The Role Of Mitochondrial Injury In Severe Shock And Treatment Of Polydatin

High mortality is a leading element of human health that following acute severe hemorrhagic shock even after transfusion and therapy. The mechanism of high morbidity in acute severe hemorrhagic shock is relatively unknown. Therefore, to find out the mechanism of high mortality in severe shock is very critical. In previous study, we showed that low ASMC ATP levels took place in relation to acute severe hemorrhagic shock, activation of ATP-sensitive potassium channels (KATP), hyperpolarization with inhibition of L-type calcium channels, as well as norepinephrine (NE) stimulated influx of Ca2+. The consequence of reduced influx of Ca2+are depressed vaso-responsiveness and persistent hypotension. Besides insufficient of oxygen and nutrients for microcirculation dysfunction in this condition of acute severe shock (bleeding2h and refusion2h), mitochondrial injury of arterial smooth muscle cells (ASMCs) led to low ATP level, which might result in difficult treatment of severe shock even after transfusion.Due to whole body ischemia-reperfusion injury in severe hemorrhagic shock, mitochondrial injury might be a common phenomenon in the later stage of acute severe shock and involved in the genesis of refractory period in severe shock. Therefore we systemic researched mitochondrial function of ASMCs, neurons and hepatocytes in severe shocked rats to prove the above hypothesis and to ascertain the role of mitochondrial dysfunction from mitochondrial ultrastructre, function, metabolism and injury mechanism. Another objective of this study is to ascertain new mitochondrial protector and to determine the role of mitochondrial injury in severe shock using mitochondrial protectors. It is known that Res and CsA are mitochondrial protectors. Resveratrol (Res) improves mitochondrial function by scavenging oxygen free radicals, or prevent their formation through inhibiting mitochondrial electron transporting chain of complex III in many pathological conditions. As an iron-chelator, another function of Res protect lysosoma and inhibit mitochondrial permeability transit pores (mPTP) opening by preventment iron-dependent, Fenton-type reactions with formation of hydroxyl radicals. Cyclosporin A (CsA) inhibits mitochondrial permeability transition pore opening through interaction with CypD and reduce mPTP sensitivity to Ca2+and thereby plays a protective role for mitochondria. Polydatin (PD) is a monocrystalline drug that can be isolated from a traditional Chinese herb(Polygonum cuspidatum). The molecular composition of PD is3,4’,5-trihydroxystibene-3-monoglucoside, which is akin to the poly-phenol resveratrol (3,4’,5-trihydroxystibene). Therefore Polydatin (PD) might be a mitochondrial protector. This study compares mitochondria protective effects of Polydatin with Res and CsA effects and alteration of three cells (ASMCs, neurons and hepatocytes) in acute severe shock to ascertain whether protection of mitochondrial against injury using an effective mitochondrial protector is necessary, which may provide a noval therapy for severe shock.Part one:The role of ASMCs mitochondrial against injury in acute severe hemorrhagic shock1. ASMCs mitochondrial injury is involve in severe shock.(1) ASMCs mitochondrial ultrastructure injury in severe shock. ASMCs mitochondria from control (sham) animals appeared sausage-shaped with normal cristae. In contrast, mitochondria from shock+NS group were spherical or irregularly shaped, apparently swollen with ruptured and poorly defined cristae, indicating ASMCs mitochondrial ultrastructure injury in severe shock. (2) Loss of ASMCs mitochondrial transmembrane potential (△Ψm) in severe shock. The mitochondrial transmembrane potential was determined by flow cytometry with JC-1. The shock+NS group contained80.34±9.01%cells with with low△Ψm, which was substantially higher than the value of13.44±7.73%in the control (sham) group (P=0.000), indicating mitochondrial electron transport chain dysfunction or opening of mPTP,which led to loss of ASMCs mitochondrial transmembrane potential in severe shock.(3) ASMCs mitochondrial dysfunction in severe shock. The mitochondrial function was evaluated using intracellular ATP content. It was again found that severe shock caused significant decrease in the ATP levels of ASMCs to17.6±7.9%of the control value (P=0.000), indicating ASMCs mitochondrial dysfunction in severe shock.