Moderate Hypothermia Ameliorates Enterocyte Mitochondrial Dysfunction In Severe Shock And Reperfusion

BackgroundHemorrhagic shock(HS)is the main reason responsible for the death of traumatic patients.Severe acute hemorrhagic shock could cause continuous low perfusion state of tissues and organs,which may further lead to a metabolic disorder of the body's internal environment,and cause multiple organ dysfunction syndrome to be life-threantening.In the progression of HS,the inadequate tissue perfusion may cause hypoxia,which can result in disfluencies in respiratory chain and disorder of mitochondrial energy metabolism.HS will also cause a series of pathophysiological changes,including distrubance of microcirculation,reduced local blood perfusion,hypoxia and metabolic disorders.Recent studies showed that,although diverse reasons could be responsible for the death of HS patients,the mitochondrial dysfuction has found to be of vital importance during the progression of the illness which has been proved based on many clinical evidence;and the extent of Mitochondrial Dysfunction is closely related to the mortality of acute severe hemorrhagic shock patients.Reperfusion and tissue oxygen supply are the main objects after HS.Even though,after the traditional objectives been achieved,patients could be died because of systemic inflammatory response(SIRS)and multiple organ dysfunction syndrome(MODS).When the patient is suffering from a serous shock,stomach and intestines will be contineously in the situation of vasoconstriction and hypoperfusion.Notably,intestines plays a key role in the pathophysiology of HS and intestinal Ischemia/reperfusion(I/R)injury.Although with lot of fluid resuscitation during the therapy,the blood flow perfusion of organs and supply of oxygen and nutrient will be recovered,the lschemia-reperfusion process will lead to a production of reactive oxygen species(ROS),which will increase the damage of mitochondrial function.Temperol severe ischemia-reperfusion injury can destroy the Mitochondrial membrane protein structure,accelerate the opening of the mitochondrial permeability transition pore,breaking the balance inside and outside the film,increase C release to the cytosolic cytochrome,which will speed up the process of cell apoptosis.Whereas,whether or not treated by surgery or fluid resuscitation,further organ damage will always be caused by the Mitochondrial Dysfunction due to the hypoxia during HS,therefore,improve the mitochondria function,reducing oxidative stress during the treatment should be the main objective in curing HS.Intestines is important digestive and absorptive organs for humans,besides,it is powerful in secretion and barrier function.The intestinal barrier function(IBF),as the important innate immune organs,is gaining more and more focus on clinical and basic research.Some literatures claimed that,gut barrier function damage in many diseases may further cause translocation and disorder of bacterial,thus leading to a systemic inflammatory response syndrome.In HS,the necrosis of intestinal epithelial cell(IEC),lint shedding,basement membrane collapse and damage of IBF caused by blood reperfusion will accelerate Intestinal dysbacteriosis,enter the systemic circulation,and eventually lead to the emergence of various inflammatory cytokines and endotoxins release.Therefore,some literatures announced that intestines are the'motor' of MOF caused by various diseases.Operating mild hypothermia therapy in the treatment has been widely reported in experimental and clinical applications,by modest cooling,the low temperature induced could protect the brain,heart m lung and intestines,which can be used to treat I/R injury and other diseases.So far,the mechanism of low temperature treatment is related to the protection of mitochondria,radical restoration and the suppression of immune responses.However,the reasons for the protection of mitochondria is mainly the cardiac effects induced by low temperature,and the mechanism of the low temperature participate in the IR of IEC mitochondria,especially under HS condition,still need to be explained.Besides,there is still controversial of the best target temperature.To explain the above questions,we used a serious HS/reperfusion(HS/R)model,and compared the general survival time of 2 kinds of low temperature treatment(32? and 34?),as