| Severe or irreversible shock is the late stage of shock with microcirculatory disturbance,persistent or refractory hypotension and multiple organ dysfunction,which is a major cause of morbidity and mortality in the world.Therefore,it is of great importance to find out the mechanism of high mortality in severe shock and to find an effective and efficient way to treat severe shock.Our previous studies have demonstrated that mitochondrial dysfunction is a common phenomenon among diverse organs,which is involved in the genesis of refractory period in severe shock and results in depression of contractile vasoresponsiveness and persistent hypotension.Mitochondrial protectors such as polydatin(PD),resveratrol(Res)and Cyclosporin A(CsA)could protect against mitochondrial injury and improve vasoresponses and mean blood pressure(MAP),and thereby prolong survival in a rat model of hemorrhagic shock.Thus,administration of mitochondrial protector might be a new approach to treatment of severe shock.Mitochondrial permeability transition pore(mPTP)is a multi-component protein aggregate in mitochondria.Molecular structure of the mPTP remains unclear and different models have been proposed.A classic model of the mPTP consists of a dimer of voltage-dependent anion channel(VDAC)in the mitochondrial outer membrane and a dimer of adenine nucleotide translocase(ANT)in the mitochondrial inner membrane forming a channel,as well as a matrix cyclophilin D(CypD),the target of CsA,facilitates mPTP opening.Accumulating evidence indicates that persistent or irreversible opening of mPTP is critical in regulation of mitochondrial functions.CypD is a prominent mediator of the mPTP,and CypD acetylation increases its binding to ANT and facilitates the opening of mPTP.Silent information regulator 2(Sir2)is a class of nicotinamide adenine dinucleotide(NAD)-dependent protein deacetylase.Mammals possess seven sirtuins(SIRTI-7)that occupy different subcellular compartments.In the last few years,SIRT1 and SIRT3 are emerging as crucial regulators of mitochondrial protein acetylation and mitochondrial biogenesis.SIRT1 is the closest mammalian homologue of yeast Sir2.It was found that activation of SIRT1 stimulates mitochondrial biogenesis and energe metabolism,induces resistance to oxidative stress.SIRT3 is a major mitochondrial deacetylase,plays a pivotal role in the regulation of mitochondrial function.Res is commonly known as a SIRT1 activator and our previous studies have demonstrated that Res could significantly inhibit the opening of mPTP and improve the depressed vasoreactivity following severe shock.However,how SIRT1 regulates mitochondrial function in arterial smooth muscle cells(ASMCs)following severe shock remained unclear,and no reports are available now in terms of the regulation of SIRT1 and SIRT3 in mitochondria function and vasoresponse in severe shock.Based on the above studies,we propose that SIRT1 and SIRT3 could regulate the opening of mPTP through deacetylation of CypD,and thereby protect against mitochondrial dysfunction by inhibiting the opening of mPTP and improve the vasoresponse in severe shock.Therefore,based on the above hypothesis,we used an in vivo rat model of acute severe hemorrhagic shock and an in vitro model of hypoxia-reoxygenation injury in human ASMCs,to ascertain the mechanisms involved in the activation of SIRT1/3 deacetylases modulates vasoreactivity and refractory hypotension through amelioration of mPTP and mitochondrial injury in ASMCs.The dissertation mainly consists of three parts:Part1:Deacetylase SIRT1 was involved in the regulation of mitochondrialdysfunction in severe shock.Part2:Mechanisms underlying the regulation of mPTP in ASMCs by SIRT1.Part3:Activation of SIRT1 protected against mitochondrial injury and improved the vasoresponse in severe shock.Part one:SIRT1 deacetylase was involved in the regulation of mitochondrial