytokines,suppress release of serum TNF-alpha and pulmonary ICAM-1 adhesion molecules,and ultimately ameliorate the lung injury.Later reports showed that PD can prevent lung injury induced by sepsis sepsis.In addition,PD can reduce the mitochondrial third complexes(complex III)oxygen free radical formation;have a direct protective role of mitochondria.Based on similarity molecular structure of PD over resveratrol(comparing with resveratrol,PD has an extra glucose molecule)and the theory that resveratrol can activate SIRT1 as a mitochondria protector,we speculate that:1.PD can attenuate renal injury after hemorrhagic shock/reperfusion;2.the therapeutic effect of PD on shock/reperfusion induced renal injury is related to its mitochondrial protection mechanism;3.PD's mitochondrial protection may be due to its deacetylation p53 pathway mediating by SIRT1,leading to cell apoptosis reduction.According to the hypothesis above,we perform the experiments following three parts below:1.Establish hemorrhagic shock/reperfusion(HS/R)animal model,detect whether mitochondrial injury is exist in of renal cell and PD treatment can reduce the mitochondrial injury;separate damaged target cells,evaluate PD effect on mitochondria;2.Explore the relationship between PD treatment and SIRT1 activation,determination SIRT1's downstream targets,p53,its acetylation level;3.Establish human renal cell line(HK-2)hypoxia/reoxygenation model,further validate PD effect on SIRT1-p53 signal.Part oneMitochondrial dysfunction to renal tubular epithelial cell exposing to hemorrhagic shock and reperfusion1.Mitochondrial damage in renal tubular epithelial cells of rats after HS/RFirst of all,we determinated mitochondrial ultrastructure of kidney cells and evaluated whether the mitochondrial damage is existing in severe hemorrhagic shock and reperfusion condition by transmission electron microscopy.We found that mitochondrial dysfunction ofRTECs is obvious;and irregular shape,obvious swelling,and cristae partly damaged or unreadable of mitochondria was detected in shock rats(vehicle group),comparing to that in normal control group with normal mitochondrial morphology,integrated mitochondrial membrane and cristae.Therefore,we determined the RETCs mitochondria as object in the follow-up study.After RTECsseparation,we determined RTECs mitochondrial permeability transition pore(mPTP),mitochondrial membrane potential(??),intracellular ATP levels and lysosomal stability by the methods as our previous reported.Bright yellow green fluorescence was found in control group by confocal calcein microscope,whereas in HS/R group(group vehicle)fluorescence is partially quenched.For the open intenstiy of mPTP quantitation,we analyzed calcein-AM fluorescence by flow cytometry.Compared with the control group,the fluorescence intensity of calcein-AM in severe shock after reperfusion(vehicle group)decreased to 34.1±2.1%(t=15.654,P=0.000).This index fluorescence probe JC-1 monomer/polymer ratio was used to reflect themitochondrial membrane potential(??).We found that JC-1 monomer/polymer increased significantly after the severe shock and reperfusion(t=-10.495,P=0.002).Using the luciferin luciferase assay for the detection of intracellular ATP levels,we found that ATP levels in vehicle group decreased by 34±7.3%of control group(t=10.939,P=0.000).Our previous studies confirmed that the mitochondria and lysosomes are closely related,the mitochondrial dysfunction may aggravate the lysosomal damage;in turn,lysosomal damage could deteriorate further mitochondrial damage.i.e.,subcellular organelles lysosomes injury is an indirect indicator of mitochondrial injury.Due to the relationship between lysosomes and mitochondria,we investigate the effect of hemorrhagic shock onRTECs lysosomal,we found that after severe shock and reperfusion the AO-red fluorescence is weakened,and even the typical "pale cell"was appeared(t=7.155,P=0.000).2.Mitochondrial proapoptosis protein of renal tubular epithelial cells in rat after HS/RIn order to investigate the mechanism of mitochondrial damage in RTECs after HS/R,we used immunohistochemical techniques to determine mitochondrial apoptosis related protein.We found that after HS/R,the proapoptosis protein Bax was significantly increased and the apoptosis anti-apoptosis protein Bcl-2 was