well as the mean arterial pressure(MAP),intestinal tissue injury and specially the mitochondria function of IEC.Inflammation is the body's key protection mechanisms,in HS,over reaction of inflammation may cause the imbalance of pro-inflammatory-anti-inflammatory,lead to a cell damage.After the infection stimulus of severe acute hemorrhagic shock,inflammatory mediators released by the internal environment will cause SIRS and pro-inflammatory cytokines like tumor necrosis factor-alpha(TNF-a),interleukin-1(IL-1),IL-6,IL-8,etc.Meanwhile,endogenous anti-inflammatory media such as IL-4,IL-10,IL-11,IL-13 will also be released,lead to compensatory anti-inflammatory response syndrome(CARS)which can result in immune dysfunction.Hypothermia can mediate each part of the inflammatory immune response,reduce the inflammatory mediators level such as ROS,RNS and multiple other rough inflammatory factors.And through the inhibition of the SCNT of transcription factor NF-kB(nuclear factor-?B,NF-kB),and further inhibit the expression of variety of lower proteins.Therefore,low temperature has a clearly inhibitory effect on inflammatory,this could also be the possible mechanism of effectiveness of hypothermia in many treatments of trauma.In the internal environment,low temperature directly affect metabolism from a variety of levels,thus effectively protecting the physiological function of tissues and organs,and promote the tissue regeneration and repair.In clinical treatment,low temperature combined with drug therapy has a broad application prospects.Nevertheless,although the protective effect of mild hypothermia has been demonstrated by a number of animal experiments,there are still many limitations for its clinical application,such as the method to select the most effective temperature of the mild hypothermia for different disease models,the way to avoid adverse reactions,the suitable time for the mild hypothermia to be involved and to evaluate the effect of mild hypothermia combined with drug treatment and so on.Although remained being controversial,but mild hypothermia treatment has been confirmed in many animal experiments,expect for the report of recovery of intestinal function after HS.To this end,we make some assumptions:(1)mild hypothermia combined with limited fluid resuscitation could reduce intestinal injury after HS and reperfusion.(2)Effects of the mild hypothermia treatment after HS is related to the mitochondria protection function.Materials and methods1 hemorrhagic shock and reperfusion injury model reproduction and injuries of mitochondria in intestinal epithelial cellIn total,42 ratswere anesthetized by intraperitoneal injection of 3%pentobarbital sodium(65 mg/kg body weight),and 0.2 mL anesthetic was added when needed.The left femoral artery was cannulated to monitor the MAP.The right femoral artery was cannulated for exsanguination;the right femoral vein was cannulated for drug administration and fluid resuscitation.After establishing arterial and venous passage,the MAP was recorded using PowerLab equipment until the animals were sacrificed or 10 h after autologous blood reinfusion.After a 30-min resting stage,the rats were bled using a syringe to produce 40 ± 5 mmHg MAP within 10 min,which was maintained for the next 2 h by blood drawing or the reinfusion of stored blood.Subsequently,the shed blood was intravenously administered within 10 min.After autologous blood reinfusion,fluid resuscitation(Ringer's solution)was performed at 15 mL/kg/h and maintained for 2 h.1.1 Observation of MAP and survival time analysis.When the fluid resuscitation was completed,24 rats(n = 6 per group)were sacrificed for mitochondrial function determination,oxidative stress tests,and analysis of apoptosis.The remaining 20 rats(n = 10 per group)were used for observation of MAP and survival time analysis.1.2 Preparation of small intestinal tissue and serum samplesLaparotomy was performed:10 cm of ileum 10 cm distal to the ligament of Treitz was carefully removed,placed on ice,rinsed thoroughly with normal saline,refilled with 10 mM dithiothreitol in enterocyte isolation buffer(17 mM HEPES,25 mM NaHCO3 in phosphate-buffered saline,pH 7.4),and tied off at both ends.In addition,2 mL arterial blood was collected and centrifuged at 2000 × g for 10 min,and the supernatant serum was stored at-20? for assessing the