(4) ASMCs hyperpolarization and KATP channel activation in severe shock. The results showed that the KATP current densities increased remarkably in severe shock at voltages ranging from-80to+80mV compared with control values (sham group)(n=10cells for each group). At+80mV, the KATP current density increased from7.0±2.2pA/pF in the sham group to15.7±7.3pA/pF in the shock+NS group (P=0.001), indicating reduced intracellular ATP content in severe shock. Consistent with the alteration of KATP current density, the ASMCs membrane potential significantly increased from-31.7±5.3mV in the control (sham) group (n=25) to-49.7±5.3mV in the shock+NS group (n=29; P=0.000), showing ASMCs hyperpolarization in severe shock.(4) ASMCs oxdative stress in severe shock. It was found that the LPO level in ASMCs was increased from10.01±1.56nmol in the sham group to15.00±2.23nmol in the shock+NS group (P=0.000), indicating ASMCs oxygen free radical activity in severe shock.(5) ASMCs lysosomal membrane permeability in severe shock. Fluorescence microscopy of ASMCs from the shock+NS group showed a population of cells with decreased intensity of AO-dependent red, granular fluorescence compared with normal cells. AO fluorescence was quantitative analyzed by flow cytometry showed 51.6±4.8%"pale cells" indicated lysosomal injury in the shock+NS group, which was much higher than the observed value of28.3±5.2%in the control group (sham group)(P=0.000), indicating ASMCs lysosomal membrane permeability in severe shock.(6) ASMCs mitochondrial permeability transit pores (mPTP) opening in severe shock. Mitochondrial permeability transition pores were evaluated by calcein-Co2+technique using confocal microscopy and flow cytometry. Fluorescence microscopy of ASMCs from shock+NS group showed decreased intensity of calcein dependent mitochondrial fluorescence compared with normal cells (sham group). Calcein fluorescence was quatitative analyzed by flow cytometry showed mean fluorescence intensity (MIF) of156.6±11.8in the sham group and48.9±7.1in the shock+NS group (P=0.000), implying ASMCs mitochondrial permeability transition pores opening in severe shock.According to this study, oxidative stress through lysosomal-mitochondrial axis led to ASMCs mitochondrial injury in severe shock. Therefore, not only microcirculation dysfunction led to ASMCs cytopathic hypoxia, but mitochondrial injury might also involve in severe shock. Therefore, protection of mitochondrial against injury might be a new therapeutic target for severe shock treatment.2. To ascertain ASMCs protective effect of new protector-Polydatin in severe shock. CsA, Res and PD were used as mitochondrial protectors in severe shock. CsA, Res and PD inhibited shock-induced mitochondrial ultrastructure injury, especially PD has the best protective effect with almost normal mitochondria; The low△Ψm ASMCs decreased from80.34±9.01%in shock+NS group to75.38±18.33%in the shock+CsA group,53.69±17.10%in the shock+Res group and31.57±6.12%in the shock+PD group and the intracellular ATP content improved from17.6±7.9%of the control value in the shock+NS group to32.7±5.4%,62.1±11.5%and90.7±7.5%in the CsA, Res and PD treated group, respectively; Along the voltage range of-80to+80mV, CsA, Res and PD decreased the KATP current density and the membrane potential compared with the shock+NS group, especially in the shock+ PD, the KATP current density at+80mV decreased from15.7±7.3pA/pF in the shock+NS group to9.4±4.2pA/pF and the membrane potential of ASMCs decreased from-49.7±5.3mV in the shock+NS group to-36.9±7.2mV; The LPO content was reduced in mitochondrial protector-treated group, especially in the shock+PD group, the value was reduced from15.00±2.23nmol in the shock+NS group to10.42±0.99nmol (P=0.000); The cells showed partial preservation of red granular lysosomal fluorescence and green mitochondrial fluorescence in mitochondrial protector-treated group, flow cytometry showed that "pale cells" was reduced from51.6±4.8%in the shock+NS group to42.0±2.8%in the shock+CsA group,47.5±3.1%in the shock+Res group and36.8±3.8%in the shock+PD group and ASMCs mitochondrial mean fluorescence intensity (MIF) were mildly preserved from48.9±7.1in the shock+NS group to59.7±13.4,62.3±24.8,79.6±8.6in the CsA-, Res-and PD-treated groups, respectively. Based on above results,3mitochondrial protectors could protect ASMCs against mitochondrial injury through inhibition of oxygen stress with lysosomal membrane permeability and subsequent mitochondrial permeability transition pores in severe shock, especially PD has the best mitochondrial protective effect with the best ATP level (90.71±7.47%of normal value). Therefore PD is the best choice for protection of mitochondrial against