| Severe irreversible shock was in the later stage of shock, after blood transfusion and various therapies which aiming to improve microcirculation, illness worse still and eventually lead to death. It’s reported that mitochondrial disease was involved in multiple organ dysfunction (MODS) which happened in shock, infection and sepsis. What’s more, it’s confirmed that smooth muscle mitochondria damaged which brought about a decrease in the ATP production in the study of severe irreversible shock. The performance was showed as mitochondrial swelling, disruption of membrane integrity with poorly defined cristae, the opening of permeability transition pore and low mitochondrial membrane potential (Δψm). After treatment, ATP in the ASMCs was still significantly reduced, but mitochondrial protectants (CsA, polydatin) appeared certain effect of protection, which pointed out that it may be a close relationship between mitochondrial function and shock. There are different reports between the relationship of platelet mitochondrial function and the outcome of sepsis. Some reported that temporal increase of platelet mitochondrial respiration was negatively associated with clinical outcome in patients with sepsis, while others believe that survivor patients showed higher cytochrome c oxidase activity per citrate synthase activity ratio and cytochrome c oxidase quantity per citrate synthase activity ratio than nonsurvivors. Our study aims to explore whether mitochondria damage of platelets occurs in rat with severe hemorrhagic shock, the relationship between the degree of mitochondria damage and the shock, and the changes of mitochondria after the application of mitochondria protecting drugs so as to offer new ideas in the diagnosis and treatment of severe irreversible hemorrhagic shock in clinical. In this study, we investigated the relationship between the degree of mitochondria damage and the shock in hemorrhagic shock rat model by setting different time and degree of shock, adopting blood transfusion treatment with different therapies and using the methods such as the determination of ATP content, transmission electron microscope observation, the detection of mitochondrial membrane potential, mitochondria permeability transition pore state, lipid peroxide and lysosome stability.MethodsSPF Wistar rats, weighing200±20g, which provided by the Southern Medical University Laboratory Animal Center, were randomly divided into sham group, shock30,60,120min group, shock120min+normal saline+blood reinfusion group (NS group) and shock120min+polydatin+blood reinfusion group (PD group). The severe hemorrhagic shock model which established by our lab was used. Anesthesia was induced with13.3%urethane and0.5%chlorine aldose (0.65ml/kg). Both sides of femoral arteries and one side of femoral venous were incised and insert catheter. Femoral arteries intubation were for bleeding and monitoring mean arterial pressure (MAP), while femoral venous intubation was for fluid resuscitation. Sham group was operated intubation without shock or resuscitation and served as negative controls. In shock groups, blood was withdraw from femoral artery in10minutes to a mean arterial pressure of30mmHg, and respectively maintained at30mmHg for30minutes,60minutes and120minutes. PD group was shocked and maintained at30mmHg for120minutes then intravenously injected polydatin0.6ml (30mg/kg),5minutes later blood reinfusion, and observed for120minutes. NS group was shocked and maintained at30mmHg for120minutes then intravenously injected NS0.6ml,5 minutes later blood reinfusion, and observed for120minutes. All the blood above was collected in a syringe with heparin anticoagulation.After the model, all the rats’ blood samples were respectively collected through abdominal aorta intubations,50UI/ml heparin anti-freezings. The platelets were separated with Percoll gradient density centrifugation. Each group’s platelets were made into electron microscope specimens then observe the mitochondria ultrastructure changes of which with transmission electron microscope. The CellTiter-GloTM Luminescent Cell Viability Assay was used to detect mitochondrial ATP levels in platelets. Estimate the mitochondrial permeability transition pore state with the intensity of Calcein fluorescence in the platelets, after the common incubation of Calcein-AM and CoCl2. JC-1probe were adopted to detect the changes of mitochondrial transmembrane potential (Δψm) in platelets. Use the percentage of JC-1monomer which Issues green fluorescence (in low Δψm cells) to illustrate the ratio of mitochondrial depolarization in platelets. Lipid Hydroperoxide (LPO) Assay Kit was used to detect the content of lipid hydroperoxide in platelets. Lysosomes’ stability was judged by acridine orange (AO). Use the percentage of "pale cells" and the intensity of AO fluorescence to illustrate the lysosomes’ stability in platelets.Results1. Swelling and damage occured in the platelets’ mitochondrialThe mitochondrial ultrastructure of platelet in rat could be observed through the transmission electron microscopy, which was shown that the more serious the shock, the more damage in the mitochondrial ultrastructure. There were integrity of mitochondrial double membrane and internal ridge structure and compact matrix density. Mitochondrial swelling with poorly defined cristas was observed in shock30min group and the situation were worse in shock60min group and NS group, with swollen and electron-lucent even cavity in the platelets in the mitochondria. The structure of platelet mitochondria were relatively integrated in PD group but with apparently swollen.2. The decrease of ATP levels in platelets Compared with Sham group, the contents of ATP in the platelets were significantly decreased in the shock60min group, shock120min group and NS group. The ATP level of platelets in the shock120min group was only63.84±8.51%in which of sham group (P=0.000). And after the treatment of NS, the ATP level of platelets was50.75±9.15%as much