| StudybackgroundHemorrhagic shock (HS) refers to more than 25%-25% of total blood volume blood loss in a short time, beyond the ability of the body compensatory,induces cardiac output and mean arterial pressure decline and shock rise. More than 45%-50%blood loss often leads rapid death.About 90000 people diefrom accidental injury in United States each year, and 66-80% of those deathsoccur from uncontrollable blood loss. Hemorrhagic shock is a systemic pathological process ofischemia and anoxic damageinduced by sharp circulating blood volume decline,acute microcirculation block, and shortage of tissueand organs perfusion. The development process of HS is roughly divided into shock compensatory period, reversible decompensation period, irreversible decompensation period. In severe refractory shock, namely irreversible decompensation period,blood pressure of patientsis still difficult to rebound, even further deteriorationand poor prognosis, though timely blood transfusion rehydration and drugs of shrink blood vessels. Patients withsevere refractory shock mainly manifest progressive drop in blood pressure, no reflow of blood capillary, a deepening of microcirculationcongestionandoccurrence of DIC.Now,refractory shock,in addition to relate with DIC,also concerns with disturbance of barrier and immune function and invading ofgerm and endotoxin into blood induced byintestinal ischemia and anoxia.Rapidblood transfusion rehydrationisprimary treatment of anti-shock, but consequent ischemia/reperfusion (I/R) injuryis a serious hit for severe shock patients once again, which not only can’t restore function of tissues and organs, also aggravate dysfunction and structural damage.Now, the phenomenon of I/R injury hasbeen confirmed in a variety of tissues and organs, bowel is not only the target organ of damage but " initating organ"of systemic inflammatory response Syndrome (SIRS). Destruction ofintestinal mucosa barrier can causetranslocation ofgerm and endotoxin, causing inflammation and damage of remote organs, eventually leading to Multiple Organ Dysfunction Syndrome (Multiple outraged Dysfunction Syndrome MODS). Therefore,intestinal protection ofreperfusion after hemorrhagic shock is important for treatment and prognosis of shock patient.With the progress of the mitochondrial research andmature of technology, a large number of studies have shown that mitochondrial dysfunction isone of the important reasons of multiple organs failureinduced bysevererefractory shock.Mitochondria are body’s energy sources and participate in the body’s normalregulation of physiological function. Energy decrease intissues and cells is not caused by anoxia due to a lack of blood perfusion and nutrients, but by ATP deficiencydue to mitochondria damage itself, namely "cell diseased anoxia, mitochondrial dysfunction is the essence of "cell diseased hypoxia".Our previous studies have shown that mitochondrial dysfunction isone of the important reasons of low vascular reactivity and refractory hypotensioninduced by severe shock, and participate in damage of kidney, neurons, vascular smooth muscle cells and liver cells in severe hemorrhagic shock. So, whether are intestinal injury caused by severe hemorrhagic shock related to mitochondrial dysfunction in intestinal cells?A wide range of mitochondrial dysfunction caused multiple organs dysfunction. Some studies have confirmed thatmitochondrialprotection could amelioratemultiple organs damage induced byhemorrhagic shock, such as mitochondria protectant resveratrol (RSV) and polydatin (PD).Treatment targetedat mitochondrial protection for severe shock is a novel strategy, as well as main study of our team.Polydatin(PD),an anti-shock drug in our laboratory, has proved to improve microcirculation and increasing the supply of nutrients, and reduce the oxygen free radical synthetic protection of mitochondrial function.Reperfusion injury of hemorrhagic shock in rats,the mitochondrial protective function of PD in artery smooth muscle cells and nerve cells is better than resveratrol (RSV), the internationally recognized mitochondrial protectant.PD is the fourth monomer extracted from rhizome of giant knotweed, a Chinese herb. Chemical structure is three, four, five-3 hydroxy3. Single. D. glucoside, and it is the combination of resveratrol and glucose, also called resveratrol glucoside, both belong to stilbene compounds of giant knotweed. Compared with RES, PD has faster absorption, most abundant content, higherintake of oxygen free radical removal efficiency. Study found thatPD could inhibit inflammation, improve microcirculation,protectmitochondrial function.After thirty years of efforts, PD has won the Chinese national class I drug clinical trial (2006L100301) approval documents and national food administration approval documents for the treatment of patients with burns and shock and entered phase II clinical trials. PD also gradually becomea widely recognized mitochondrial protectant, but underlying mechanism is not still clear.In theregulatory mechanism of oxidative stress, SIRT3 (namely deacetylation enzyme 3 of sirtuins family) deacetylates related targeted proteins in mitochondria, suppresses the accumulation of ROS in the mitochondria by increasing removal enzyme activity