dysfunction in acute severe shockObjective:To determine the changes of protein expression and activity of SIRT1/3 following severe shock,and to ascertain the association between SIRT1/3 alterations and mitochondrial dysfunction in severe shock.Methods:1.Rats were randomly divided into six groups,namely sham group,shock 60 min group,shock 120 min group,reinfusion 10 min group,reinfusion 60 min and reinfusion 120 min group.A rat model of severe hemorrhagic shock was reproduced and mesenteric arterioles were collected.2.Western blot was used to determine the protein expression of SIRT1 and SIRT3 at different groups following severe shock.CycLex SIRT1/sir2 and SIRT3 Deacetylase Fluorometric Assay Kit were used to measure SIRT1 and SIRT3 activity following severe shock,respectively.The location of SIRT1 and SIRT3,acetylation of CypD in ASMCs at 120 min postreinfusion were detected by immunofluorescence staining.3.To further determine the relationship between SIRT1/3 changes and mitochondrial injury in severe shock.Mitochondrial dysfunction in arterioles at different time point was detected by Transmission electron microscopy(TEM)and flow cytometry.Intracellular ATP content was measured by CellTiter-Glo Luminescent Cell Viability kit to evaluate mitochondrial function.4.Statistical analysis:All variables are presented as means±SD(?±s).Differences between groups were determined using the one-way ANOVA with LSD test(Dunnett's test for unequal sample size was employed when appropriate).Values were considered significant when P<0.05.Results:1.The activity of SIRT1 and SIRT3 in differernt groups showed great statistical significance(F=6.100,P=0.001;F=5.377,P=0.002).The activity of SIRT1 and SIRT3 progressively decreased at the beginning of shock 60 min,Western blot analysis showed that the protein expression of SIRT1 and SIRT3 had great difference among groups(F=3.846 and 2.848,P=0.015 and 0.046).Compared with sham group,the protein expression of SIRT1 and SIRT3 were reduced by 42.25±0.06%(P=0.002)and 40.67±4.08%(P=0.002)at 120 min postreinfusion,respectively.Immunofluorescence staining of ASMCs showed the increased acetylation of CypD and decreased expression of SIRT1/3 at 120 min postreinfusion in severe shock,but no translocation of SIRT1/3 was obviously observed.2.The mitochondrial ulstructure in ASMCs was injuried at the beginning of shock 120min.Flameng,scales indicated great significance among different time points in severe shock(F=77.189,P<0.001).The mitochondria of ASMCs in sham group appeared sausage or round-shape with normal cristae.From the time point of shock 120 min,some mitochondria swelled,crista lightly ruptured.After reinfusion,most mitochondria were irregularly shaped,apparently swollen with ruptured and poorly defined cristae even vacuolization.3.The ROS level increased from the time point of shock 120 min.The intracellular ROS level at differernt timepoint showed great statistical significance(F=34.013,P<0.001).Compared with sham group,the intracellular ROS level increased by 48.12± 10.13%(P=0.035)at shock 120 min,88.04 ± 18.67%at 10 min postreinfusion(P=0.005),120.91 ±26.29%(P=0.001)at 60 min postreinfusion and 152.71±24.20%at 120 min postreinfusion(P<0.001).4.The mitochondrial permeability transition pores persistently opend at the 10 min postreinfusion.The mean fluorescence indensity(MFI)of calcein detected at different timepoint showed great statistical significance(F=6.946,P<0.001).Compared with the sham group,the mean fluorescence indensity(MFI)of calcein was decreased by 29.94±9.12%(10 min postreinfusion,P=0.001),23.1518.53%(60 min postreinfusion,P=0.011)and 45.29±7.16%(120 min postreinfusion,P<0.001),respectively.5.The mitochondrial membrane potential was decreased at the beginning of shock 120 min.The cells with a depolarized membrane potential in different groups showed great statistical