inflammatory cytokines.1.3 Hematoxylin-eosin(HE)tissue staining The pathogenesis of small intestinal mucosal injury was observed under a microscope and evaluated using the Chiu scoring system(w = 6 per group).Ten fields were observed for each sample;the average score was recorded as the histopathology score of the small intestinal tissue.1.4 Measurement of mitochondrial membrane potential and cellular adenosine triphosphate(ATP)Data were analyzed using FlowJo 7.6(Flowjo.LLC,Ashland,OR).The results are expressed as the ratio of green to red(monomers/aggregates)fluorescence(n = 6 per group).Partial enterocyte ATP content was measured using the CellTiter-Glo luciferase-based assay.The plates were incubated at room temperature for 10 min and the luminescence was recorded in an automated microplate reader(n = 6 per group).1.5 Western blottingAfter extensive washing,the secondary antibody was added,and immunocomplexes were detected using an enhanced chemiluminescence western blotting detection kit.The relative densities of the protein bands were analyzed using ImageJ software.Data from at least three independent experiments are presented in relative units as compared to the sham group.1.6 Statistical analysisData were analyzed by SPSS 20.0,results are expressed as the mean ± standard deviation.Statistical analysis was performed by one-way analysis of variance followed by Tukey's multiple comparisons test.P<0.05 was considered to indicate statistical significance.2.Moderate hypothermia ameliorates enterocyte mitochondrial dysfunction in severe shock and reperfusion88 ratswere anesthetized by intraperitoneal injection of 3%pentobarbital sodium(65 mg/kg body weight),and 0.2 mL anesthetic was added when needed.The left femoral artery was cannulated to monitor the MAP.The right femoral artery was cannulated for exsanguination;the right femoral vein was cannulated for drug administration and fluid resuscitation.After establishing arterial and venous passage,the MAP was recorded using PowerLab equipment until the animals were sacrificed or 10 h after autologous blood reinfusion.After a 30-min resting stage,the rats were bled using a syringe to produce 40 ± 5 mmHg MAP within 10 min,which was maintained for the next 2 h by blood drawing or the reinfusion of stored blood.Subsequently,the shed blood was intravenously administered within 10 min.After autologous blood reinfusion,fluid resuscitation(Ringer's solution)was performed at 15 mL/kg/h and maintained for 2 h.The animals were randomly divided into the following groups using different control temperatures(using ice packs on the abdomen or a thermal mattress)in the 2-h resuscitation periods:(1)control(sham);(2)320C resuscitation;(3)34? resuscitation;(4)37? resuscitation.The temperature of the animals in each group was monitored rectally.When the fluid resuscitation was completed,24 rats(n=6 per group)were sacrificed for mitochondrial function determination,oxidative stress tests,and analysis of apoptosis.The remaining 64 rats(n = 16 per group)were used for observation of MAP and survival time analysis.2.1 Analysis of survival timeWe used 64 rats(n = 16 per group)to study survival time.The rats were sutured and returned to their cages to observe their survival time.All animals had ad libitum access to food and water.Apnea lasting>1 min was considered to indicate death.The remaining animals that survived>72 h were euthanized by cervical dislocation.2.2 Preparation of small intestinal tissue and serum samplesLaparotomy was performed:10 cm of ileum 10 cm distal to the ligament of Treitz was carefully removed,placed on ice,rinsed thoroughly with normal saline,refilled with 10 mM dithiothreitol in enterocyte isolation buffer(17 mM HEPES,25 mM NaHCO3 in phosphate-buffered saline,pH 7.4),and tied off at both ends.In addition,2 mL arterial blood was collected and centrifuged at 2000 × g for 10 min,and the supernatant serum was stored at-20? for assessing the inflammatory cytokines.2.3 Hematoxylin-eosin(HE)tissue stainingThe pathogenesis of small intestinal mucosal injury was observed under a microscope and evaluated using the Chiu scoring system(n = 6 per group).Ten fields were observed for each sample;the average score was recorded as the histopathology score of the small intestinal tissue.2.4 Measurement of mitochondrial membrane potential and cellular adenosine