Inhibition Of Mitochondrial Permeability Transition Pore Opening Exerts Organ Protection Against Ischemia-hypoxia Injury In Rats And Its Mechanisms

Ischemia-hypoxia plays a crucial role on the organ injury and dysfunction appearedafter multiple critical illnesses. Traumatic hemorrhagic shock is one of the most commoncauses of death in critically patients for insufficient circulating volume and inadequatetissue level of oxygen. The key management for the treatment of shock is to restore theeffective circulating blood volume and microcirculation perfusion, and increase oxygensupply of tissue. Although volume resuscitation is the ideal treatment for traumatichemorrhagic shock, for some patients simple fluid resuscitation cannot restore the dereasedideal hemodynamic parameters. Therefore, searching for an anti-shock agent that have theintrinsic benefits both in hemodynamic performance and organ function, is very importantfor the treatment of traumatic hemorrhagic shock. It has been well documented that themitochondrial permeability transition pore （MPTP） has a crucial role in ischemia, hypoxiaand reperfusion injury of the heart and brain. Using pharmacological inhibitors such ascyclosporine A （CsA）, inhibition of MPTP opening has been shown cardioprotection againstmyocardial ischemia-reperfusion injury. However, rarely studies have examined whetherCsA is suitable for the treatment of shock and via inhibition of MPTP opening and theprecise mechanisms. Studies have shown that the vascular function disorder （vasodilationand constriction function） appeared after severe trauma/shock severely interferes with thedevelopment and therapy of these disorders. Our previous studies showed that proteinkinase C （PKC） and Rho associated serine/threonine kinase （ROCK） took part in theregulation of vascular constriction/relaxation function after shock. But, it is unknownwhether inhibiting mitochondrial permeability transition pore opening with CsA couldimprove the decreased vascular function after shock, restore the tissue perfusion and protectorgan function, and its relationship to PKC and ROCK. So with a model of traumatic hemorrhagic shock in rats and hypoxia-treated VSMC, we investigated whether CsA, apowerful inhibitor of MPTP opening, is beneficial for traumatic hemorrhagic shock viaobserving the effects of CsA on animal survival, hemodynamic parameters, blood gases,vital organ and mitochondrial function, and the effects of CsA on the vascularconstriction/relaxation function and its relationship to PKC and ROCK.Methods:The experiments were conducted in two parts.Part I. Beneficial effects of inhibiting MPTP on traumatic hemorrhagic shock in rats.The traumatic hemorrhagic shock model was induced by femur fracture and the meanarterial pressure （MAP） maintained at40mmHg for3h. SD rats were randomly dividedinto six groups: normal control （sham-operated） group, shock control group, shock+LRgroup, and shock+LR+CsA1,5,10mg/kg groups. At the end of shock, rats at the CsAgroups received a continuous infusion of CsA （1,5,10mg/kg, respectively） with twovolumes of LR. The LR group was just infused with equal volume LR. The shock controlgroup did not receive any treatment after shock model was established. The sham operatedgroup experienced the same operation but no hemorrhage and no fluid infusion. The effectsof CsA on the animal survival time and24-h survival rate were observed. And at baseline,the end of shock, and at1-h and2-h after resuscitation, the hemodynamic parameters（including MAP, HR, LVSP, LVEDP,±dp/dtmax） and blood gases （including blood pH,lactate, HCO₃^-, BE, PaO₂, PaCO₂, SaO₂） were observed. In addition, at each time point, ratsunderwent a laparotomy and the blood flow in liver and kidney were measured, and bloodwas sampled to measure the function of the liver and kidney （ALT, AST, BUN, CREA）,then rats were killed to remove the liver, kidney and small intestine for the measurement ofmitochondrial function. In this experiment, we observed the effects of CsA on animalsurvival, hemodynamic parameters, blood gases, vital organ and mitochondrial functionafter traumatic hemorrhagic shock.Part II. The effects of inhibiting MPTP on vascular constriction/relaxation functionand its relationship to PKC and ROCK.1. In isolated SMA—Rats were randomly divided into ten groups: sham-operatedgroup, shock control group, LR group, CsA1mg/kg group, CsA5mg/kg group, CsA10mg/kg group, CsA5mg/kg+U46619（ROCK agonist） group, CsA5mg/kg+Y27632 （ROCK inhibitor） group, CsA5mg/kg+PMA （PKC agonist） group and CsA5mg/kg+Staurosporine （PKC inhibitor） group. After the traumatic hemorrhagic shock