for active oxygen radicals (ROS) and stabilizing mitochondrial function. Its high expression has the effect of anti-oxidative stress. Certain experiments in vivo have proved that SIRT3 relying on SOD2 mechanism regulated a variety of pathological processes and diseases, such as cancer, aging, radiation damage, calories restriction, nonalcoholic fatty liver disease, diabetes and so on. Ourlaboratory had found that RSV could suppress the opening of membrane permeability transition pore (mPTP) by activation of SIRT1/SIRT3-CyPD(astructural protein of mPTP) thus improvingthevascular reactivity of hemorrhagic shock. As PD is a mitochondrial protectantinvolved inanti-oxidative stress, and SIRT3 is a regulatory protein of anti-oxidative stress, sowhether is mitochondrial protection of PDrelevant to SIRT3,and how?Objective Oxidative stress model of intestinal epithelial cell IEC-6 and hemorrhagic shock rat model was to further probe the effect and underlying mechanism of mitochondrial protectant PD for small intestinal injury induced by hemorrhagic shock, providing new theoretical basis forclinical application of PD in patients with severe shock.Methods1. Models and groups1.1 Cell model of oxidative stress damageand groupsThe most suitable stimulus condition of H2O2:EEC-6 cells with 90% cellconfluence in 96-well plates were stimulated by different concentration (0,25, 50,100,250,500 um/L) of hydrogen peroxide solution (H2O2) for various times(0,15, 30,60,120min), and then Cell Counting Kit-8 was to test cell viability of different groups.20-30% cell activity decline as the ideal standard, all experiments were repeated in triplicate to determine the most suitable concentration of H2O2.Optimum protective concentration of PD:IEC-6 cells were firstly pretreatmented with different concentrations (0,25,50,100 um/L) of PD for 12h, and then stimulated with H2O2 solution. CCK8 kit was to detect cell viability of different groups.Independent three times experiments repeated to determine optimum concentration of PD.Different treatment groups:IEC-6 cells were randomly divided into 5 groups (n = 6). Namely, (ⅰ)blank control group; (ⅱ) vehicle pretreatment group; (ⅲ)PD pretreatment group; (ⅳ) PD+3-TYP (selective inhibitor of SIRT3, 50uM/L)pretreatment group; RSV pretreatment group. After pretreatment for 12h and thenH2O2 stimulus, collect cells of different treatment groups to detect following observation indices.1.2 Rat model of hemorrhagic shockand groupsExperiments conform to international and Chinese administration of experimental animals, and approval animal feedingand using committeeofsouthern medical university. Rat modelshock referred toprevious experiment scheme in our laboratory. Femoral artery bleeding to 30mmHg, maintainingfor 2h, and then blood transfusion back, continue observation for 2h.Experimental animals were randomly divided into 5 groups (n= 14):(ⅰ) sham operationcontrol group; (ⅱ)vehicle group; (ⅲ)PDpretreatment group; (ⅳ)PD+3-TYP pretreatment group; (ⅴ) RSV pretreatment group. Six rats in each groupwere executedwith broken neck method forprotein extraction and observation of mitochondrial morphology, and eight ratsremaining wereremovedcatheter and sutured incision for survival analysis.2. Tested indices2.1 The influence of PD onmitochondria in damaged cells induced by oxidative stressDetecting ofmitochondrial transmembrane potential (ΔΨm), mitochondrial permeability transition pore (mPTP) opening and ATP content in cells were to evaluate the effect of oxidative stress on mitochondrial structure and function, and the influence of PD interventions.Lower ΔΨm, more mPTP opening and less cellular ATP contentmanifest more serious mitochondrial damage.2.2 Theinfluence of PD on of oxidative stress level in damaged cellsDCF fluorescence detected byFlow cytometry was to assess ROS levels in different groups, the strongerfluorescence, the higher level of ROS was, and oxidative stress damage was more serious.2.3 The influence of PD on mitochondrial morphology in small intestine cellsof hemorrhagic shock ratsSmall intestine tissueof hemorrhagic shock rats were made into ultrathin tissue biopsies,mitochondrial morphology was observed with transmission electron microscope.An elliptical shape with well-developed cristae and electron-dense matrices was noted in normal mitochondria, andirregularly shaped, swollen, and disruptedwith poorly defined cristae and electron-lucent matrices were observed in damaged mitochondria. Comparison of mitochondrial damage degree was to evaluatethe influence of PDintervention for intestinal injury induced by hemorrhagic shock.2.4 The influence of PD on oxidative stress level in small intestine of hemorrhagic shock ratsSmall intestine tissue homogenate was detected the ratio of reduced glutathione/oxidation glutathione (GSH/GSSG) and malondialdehyde (MDA) content with test kits to assess oxidative stress levels in groups. Lower GSH/GSSG ratio and higherMDA content, the higher level of oxidative stress was.Comparison of oxidative stress levels was to evaluateintestinal injury induced by hemorrhagic shock and the influence of PD intervention.2.5 The influence of PD onapoptosis of small intestine cell and survival time of ratsSmall intestine of hemorrhagic shock rats was