s:ignificance(F=10.667,P<0.001),The intensity of green fluorescence emitted by JC-1 monomer increased from the time point of shock 120 min.Compared with the sham group,the cells with depolarized ??m increased by 62.35±22.65%at shock 120 min(P=0.005),88.34±45.64%at 10 min postreinfusion(P<0.001),66.84±28.14%at 60 min postreinfusion(P=0.003)and 133.56±15.89%at 120 min postreinfusion(P<0.001),respectively.6.The intracellular ATP content decreased progressively at the beginning of shock 120 min.The ATP content in different groups showed great statistical significance(F=17.392,P<0.001).Compared with sham group,the intracellular ATP level decreased by 34.37±10.59%(shock 120 min),26.52±4.57%(10 min postreinfusion),31.47±2.39%(60 min postreinfusion,)and 49.30±5.36%(120 min postreinfusion),respectively.Part two:The underlying mechanism involved in the opening of mitochondrial permeability transition pore regulated by SIRT1Objective:To determine the possible mechanism by which SIRT1 regulated the opening of mPTP in ASMCs following severe shock,an in vitro model of hypoxia and reoxygenation in human ASMCs was performed.Methods:1.An in vitro model of hypoxia-reoxygenation injury in human ASMCs was performed.Western blot and co-immunoprecipitation(CoIP)techniques were used to determine the protein expression of SIRT1/3 and CypD acetylation.2.To explore the effect of SIRT1 on CypD acetylation in human ASMCs exposed to hypoxia and reoxygenation,Lentivirus that express SIRT1 was constructed to infect human ASMCs.Based on this,Knockdown of SIRT3 by siRNA interference was next used to determine the involvement of SIRT3 in the regulation of CypD acetylation mediated by SIRT1 during hypoxia and reoxygenation.3.In order to further explore whether PGC1? was involved in the regulation of SIRT3 by SIRT1 in human ASMCs submitted to hypoxia and reoxygenation,we knockdowned PGC1? by siRNA interference,and then measured SIRT3 activity according to the kit described in Part one.4.Statistical analysis:The data was analysed by SPSS 13.0 software as described in Part one.Results:1.GFP expression showed high lentiviral transfection efficiency up to 90%,Lentivirus-mediated overexpression of SIRT1 in human ASMCs detected by Western blot was nearly 4.8 times that of control(P<0.001).2.SIRT3 mediated SIRT1-downregulated acetylation of mitochondrial CypD in human ASMCs exposed to hypoxia and reoxygenation.Compared with control,acetylation of mitochondrial CypD significantly increased in human ASMCs exposed to hypoxia and reoxygenation.Overexpression of SIRT1 suppressed acetylation of CypD.Overexpression of SIRT1 combined with knockdown of SIRT3 prevented the decrease of CypD acetylation induced by SIRT1 during hypoxia and reoxygenation.3.PGC-la was involved in the regulation of SIRTI on SIRT3 activity in human ASMCs exposed to hypoxia and reoxygenation.SIRT3 deacetylase activity was significantly reduced by 46.34±7.50%(n=6,P<0.001)in ASMCs exposed to hypoxia and reoxygenation,overexression of SIRT1 partly restored SIRT3 activity by 36.11 ±9.40%(n=6,P<0.001),Overexpression of SIRT1 combined with knockdown of PGC-1? prevented the restoration of SIRT3 activity.Compared with SIRT1 overexpression group,SIRT3 activity decreased by 19.56±6.08%(n=6,P=0.023).Part Three:Activation of SIRT1 protected against mitochondrial injury and improved the vasoresponse in severe shockObjective:To explore the effect of SIRT1 activator SRT1720,resveratrol and polydatin on mitochondrial structure and function,vasoreactivity,MAP and survival of rats with hemorrhagic shock.Methods:1.Rats were randomly divided into 8 groups:sham group,vehicle control(shock)group,Resveratrol(Res)group,SRT1720(SRT)group,polydatin(PD)group,Ex527+Res group,Ex527+SRT group,Ex527+PD group.A sham operated group without bleeding.The Res,SRT and PD group,in which rats were subjected to hemorrhage to maintain MAP at 30 mmHg for 120 