reduced in vehicle group that that in control group.In order to further quantification of apoptosis related proteins Bax and Bcl-2,the relative content of the two kinds of protein was determined using immunoblotting technique,followingthe extracted cytoplasmic and mitochondrial protein from renal cortex for cytochrome c determination.In consist with immunohistochemistry results,after the HS/R,the Bax content in vehicle group was increased by 30±14%than that in control group(t=-3.682,P=0.006).The corresponding Bcl-2 protein content showed the reverse phenomenon,while in vehicle group the Bcl-2 protein reduced by 29±9.8%of control group(t=4.692,P=0.002).As cytochrome c determination,the cytoplasmic/mitochondria ratio of cytochrome c in vehicle group increased by 115±32.2%of control group(t=-6.817,P=0.000).These results show that in mitochondrial damage condition,mitochondrial apoptosis related proteins were changed.3.Acetylated p53 protein of renal cortex in rats was increased after HS/RImmunoblotting results found that in vehicle group the p53 protein significantly increased than in control group;in addition to p53 protein,the acetyl-p53 was also raised than that in control group(t=-3.063,P=0.039),these results suggest that p53 may be involved in the regulation of mitochondrial apoptosis after HS/R.4.SIRT1 protein and activity of renal cortex in rats were reduced after HS/RWe measured the protein expression and activity of SIRT1 in renal cortex of rats after HS/R shock.We found that,comparing with conrol group,protein expression and activity of SIRT1 in renal cortex of shock in rats were decreased by 48.6±5.3%(t=8.859,P=0.002)and 58.2±8.9%(t=8.329,P=0.001).Part twoPolydatin attenuatehemorrhagic shock/reperfusion induced mitochondrial damage of renal tubular epithelial cell in rats through SIRT1 activation1.Influence of PD and Ex527 on SIRT1 activity of renal tissue inhealthy rats.Aftedrug administration for seven days,the SIRT1 protein expression and activity in kidney tissue was significantly different among the groups(SIRT1 protein:F=30.233,P=0.000;SIRT1 activity:F=56.636,P=0.000).PD increased the SIRT1 expression and SIRT1 activity by 25.3±11.8%and 30.5±13.6%,respectively,than that in control group;In contrast,the SIRT1 protein expression and activity were decreased 30.5±13.6%and 24.3±5.4%in Ex527 group,respectively,comparing with that in control group,and the difference of two values between two groups was statistically.2.PD upregualte the SIRT1 activity of renal tubular epithelial cell After PD administration,the SIRT1 protein expression and activity in kidneytissue was significantly different among the groups(SIRT1 protein:F=22.236,P=0.002;SIRT1 activity:F=11.006,P=0.002).The SIRT1 protein expression and activity were increased by 60.5±10.5%and 65.1±27.9%compared to vehicle group;when Ex527 is added to the PD/Ex527 group,SIRT1 activity and protein expression was decreased by 33.8±14.1%and 30.1±8.8%than that in PD group.3.The p53 deacetylated effect of PD onrenal epithelial cells after HS/RImmunoblotting assay results showed that no significantly difference of p5 3 expression was found among the groups(F=0.836,P=0.478).However,the acetyl-p53 level was differed significantly(F=20.778,P=0.002).In PD group the acetyl-p53 was reduced by 40.8±7.9%of vehicle group.However,when Ex527 was added,the decreased acetyl-p53 in PD group was elevated,which was even exceeding over the velue in vehicle group.These results suggest that PD could effectively reduce acety-p53 level and PD possese deacetylated effect on p53.4.PD inhibits mitochondrial apoptotic signalThe results found that,comparing with vehicle,PD administration partially reduced the expression level of Bax and increase the expression of Bcl-2.In order to further quantitative PD effect on Bax and Bcl-2,immunoblotting technique was used.The Bax,Bcl-2 and cytochrome c were all differed significantly among the four groups(Bax:F=7.423,P=0.008;Bcl-2:F=6.270,P=0.014;cytochrome c:F=14.109,P=0.001).In consist with results of immunohistochemistry,PD administration significantly reduced the Bax protein and increased Bcl-2 protein expression than vehicle treatment.In additon,cytochrome c protein in cytoplasma and mitochondria of renal cortex were determined and PD treatment significantly reduced cytochrome c