made into paraffin embedding tissue section, and then pinpointedapoptosis of small intestinal cellsusing Tunel apoptosis detection kit, and then record number of apoptosis cells under a microscope. The more apoptosis cell, the serious small intestinal injury.Comparison ofthe number of apoptotic cellswas to evaluate intestinal injury induced by hemorrhagic shock and the influence of PD intervention. Hemorrhagic shock rats with catheterremoved and incision sutured were given enough food and water, then observe and record survival time for 48h.The shorter survival time,the more serious hemorrhagic shock damage was.Comparison ofsurvival timewas to evaluate hemorrhagic shock and the influence of PD intervention.2.6 The relation of PDintervention and protein expression and activity ofSIRT3 in the tissue and cellsProtein expression of SIRT3 were detected with Western blot,activity of SIRT3 with specific kits. Comparison ofprotein expression and activityof SIRT3 was toevaluate the influence of hemorrhagic shock on SIRT3and PD intervention.2.7 The relation of PD intervention and protein expression and activity ofSOD2 in the tissueand cellsProtein expression ofSOD2, acetyl-SOD2 were detected with Western blot, activity of SOD2with specific kits. Comparison ofProtein expression and activityof SOD2 was to evaluate the influence of hemorrhagic shock on SOD2and PD intervention.Results1. The establishment of cell model for oxidative stress20-30% cell activity decline as the ideal standard, H2O2 250uM/L,30min was established the optimal incentive condition, cell activity of which was 74±2.2% vs control group. The optimal concentration of PD was 50 um/L.2. The influence of PD on mitochondria in damaged cells induced by oxidative stressCompared with control group, the structure and function of mitochondrial in intestinal epithelial cells in H2O2groupwere damaged, ΔΨmreduced, mPTP opened, cellular ATP contentlowered.Pretreatment ofPD or RSV couldameliorate mitochondrialdamage induced by oxidative stress, improve decline of ΔΨm,suppress opening of mPTP, promoteATP synthesis, moreover PD and RSV affected equally.3-TYP, a selective inhibitors of SIRT3 could partially inhibitmitochondrial protection of PDfrom oxidative stress.2. Theinfluence of PD on of oxidative stress level in damaged cellsCompared with control group, ROS increased dramatically and the level of oxidative stress boosted in H2O2 group. PD or RSV pretreatment could reduce the production of ROS and the level of oxidative stress, moreover PD and RSV affected equally.3-TYPcould partially inhibitthe influence of PDonoxidative stress level.4. The influence of PD on mitochondrial morphology in small intestine cells of hemorrhagic shock ratsCompared with control group,mitochondrial morphology was significantly impaired insmall intestine cells of hemorrhagic shock rats,irregularly shaped, swollen, and disruptedwith poorly defined cristae and electron-lucent matrices were observed in damaged mitochondria. PD or RSV pretreatment couldamelioratethe change of mitochondrial morphology, even close to normal morphology.3-TYPcould partially inhibitmitochondrial protectionof PD.5.Theinfluence of PD on oxidative stress level in small intestine of hemorrhagic shock ratsCompared with control group, GSH/GSSG ratio decreased and MDA content increased in small intestine cells of hemorrhagic shock rats,hemorrhagic shock caused excessive oxidation.PD or RSV pretreatment couldimprove the change ofGSH/GSSG ratio and MDA content.3-TYP could partially inhibitanti-oxidative stress of PD.6. The influence of PD on apoptosis of small intestine cell and survival time of ratsCompared withcontrol group, more cell apoptosisof small intestine cells and shorted survival time in hemorrhagic shock rats.PD or RSV pretreatment could decrease cell apoptosis and prolong survival time of rats.3-TYP could partially inhibitthe protection of PD.7. The relation of PDintervention and protein expression and activity of SIRT3 in the tissueand cellsCompared with control group, protein expression and activity of SIRT3 decreased in model group.PD or RSV pretreatment could decrease the effect of H2O2 stimulus or hemorrhagic shock on SIRT3, protein expression and activity increased (vs model group).3-TYP could partially inhibit the influenceof PD for SIRT3.8. The relation of PDintervention and protein expression and activity ofSOD2 in the tissueand cellsCompared with control group, expression of SOD2 decreased, acetyl-SOD2 increased, resulting in decline ofactivityof SOD2 in model group. PD or RSV pretreatment could decrease the effect of H2O2 stimulus or hemorrhagic shock on SOD2, causing increased expression of SOD2, decreased acetyl-SOD2, enhanced activity of SOD2(vs model group).3-TYP could partially inhibit the influence of PD for SOD2.Conclusions1. Mitochondrial damage existed inintestinal cells of hemorrhagic shock rat2. PDameliorated intestinal injury and prolongedsurvival time of hemorrhagic shock rats.3. The protection of PDfor intestinal injury induced by hemorrhagic shock was related to mitochondrial protection.4. Mitochondrial protection of PD may be related to the activation ofSIRT3-SOD2 pathways.5. PD may be an activator of SIRT3. |
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