min,followed by administration of Res,SRT or PD,respectively,and then reinfusion of the shed blood with an observation period of 120 min posttreatment.The rats in shock group were administered with same volume of saline vehicle instead of the reagents.In Ex527 pretreatment group,Ex527 was administered via femoral vein 1 h before bleeding,then hemorrhagic shock model was reproduced.For survival analysis,at the end of observation period,catheters were removed and skin wounds were sutured.Survival time was recorded.2.To determine vascular reactivity,the spinotrapezius muscle of rat was isolated and prepared for intravital microscopy using the technique described in our lab.The arteriolar reactivity to norepinephrine(NE)was measured by topical application of increasing concentration of NE until a threshold concentration was reached resulting in complete cessation of blood flow in the transverse arteriole for 10-20 s.Throughout the surgical preparation and experimental procedures,the exposed muscle was continuously superfused with balanced Kreb's solution to maintain stable temperature,humidity and pH.3.To further explore the effect of SIRT1 activators on SIRT1/3 activity and mitochondrial injury in severe shock.SIRT1 and SIRT3 activity were determined according to the methods described in part one.Mitochondrial ultrastructure and function were determined in ASMCs freshly isolated at 120 min post reinfusion,according to the methods described in Part One.4.Statistical analysis:All variables were presented as means±SD.Differences between groups were determined using the one-way ANOVA with LSD test(Dunnett's test for unequal sample size was employed when appropriate).Values were considered significant when P<0.05.The MAP and NE threshold were analysed by Repeated Measures and Multivariate analysis,Survival was analysed by Kaplan-Meier mehod and Log-rank test.P<0.05 was conidered significance.Results:1.SIRT1 activators significantly attenuated the decrease of SIRT1 and SIRT3 acvitity following severe shock.Pretreatment with Ex527 attenuated the activation effect of three reagents(F=25.142 and 16.669,P<0.001).Compared with the shock group,SIRT1 and SIRT3 activity was increased by 38.24±5.10%and 48.21±15.56%in Res group,increased by 36.77±11.22%and 34.16±14.52%in SRT1720 group,increased by 33.89±16.56%and 47.36±26.49%in PD group(P<0.001).2.SIRT1 activation protected ASMCs against mitochondrial ultrastructure injury.The Flameng's scale in different groups showed great statistical significance(F=86.135,P<0.001).Compared with the shock group,the structure of mitochondria restored in different degree after treatment with these three protective reagents.The mitochondrial injury was scored by the Flameng,scale,which showed that in PD,SRT1720 and Res group,the scale decreased by 64.30±3.72%,57.78±2.89%and 54.18±8.46%(P<0.001),respectively.3.SIRT1 activation improved the intracellular ATP content.Ex527 supressed the protective effects of three reagents(F=38.757,P<0.001).Compared with the ATP level in shock group,treatment with SRT1720,Res and PD significantly increased the ATP level by 73.60±32.30%,64.91±22.74%and 80.90±23.04%.Pretreatment with Ex527 prevented the elevation of intracellular ATP content induced by the three reagents(F=38.757,P<0.001).Compared with the three activators,the ATP content in Ex527+Res,Ex527+SRT1720 and Ex527+PD group significantly decreased by 32.76±12.02%(P=0.019),33.38±16.01%(P=0.036)and 42.86±7.88%(P<0.001).4.SIRT1 activation greatly inhibited the opening of mPTP in severe shock,Ex527 prevented the protective effect of three reagents(F=25.318,P<0.001).SIRT1 activator SRT1720,Res and PD increased the mean fluorescence idensity of calcein by 54.00±20.44%,62.86119.21%and 68.40±20.79%(P<0.001),relative to that in shock group;Compared with that in activator groups,pretreatment with Ex527 also attenuated the increase of