injury in severe shock treatment. Part two:The role of multiple organs mitochondrial injury in acute severe hemorrhagic shock**P＜0.01versus sham group.Mitochondrial injury of ASMCs, neurons and hepatocytes took placed in severe shock.1. Mitochondria in the shock+NS group were apparently swollen with ruptured cristae, indicating severe shock induced multiple organs mitochondrial ultrastracture injury.2. The ATP level were decreased in the shock+NS group, especially in ASMCs, the value decreased to17.61±7.87%of normal cells, indicating multiple organs mitochondrial dysfunction in severe shock.3. Loss of mitochondrial transmembrane potential (△Ψm) was showed in the shock+NS group, indicating multiple organs mitochondrial electron transporting chain dysfunction and mPTP opening in severe shock.4. Mitochondrial injury of ASMCs, neurons and hepatocytes have similar mechanism. LPO level in ASMCs, neurons and hepatocytes increased with lysosomal membrane permeability and subsequent mPTP opening, implying oxidative stress induced lysosomal injury and subsequent mPTP opening might be involved in the genesis of multiple organs mitochondrial injury in severe shock.Part three:The protective effect of mitochondrial protectors against multiple organs mitochondrial injury in acute severe hemorrhagic shock1. Mitochondrial protectors attenuate multiple organs mitochondrial ultrastructure injury in severe shock. Transmission electron microscopy (TEM) was used to examine mitochondrial morphology. Control cells showed normal mitochondria with preserved membranes and cristae. In contrast, mitochondria of ASMCs, neurons and hepatocytes from the shock+NS group appeared swollen and irregularly shaped with disrupted and poorly defined cristae. The mitochondrial alterations in the shock+NS group were partially protected by3mitochondrial protectors, especially by PD. It is indicated that mitochondrial protectors might protect multiple organs mitochondrial against injury in severe shock and PD has the best protective effect with almost normal mitochondria.2. Mitochondrial protectors inhibit loss of multiple organs mitochondrial transmembrane potential (△Ψm) in severe shock. We determined the mitochondrial transmembrane potential (△Ψm) with JC-1. ASMCs with low△Ψm mitochondria decreased from65.86±10.88%in the shock+NS group to59.46±8.23%,56.32±13.70%and26.22±7.73%after treatment with CsA, Res and PD, respectively. Meanwhile, the value decreased from65.86±10.88%in the shock+NS group to59.46±8.23%in the shock+CsA group,56.32±13.70%in the shock+Res group and26.22±7.73%in shock+PD group in neurons, and from37.21±4.98%in the shock+NS group to23.09±4.06%、25.12±3.31%and12.49±3.06 %after treated by CsA,Res and PD in hepatocytes,respectively,indicating inhibition of mitochondrial electron transition chain dysfunction and mitochondrial permeability transition pores opening by mitochondrial protectors in severe shock and PD has the best protective effect.3.Mitochondrial protectors improve multiple organs mitochondrial function in severe shock.Intracellular ATP content increased from17.61±7.89%in the shock+NS group to32.69±5.45%.62.12±11.49%and90.71±7.47%in ASMCs,and from44.14±13.81%of normal value in the shogk+NS group to54.93±13.79%,63.74±16.06%and89.57±9.21%in the CsA,Res and PD-treated groups, respectively in neurons.Meanwhile,the value also increased from48.84±3.84%in the shock+NS group to63.03±6.81%in shock+CsA group,57.56±7.08%in the shock+Res group and87.17±17.29%in shock+PD group,showing that PD is a better protective effect for multiple organs mitochondrial function than CsA and Res in severe shock.4.Mitochondrial protectors inhibit multiple organs oxidative stress in severe shock. The LPO levels were significantly increased in shock+NS group compared with the sham group,but reduced among the three protector-treated groups,especially in PD-treated group,LPO levels decreased from149.85±22.27%of normal value to104.00±9.89%in ASMCs and from139.93±17.67%of normal values in the shock+NS group to102.25±9.43%in neurons,meanwhile the value decreased from211.87±21.05%of normal value to102.78±19.61%in hepatocytes,indicating that inhibition of multiple organs oxidative stress with lysosomal and mitochondrial injury by protectors in severe shock,especially by PD.5.Mitochondrial protectors inhibit multiple organs lysosomal membrane permeability in severe shock.The percentage of "pale cells",which indicated lysosomal membrane permeabllity,decreased after treatment with CsA,Res and PD in severe shock, especially in the shock+PD group.The value decreased dramatically