cytoplasmic/mitochondrial ratio in vehicle group.While the Ex527 addition blockedbenifiteffect of PD on mitochondrial apoptosis related protein that Ex527 promoted the expression ofpro-apoptosis protein Bax andcytoplasmic cytochrome c,inhibits the expression of anti apoptotic protein Bcl-2.These results suggest that PD may play a protective role of mitochondria through its activation of the SIRT1 signaling pathway.5.PD attenuated the mitochondrial damage of renal tubular epithelial cells after HS/R.Through TEM observation,we found that RTECs' mitochondrial swelling was reduced and mitochondrial cristae morphology were more veisible in PD group than in vehicle group;Through flow cytometry determination,we found that the indexes calcein-AM,JC-1,ATP and AO-red were all differed significantly among the groups(Calcein-AM:F=115.480,P=0.000;JC-1:F=11.078,P=0.002;ATP:F=5.406,P=0.013;AO-red:F=11.158,P=0.002).Comparing with vehicle group,PD partially inhibited the opening of permeability transition pore.In PD group,the calcein fluorescence intensty restored to 81.1±5.6%of control group,which was 238.2±16.3%of vehicle group;After PD treatment,JC-1 monomer/polymer ratio was partial recovery,which indicated that PD can effectively stabilize transmembrane potential of RTECs after HS/R.In addition,PD administration significantly increased ATP levels than that in vehicle group.However,when Ex527,a selective inhibitor of SIRT1 was added in PD/Ex527 group,all the benifit effects of PD were attenuated in different intensity evidencing by swollen mitochondria,disappeared mitochondrial cristae,reduced calcein fluorescence,increased JC-1 monomer/polymer ratio,decreased ATP and AO-red fluorescence intensity comparing to PD group.Part threeThe mitochondrial protective effect of PD on HK-2 cell exposing to hypoxia/reoxygen1.PD and Ex527 effect on SIRT1 protein and activityTo further confirm the PD effect on SIRT1,a HK-2 cell line was enrolled.First of all,we explored the effect of PD,Ex527 and pifithrin-?(PFT-?,a special inhibitor of p53)on SIRT1 in normal HK-2 cell.Both the SIRT1 protein and SIRT1 activity were differed significantly among the groups(SIRT1 protein:F=26.202,P=0.000;SIRT1 activity:F=36.651,P=0.000).As expected,the SIRT1 protein and activity in PD group was increased by 34.4±9.8%and 60.0±15.0%of control group,respectively.In contrast to PD group,the SIRT1 protein and activity was decreased by 52.9±8.6%and 39.7±6.6%of control group,respectively.However,no significant difference was found between PFT-? group and control group as to SIRT1 protein and activity determination.2.PD activate SIRT1-p53 signal pathwayTo further explore the exact mechanism of PD effect,we reproduced hypoxia/reoxygen(H/R)model in HK-2 cell.The H/R model possesses a similar pathophsiology as HS/R animal model.First of all,we confirm that 48 h hypoxia time and 50 ?M concentration of PD administration through time-and dose-effect curve.In addtion,we set a PD/Ex527 group and PD/Ex527/PFT-? group.We determined SIRT1 protein expression and SIRT1 activity of HK-2 cell and significant differences were found among the groups(SIRT1WB:F=23.553,P=0.002;SIRT1 activity:F=34.362,P=0.000).As expected,the SIRT1 protein and activity all decreased comparing with vehicle group.However,in PD group,the values were inceased to 2.1±0.5 times,1.7±0.3 times,and 2.2±0.6 times over vehicle group.Interestingly,the SIRT1 protein and activity in PD/Ex527 group was again reduced comparing with PD group.These results indicated that Ex527 administration effectively abolised PD effect on SIRT1.In PD/Ex527/PFT-? group,although PFT-? was added,the SIRT1 protein and activity did not increased significantly.Based on the results that PD could activate SIRT1,we next analyzed the downstream target,p53.We found that p53 protein expression,mitochondrial translocation and acetyl-p53 were all differed significantly among the groups(p53 expression:F=53.134,P=0.000;p53 mitochondrial transfer:F=108.571,P=0.000;Acetyl-p53 WB:F=55.264,P=0.000;Acetyl-p53 FCM:F=36.450,P=0.000).Comparing with vehicle group,although the p53 protein expression was not signifcantly decreased in PD group,the p53 mitochondrial translocation was reduced and acetyl-p53 was decrease.When Ex527 was added in PD/Ex527 group,both the p53 mitochondrial translocation