triphosphate(ATP)Data were analyzed using FlowJo 7.6(Flowjo.LLC,Ashland,OR).The results are expressed as the ratio of green to red(monomers/aggregates)fluorescence(n = 6 per group).Partial enterocyte ATP content was measured using the CellTiter-Glo luciferase-based assay.The plates were incubated at room temperature for 10 min and the luminescence was recorded in an automated microplate reader(n = 6 per group).2.5 Determination of mPTP and oxidative damageScraped enterocytes were incubated,protected from light,at room temperature for 15 min in DMEM containing 1 ?M calcein-AM and 2 mM CoCl2 The cells were then quantitatively measured and analyzed by flow cytometry(n = 6 per group).The mitochondrial GSH,GSSG,and MDA content and serum MPO activity were measured according to the manufacturer's instructions(w = 6 per group).2.6 TUNEL assayIntestinal tissue sections(4-?m thick)were labeled and detected using a DeadEndTM Fluorometric TUNEL System to evaluate cell death.The apoptotic indices in five non-overlapping fields per section were averaged(n=6 per group).2.7 Western blottingAfter extensive washing,the secondary antibody was added,and immunocomplexes were detected using an enhanced chemiluminescence western blotting detection kit.The relative densities of the protein bands were analyzed using ImageJ software.Data from at least three independent experiments are presented in relative units as compared to the sham group.2.8 Statistical analysisData were analyzed using GraphPad Prism 6.04(GraphPad Software,Inc.,La Jolla,CA).The survival curve trend was analyzed and compared using the log-rank test.Other results are expressed as the mean ± standard deviation;statistical analysis was performed by one-way analysis of variance followed by Tukey's multiple comparisons test.P<0.05 was considered to indicate statistical significance.Results1 Mitochondria dysfunction and significantly reduced survival time after Hemorrhagic shock1.1 Effects of hypothermia on MAP and survival timeTo verify the effect of induced hypothermia on shock-induced intestinal injury,we evaluated the general condition of the rats,i.e.,MAP and survival time.The control group(normal group),10 rats in 24h,36h and 48h at three time points were survival status,the survival rate was 100%.Rats HS/R are processed at different times of death.Among them,24 hours before the death of six rats,eight rats within 48 hours of death,the survival rate is divided into two time points of 40%and 20%,with an average survival time of 72h observation period was 34.00 ± 5.18h,and the survival curves were significantly different(P<0.01).1.2 Changes in rat intestinal epithelial cell mitochondriaTo explore the possible mechanism of hypothermia on intestinal cell injury,we first measured mitochondrial function,namely mitochondrial membrane potential,cellular ATP,and mPTP.The increased JC-1 aggregates/monomers in the H/S group indicated sharply decreased mitochondrial membrane potential.In contrast,JC-1 aggregates/monomers were reduced significantly(t=9.13,p=0.00).In addition,the enterocyte ATP content in the H/S group was reduced as compared to the sham group(t=6.27,p=0.00).A similar result was observed in the mPTP measurement,where the calcein fluorescence was reduced by 29%as compared to the sham group(t=16.63,p=0.00).2 Moderate hypothermia ameliorates enterocyte mitochondrial dysfunction in severe shock and reperfusion2.1 Effects of hypothermia on enterocyte mitochondrial functionTo explore the possible mechanism of hypothermia on intestinal cell injury,we first measured mitochondrial function,namely mitochondrial membrane potential,cellular ATP,and mPTP.The increased JC-1 monomers/aggregates in the 37? group indicated sharply decreased mitochondrial membrane potential.In contrast,JC-1 monomers/aggregates were partially restored in both 32? and 34? hypothermia treatment groups(all,P<0.01,Fig.1A).Comparing the JC-1 monomer/aggregate ratios of the two treatments,32? hypothermia treatment had better therapeutic effects.In addition,the enterocyte ATP content in the 37? group was reduced by 48%as compared to the sham group,and was restored to 59%and 76%,as compared to the sham group,in the 34?and 32? groups,respectively(P<0.05 and P<0.01,respectively).A similar result was observed in the mPTP measurement,where the calcein fluorescence was reduced by 48%as