from51.63±4.77%in the shock+NS group to36.85±3.84%in ASMCs.Meanwhile the value reduced from18.89±2.03%in the shock+NS group to10.12±1.00%in neurons and from30.14±5.73%in the shock+NS group to 5.79±1.28%in hepatocytes, indicating inhibition of multiple organs lysosomal membrane permeability in protector-treated groups, especially PD has the best effect.6. Mitochondrial protectors inhibit multiple organs mitochondrial permeability transit pores (mPTP) opening in severe shock. Mean mitochondrial fluorescence were improved in protector-treated groups and PD showed the best effect. After treated by PD in severe shock, the fluorescence intensity increased from31.16±0.05%of normal value in the shock+NS group to50.83±5.49%in ASMCs, and from60.37±10.06%of normal conditions in the shock+NS group to91.32±18.57%in neurons, meanwhile increased from23.68±2.41%in the shock+NS group to46.69±2.89%in hepatocytes, indicating inhibition of multiple organs mPTP in severe shock by protectors, especially by PD.7. Mitochondrial protectors improve Vaso-reactivity and MAP in severe shock. The results showed that the NE threshold concentration increased to29.3times of that of the prehemorrhage level at the end of a period, including2h of hemorrhage and2h of treatment and MAP had decreased to47.23±11.28mmHg in the shock+NS group. In the shock+CsA and shock+Res groups, the NE threshold concentration increased to10.4and11.8times of the prehemorrhage level and MAP increased to55.23±9.92and57.10±15.74mmHg, respectively, at the same time points. Meanwhile, in the shock+PD group, the NE threshold concentration increased to4.8times of the prehemorrhage level during the same period, while MAP increased to89.38±16.31mmHg, indicating improvement of shocked rats vaso-reactivity and resistant hypotension in PD treated group.8. Mitochondrial protectors decrease severe shocked rat cerebral cortex NADH. It was found that NADH level increased by38.57±6.52%compared with the prebleeding value in shock+NS group, implying cerebral cortex mitochondrial electron transporting respiratory chain dysfunction. The value significantly decreased to15.03±3.06%in shock+PD group, indicating improvement of severe shocked rat cerebral cortex mitochondrial electron transporting chain by PD.9. Mitochondrial protectors prolong shocked rats survival time. A mean survival time was only5.4±2.6h and100%animal death within24h after reinfusion of shed blood in the shock+NS group. The survival time in the shock+CsA and shock+Res groups was prolonged2.05and1.95times that of the shock+NS group, respectively, which was significantly longer than that in shock+NS group (P<0.000), but all shocked animals died within24h. In the shock+PD group, the survival time was significantly prolonged to4.35times that of the shock+NS group, and the24-h survival rate was significantly increased to5/8, it is indicated that the survival time of shocked rat was significantly prolonged by PD.According to the above results, mitochondrial protectors could protect mitochondrial against injury through inhibition of oxygen stress with subsequent lysosomal membrane permeability and mitochondrial permeability transition pores, finally improve resistant hypotension, low vaso-reactivity and cerebral cortex mitochondrial electron transporting chain function in severe shock, and prolonged the survival time of shocked rats, especially PD has the best effect with survival time (23.7±3.7h) and the24h survival rate (5/8). Therefore protection of mitochondrial against injury using PD is an ideal choice for severe shock treatment, which might provide a novel clinical therapy for severe shock.Conclusions1. Mitochondrial injury of arterial smooth muscle cells (ASMCs) might lead to vaso-responsiveness and persistent hypotension with finally high mortality and refractory in severe shock.2. ASMCs mitochondrial injury might be come from lysosomal-mitochondrial axis and oxidative stress in severe shock.3. Mitochondrial injury not only appeared in ASMCs, but also in neurons and hepatocytes during severe shock, implying that multiple organs mitochondrial injury might be a common phenomenon in the genesis and development of severe shock.4. Mitochondrial protectors could attenuate mitochondrial injury in severe shock and PD has the best protective effect among3protectors in the study. Therefore, we present the conclusion that treatment of severe shock should not only improve microcirculation, but also protect mitochondria against injury with a noval protector-Polydatin in which independent intellectual property rights was owned by our country.