and acetyl-p53 were increased,which were almost equal to control group.Notably,the increased p53 mitochondrial translocation was again reduced when PFT-? was added in PD/Ex527/PFT-? group comparing with PD/Ex527 group,however,the acetyl-p53 did not decreased significantly.The above results indicated that PD effect is associated to SIRT1 activator.When SIRT1 expression was inhibited,acetyl-p53 increased.Although PFT-? could reduced p53 protein,it could not significantly reduce the acetyl-p53 level.3.PD redueced H/R induced HK-2 cell apoptosisAfter H/R,the posistive apoptosis cell among the groups(F=149.025,P=0.000);PD administraion inhibited apoptosis in vehicle group(9.8±0.4%vs 3.5±0.4%);Comparing with PD group,the apoptotic cell increased in PD/Ex527 group(P=0.000);when PFT-? was added,cell apoptosis was reduced comparing with PD/Ex527 group,which was equal to PD group.Then we determined the expression of apoptosis related protein.The difference among the groups were significantly(Bax:F=67.627,P=0.000;Bcl-2:F=40.551,P=0.000;cytochrome c:F=134.290,P=0.000).We found that pro-apoptosis protein Bax,cytochrome c cytoplasma/mitochondria ratio were significantly increased and anti-apoptosis protein Bcl-2 was reduced;Comparing with vehicle group,the Bax expression and cytochrome c cytoplasma/mitochondria ratio was reduced but Bcl-2 was increased;When Ex527 was added,Bax expression and cytochrome c cytoplasma/mitochondria ratio was again increased but Bcl-2 was significantly decreased;In PD/Ex527/PFT-? group,the pro-apoptosis protein was increased but anti-apoptosis protein was decreased comparing with Ex527/PD group.4.PD attenuate hypoxia/reoxyge induced mitochondrial dysfunction in human proximal tubular epithelial cellThe indexes of mitochondrial function were all differed significantly(Calcein-AM:F=87.36,P=0.000;JC-1:F=268.800,P=0.000;ATP:F=76.620,P=0.000;AO-red:F=152.809,P=0.000).The cailcein fluorescence in vehicle group was decreased by 59.7±2.4%of control group after H/R.In PD group,the decreased flurescence restored to 66.1±4.2%of control group.What's more,the JC-1 monomer/polymer ratio increased to 23.5±1.8 times over control group and ATP levels dropped to 34±5.9%of control group,PD administration reduced the JC-1 monomer/polymer ratio to 2.5±0.5 times of control group and ATP in control group increased to 80±6.5%of control group;As lysosomal stability determination,the AO-red fluorescence intensity in vehicle group dropped to 21.1±2.5%of control group.In PD treatment group,the decreased value in vehicle group increased to 56.3±4.8%of control group.These results suggest that PD can improve the mitochondrial damage after H/R.However,when Ex527 was added in PD/Ex527 group,the protective effect of PD on mitochondria were decreased and the difference was significant.These results indicate that the PD effect is likely related to its activation of SIRT1.While in PD/Ex527/PFT-? group the mitochondrial function was effectively restored than in PD/Ex527 group,indicating that although the effect of PD on SIRT1 was blocked,PFT-? could still reduce apoptosis mediated by mitochondria by inhibiting p53.Conclusion1.Mitochondrial dysfunction exist in renal tubular epithelial cells after severe shock/reperfusion.The decreased SIRT1 protein expression and activity and increased p53 acetylation levels may be the mechanisms of mitochondrial dysfunction in renal tubular epithelial cell.i.e.,severe shock and reperfusion induced reduction of SIRT1 protein and SIRT1 activity,following reduced deacetylation activity.The decreased SIRT1 activity causes acetylated p53 and p53 mitochondrial translocation reduced,leading to increased mitochondrial proapoptotic proteins and decreased antiapoptosis,resulting in increased mitochondrial outer membrane permeabilization.Finally,cytochrome c release into the cytosol and initiate cell apoptosis;2.PD is a SIRT1 agonist,it may be one of the mechanisms to alleviate mitochondrial damage of renal tubular epithelial cells following severe shock and reperfusion.Through SIRT1 upregulation,PD reduce acetylated p53 level,inhibit p53 non-dependent mitochondrial apoptosis pathway;3.PD reduced the apoptosis of HK-2 cells induced by H/R through the up regulation of SIRT1-p53 signal pathway. |
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