calcein by 40.83±8.46%,30.79±22.41%and 30.40±5.39%(P<0.001),respectively.5.SIRT1 activation inhibited the loss of mitochondrial transmembrane potential(??m)in ASMCs following severe shock(F=28.759,P<0.001).ASMCs with low??m decreased by 62.13±5.73%in SRT1720 group,59.32±4.66%in Res group and 68.45±5.73%in PD group,respectively(P<0.001).Similarly,Ex527 pretreatment prevented the decrease(P<0.001).Compared with that in the three activator group,the ASMCs with low ??m increased by 78.66±19.27%in Ex527+Res group,50.55±7.80%in Ex527+SRT1720 group and 83.66±9.14%in Ex527+PD group,respectively.6.SIRT1 activation improved vasoreactivity and MAP of rats in severe shock.Significant difference existed in different treatment groups and different timepoint(F=154.320 and 713.811,P<0.001).The results showed that the NE threshold concentration at 120 min post reinfusion in shock group increased to 22.7 times of that in the prebleeding period,and MAP decreased to 39.5±12.43 mmHg in the shock group(P=0.004 and 0.001).In the SRT1720,Res and PD groups,the NE threshold concentration increased to 5.68,8.72 and 3.6 times of the prehemorrhagic level(P=0.008,0.025 and 0.006)and MAP increased to 83.45 ±11.74mmHg(P=0.002),71.40±6.30mmHg(P=0.012)and 87.98±8.48mmHg(P=0.001),respectively.Ex527 pretreatment significantly prevented the elevation of MAP mediated by the activators(P<0.001).The MAP in EX527+Res,Ex527+SRT1720 and Ex527+PD groups decreased to 37.90±5.47mmHg,45.72±18.16 mmHg(P=0.043)and 53.13±9.05 mmHg(P=0.001),respectively.7.SIRT1 activation prolonged the survival of rats with hemorrhagic shock.The median survival times in different groups showed great significant difference(Log Rank:?2=69.925,P<0.001).The median survival time was only 7 h and 100%animal died within 24 h after reinfusion of shed blood in the shock group.The median survival time in the SRT1720 and Res groups were 14 h and 13.5 h,respectively,which was significantly prolonged than that in shock group(P<0.001).In the PD group,the median survival time was 26 h,which was significantly prolonged to 3.25 times that of shock group.However,the median survival time in Ex527 pretreatment groups was greatly shortened than that in the activator groups,which was 7.5 h,6 h and 10 h in Ex527+Res group,Ex527+SRT group and Ex527+PD group,respectively.Conclusions1.Both the activity of SIRT1 and SIRT3 in ASMCs were progressively decreased following severe shock,which contributed to the opening of mPTP,and thereby lead to mitochondrial dysfunction.2.Hypoxia and reoxygenation suppressed protein expression and activity of SIRT3,concomitantly increased acetylation of CypD in ASMCs.Overexpression of SIRT1 restored SIRT3 activity via PGCla and prevented hyperacetylation of CypD.Thus,it was further demonstrated that SIRT1/3 mediated CypD acetylation in ASMCs exposed to hypoxia and reoxygenation,suggesting SIRT1-PGCla-SIRT3-CypD signal pathway was involved in the regulation of mPTP opening.3.Activation of SIRT1 by SRT1720,Res and PD protected against low vasoreactivity and refractory hypotension by preventing mitochondrial injury in severe shock,which was characterized with decreased mPTP opening,restoration of mitochondrial membrane potential,and increased intracellular ATP content.SIRT1 activation maybe an important target to prevent mitochondrial dysfunction and a new approach to treatment of severe shock.In summary,the present study demonstrated,by in vivo animal experiment ana in vitro cen cuitivation experiment,that the activity of SIRT1 i and SIRT3 decreased in severe hemorrhagic shock,which caused an increase in the aceylation and binding of CypD to ANT,promoted the opening of mPTP,thereby contributing to mitochondrial injury and dysfunction in ASMCs.Activation of SIRT1 could protect against mitochondrial injury by suppressing the opening of mPTP. |
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