modelwas established and the treatment with LR/CsA as described above, at2-h afterresuscitation, the superior mesenteric artery （SMA） were isolated, the effects of CsA onvascular constriction/relaxation response to norepinephrine （NE）/acetycholine （ACh） andthe effects of ROCK/PKC were observed with an isolated organ perfusion system.2. In VSMC—With hypoxia-treated vascular smooth muscle cell （VSMC）, the effectsof ROCK agonist/inhibitor on the opening of VSMC mitochondrial permeability transitionpores （MPTP） after3-h hypoxia was determined by the calcein-CoCl2method using laserscanning confocal microscopy.Results:1. As compared with the LR alone group, CsA （5mg/kg and10mg/kg） infusionsignificantly prolonged the survival time and improved the survival rate of shock rats. The24-h survival rates in the sham-operated group, shock control group, LR group, and CsA1,5,10mg/kg groups, were16/16,0/16,4/16,5/16,9/16and9/16, respectively.2. After shock, the hemodynamic parameters were significantly decreased includingMAP, LVSP and±dp/dtmax. LR infusion only slightly increased these parameters. CsA （5mg/kg and10mg/kg） significantly increased MAP, LVSP and±dp/dtmax, which weremarkedly higher than that in LR group at2-h after resuscitation （P<0.05）. CsA1mg/kgonly had a slight effect on these parameters, and there were no significant differencesbetween CsA1mg/kg group and LR group. CsA infusion also restored the decreased HR ofshock animals, but there were no significant differences between three CsA groups and LRgroup. LVEDP was not different in all groups at each time-point.3. At the end of the shock period, blood pH value, HCO₃^-and BE significantlydecreased, and the level of blood lactic acid was significantly increased. CsA couldincreased the level of HCO₃^-and BE, and decreased the level of lactic acid in arterial blood.Among these doses,5mg/kg of CsA showed a better effect, which were markedly higherthan that in LR group （P<0.05or0.01）. And SaO₂did not show significant changes in allgroups during the entire experiment period.4. After shock, blood flow in liver and kidney was significantly reduced, the liver andkidney function parameters including AST, ALT, BUN and CREA were increased. CsA （5 mg/kg and10mg/kg） significantly increased blood flow （P<0.05or0.01）, and CsA （1mg/kg and5mg/kg） significantly decreased the level of ALT and CREA as compared to LRgroup （P<0.05or0.01）. LR or CsA had no significant influences on blood AST and BUN.5. The respiratory control rate in the liver, kidney and intestine was significantlyreduced after shock. CsA （5mg/kg） infusion could better protect the mitochondrial functionas compared to alone LR resuscitation, and CsA1mg/kg also improved the mitochondrialfunction in kidney and intestine, which all were higher than those in LR group （P<0.05or0.01）.6. The vascular constriction reactivity of SMA to NE and vasodilator reactivity ofSMA to ACh were significantly reduced after shock. LR only slightly increased theconstriction/relaxation reactivity. CsA markedly restored the decreased constriction andvasodilator reactivity of SMAs after shock （P<0.05or0.01）, among these doses,5mg/kg ofCsA showed a better effect.7. ROCK inhibitor Y27632significantly antagonized CsA-induced increase ofvascular constriction reactivity, and ROCK agonist U46619further increased the contractileresponses of SMA. While the agonist and inhibitor of PKC had no significant influences onthe effects of CsA in the regulation of the vascular function after shock.8. The mean intensity of calcein-related mitochondrial fluorescence was significantlydecreased after hypoxia, which indicating that hypxia induced opening of MPTP in VSMCs.ROCK agonist U46619inhibited the open of MPTP after hypoxia, and ROCK inhibitorY27632further increased the open of MPTP in VSMCs.Conclusions:1. Cyclosporine A, the inhibitor of MPTP opening, was beneficial for the treatment oftraumatic hemorrhagic shock in rats. CsA could improve and stabilize hemodynamicparameters, correct acid-base imbalance, increase tissue blood flow, and protect the vitalorgan function and their mitochondrial function in shock rats, and prolong the survival timeand24-h survival rate of shock animals. It suggested that inhibition of MPTP opening is apromising therapeutic strategy for traumatic hemorrhagic shock.2. The vascular contractile/relaxation function after severe shock is impaired.Inhibition of MPTP opening could restore the decreased vascular function after shock, andwhich contributed to the improvements on the tissue perfusion and organ function for shock animals, and ROCK may participate in the regulation process.