compared to the sham group,and was restored to 67%and 79%,as compared to the sham group,in the 34 ? and 32 ? groups,respectively P = 0.09 and P<0.01,respectively).2.2.Effects of hypothermia on oxidative stressAn array of cellular pathways participates in the genesis of anoxic injury;hypothermia may interrupt one or more of them,preventing free radical injury and membrane damage.Therefore,we measured the mitochondrial antioxidative enzyme content,i.e.,GSH,GSSG,and MDA.In the small intestinal tissue homogenate in the 37? group,GSH was decreased,GSSG was increased,and the GSH/GSSG ratio was decreased;MDA was increased.The induced hypothermia treatments partially repressed the increase in pro-oxidative enzymes and restored the decreased antioxidative enzymes,especially in the 32? group,in which antioxidative enzyme activity was effectively restored.2.3.Effects of hypothermia on intestinal pathological changes and apoptosisThe intestinal pathological changes were observed via HE staining.There was severe intestinal injury in the 37? group,as evidenced by epithelial sloughing,lamina exposure,and hemorrhage on denuded villi.As expected,there was only extended subepithelial spaces and focal epithelial disruption in the 32? group;the pathological change was slightly worse in the 34? group.In agreement with the histological observation,the Chiu score was significantly lower in the 32? group as compared with the 37? and 34? groups(P<0.01 and P<0.05,respectively).We analyzed the level of apoptosis and calculated the apoptotic indices.Remarkably,there were increased apoptotic small intestinal cells in the 37? group,which,following comparison,was decreased by 48.2%in the 32? group(P<0.01).Similarly,there were decreased apoptotic cells in the 34? group;however,the difference between the 32? and 34? groups was not significant.2.4.Effects of hypothermia on systemic inflammatory responsePrevious studies have implicated polymorphonuclear neutrophils as major inflammatory cells in I/R injury,and increased MPO is an index of damaged villi after reperfusion injury.Herein,we determined the MPO concentration of small intestinal tissue,finding that MPO levels were significantly increased in rats in the 37? group.In contrast,induced hypothermia treatment partially decreased the elevated MPO(all,P<0.01),and MPO in the 32? group was decreased by 40%of the 34? group(P = 0.01).Based on the report that hypothermia modulates the downstream inflammatory pathways that cause delayed cell death,we measured the serum levels of the proinflammatory cytokines TNF-a,IL-1?,and IL-6.There was an apparent increase in the proinflammatory cytokine levels after 2 h of reperfusion,which the 32?induced hypothermia treatment reduced effectively(all,P<0.01).Notably,there was no significant inflammatory response suppression in the 34? group.2.5.Effects of hypothermia on MAP and survival timeTo verify the effect of induced hypothermia on shock-induced intestinal injury,we evaluated the general condition of the rats,i.e.,MAP and survival time.As expected,induced hypothermia,especially 32?,increased the MAP at 2 h,4 h,and 10 h after reperfusion.Furthermore,the induced hypothermia significantly prolonged survival time,as evidenced by the increased survival rate(320C vs.340C vs.37?=12/16 vs.10/16 vs.8/16),and the survival curves were significantly different(P<0.01).2.6 Effects of hypothermia in CytCIt is widely acknowledged that the release of cytochrome c from the mitochondrial inner membrane into the cytosol is the initial signal of the intrinsic apoptotic pathway.Therefore,we analyzed the cytoplasmic and mitochondrial cytochrome c content via western blotting.The induced hypothermia treatments partially restored mitochondrial cytochrome c(mito-cytc)content and decreased cytoplasmic cytochrome c(cyto-cytc)content;32? treatment had superior therapeutic effects as compared to the 34? treatment evidencing by 14%increased mitochondrial cytochrome c content(P = 0.16)and 14%decreased cytoplasmic cytochrome c content(P<0.05).Conclusion1.Induced hypothermia,especially moderate hypothermia,ameliorates gut injury induced by hemorrhagic shock and reperfusion.2.The mechanism of the hypothermia effect is associated with enterocyte mitochondrial protection,oxidative stress injury reduction,and the inhibition of apoptosis and the systemic inflammatory response.