as the sham group (P=0.000). It was shown that the ATP level was79.57±8.48%of sham control group in PD group (P=0.011), which was significantly higher than shock120min group (P=0.045) and NS group (P=0.001). It’s suggested that as the more time in shock or the severer of shock degree, the more damage in platelet mitochondrial function, under the therapy of polydatin, the damage could be reduced.3. The opening of platelets’ mitochondrial permeability transition poreThe fluorescent confocal results of the commonly dyeing with Calcein-AM and CoCl2shown that calcein fluorescence in platelets mitochondria was gradually weakened with the severer of shock degree. It was the same in the flow results, that the calcein fluorescence was significantly reduced with the severer of shock degree. Compared with the calcein fluorescence in the sham group (323.60±46.51AU), the calcein fluorescence in the shock60min group, the shock120min group and the NS group were apart197.20±56.15,175.16±58.33,165.09±1.44AU which were significantly weakened (P=0.000, P=0.000, P=0.000). The calcein fluorescence in PD group (234.76±40.97AU) was significantly higher than NS group (P=0.008). It suggested that polydatin may protect platelets mitochondrial permeability transit pore from opening.4. The dropping of platelets’ mitochondrial membrane potentialUse the percentage of JC-1monomer which launching green fluorescence (in low Δψm cells) to illustrate the ratio of mitochondrial depolarization in platelets. Compared with the percentage of low Δψm cells in the sham group (11.72±4.43%), the percentage of low ΔΨm cells in the shock30min group was23.85±6.05%which was markedly increased (P=0.003). And the low Δψm cells in shock60min and120min group were32.40±3.27%,37.00±9.97%which suggested that the polarization of platelets’ mitochondrial membrane potential occurred early in shock30min and the polarization was increased with the severer of shock degree. It’s significantly that the percentage of low Δψm cells in the PD group (16.09±4.50%) was less than that in NS group (34.13±7.72%)(P=0.000). Polydatin may protect the platelets mitochondrial membrane from depolarization.5. The increase of lipid peroxide content in plateletsCompared with Sham group, the content of lipid peroxide in platelets has no statistical differences in Shock30min group, Shock60min group and Shock120min group (P=0.848, P=0.875, P=0.951). The content of lipid peroxide in NS group was (4.33±1.09nmol/1.0×108) which was significantly higher than that in sham group (3.06±0.42nmol/1.0×108)(P=0.006) and the content of lipid peroxide in PD group was significantly less than that in NS group (P=0.007). It was implied that lipid peroxide could be produced after blood transfusion not during the bleeding shock, and the therapy of polydatin may reduce the production.6. The decreased of lysosomes’ stability in plateletsThe fluorescent confocalresult of AO dyeing shown that AO fluorescence in platelets was gradually weakened with the severer of shock degree. The flow results displayed that the percentages of pale cells in shock30min group, shock60min group, shock120min group and NS group were8.30±2.45,19.96±2.95and22.68±3.37%which were significantly increased compared with that in the sham group (3.97±0.62%)(P=0.011,P=0.000, P=0.000, P=0.000). It implied that the severer the shock degree, the more percentage of pale cells, the lower stability of lysosome in platelets. The percentage of pale cells in the PD group was7.12±2.90%which was decreased compared with that in the NS group (21.35±1.44%)(P=0.000) which suggested that the polydatin defended the stability of lysosome in platelets under shock and reperfusion.7. Weight, Bleeding volume and MAP of ratsThere were no statistical difference in the weight of rats and basic mean arterial pressure (P=0.223,P=0.402). The bleeding volume of Shock120min group, NS group and PD group had no statistical difference (P=0.669, P=0.821, P=0.997). At 30minutes after the dose and blood reinfusion the MAP of NS group and PD group were respectively51.40±10.80mmHg and71.06±5.73mmHg, between which had statistical difference (t=-3.937, P=0.003). The MAP of PD group was obviously above the NS group, with the observation going, the difference was greater (t=-7.797, P=0.003). At120min after blood reinfusion the MAP of rat were apart41.57±11.34,90.67±10.46mmHg in the NS group and PD group.1. ATP content in platelets of rats was significantly reduced (P=0.002) in shcok60min group, which was further decreased with the time going and even more serious in the NS group (50.75±9.15%of normal value).2. Mitochondrial swelling with poorly defined cristas and the increased number of platelets with low mitochondrial membrane potential (Δψm) and low lysosome stability (pale cells) were observed in shock30min group, and the calcein fluorescence in mitochondria dropped in shock60min group (P=0.000), which indicated that the low ATP level in platelets resulted from not only microcirculatory disorder but also Mitochondrial dysfunction in shock. However, in shock120min+PD+blood reinfusion group, all the variables mentioned above were recovered markedly, especially the ATP level returned to near80%of normal value.3. The mechanisms of platelets’ mitochondrial ATP synthesis function abating in severe hemorrhagic shock included lipid peroxidation of free radical, the damage of lysosomes (which occurred in shock30min), the opening of mitochondrial permeability transit pore (which occurred in shock60min), the dropping of platelets’ mitochondrial membrane potential and the swelling and damage occured in the platelets’ mitochondria (which occurred in shock30min).4. The treatment of polydatin could reduce the changes above and make the blood pressure of rats rising.In a word, the platelet mitochondrial dysfunction takes place at30-60min of severe shock, which may be served as an invasive variable for the diagnosis and treatment of severe shock. |
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