Changes Of Platelet Mitochondrial Function In Acute Pancreatitis And Clinical Study

BACKGROUND: Acute pancreatitis(AP)is a common clinical acute illness.In most cases,it can be cured by medical treatment.About 20%-30% of AP patients develop severe pancreatitis(SAP),causing multiple organ function.Obstacles lead to death.Systemic inflammatory response(SIR)is a key component of AP's multiple organ dysfunction.Therefore,to explore the pathophysiological mechanism of SIR in acute pancreatitis,it is helpful to inhibit the aggravation and treatment of pathophysiological processes of acute pancreatitis.Multiple organ dysfunction syndrome is caused by SIR.Systemic inflammatory response syndrome(SIRS)is a common pathological process of cascading reactions,common in sepsis,trauma,burns,acute pancreatitis and other diseases.Studies have shown that mitochondria play a key role in this process.The main function of mitochondria is to produce adenosine triphosphate(ATP),an essential energy element for cell metabolism,through oxidative phosphorylation.In addition,mitochondria mediate the production and elimination of oxygen free radicals.Mitochondria are the main site for the production of reactive oxygen species(ROS).When mitochondria are damaged,oxidative stress is overactivated,active oxygen synthesis is increased,and mitochondrial biosynthesis and stability are inhibited.Maintenance,damage to the mitochondrial membrane causes membrane potential to decrease,resulting in decreased ATP production and intracellular environmental disorders,ultimately leading to cell death.Therefore,mitochondrial dysfunction will inhibit cell energy production and abnormal increase of oxygen free radicals,leading to oxidative damage of cells,expanding and aggravating the inflammatory process.Platelets have a greater number of functional mitochondria.Studies have shown that mitochondrial function in platelets is not only involved in the regulation of thrombosis [5],but significant changes in platelet function during AP.Alessandro et al found that platelet reactivity was reduced in patients with sepsis and cardiogenic shock,and mitochondrial respiratory enzyme function was inhibited.Yamakawa et al confirmed that platelet mitochondrial membrane potential decline and platelet apoptosis were significantly increased in patients with systemic inflammatory response syndrome.Platelet counts and mitochondrial membrane potential were significantly reduced in patients with severe sepsis,and Gründler et al.found a correlation between dysfunction of platelet mitochondrial membrane potential and sepsis severity score(APACHE II,etc.).Therefore,this study provides an experimental basis for the assessment and treatment of AP disease by detecting changes in mitochondrial function in platelets in patients with acute pancreatitis and analyzing the association with disease severity.Objective: To investigate the changes of platelet mitochondrial function and its relationship with disease severity in patients with acute pancreatitis by detecting changes in platelet mitochondrial function in patients with acute pancreatitis(AP)of different severity.Method: 1.Selenium(superoxide dismutase),MDA(malondialdehyde),GSH-Px(glutathione peroxidase),SDH(succinate dehydrogenation)in platelets of patients with acute pancreatitis and normal persons with different severity Changes in platelet mitochondrial function in patients with acute pancreatitis were observed by changes in enzyme and ATPase levels and mitochondrial swelling.2.The SOD,MDA,GSH-Px,SDH,ATPase,and BCA protein detection kits were purchased from Nanjing Jiancheng Bioengineering Research Institute.3.Preparation of platelets: AP patients received 10 ml of venous blood on an empty stomach within 24 hours after admission.The control group received 10 ml of venous blood 12 h after fasting,placed in an EDTA anticoagulant tube,centrifuged at 150 °C for 20 min at 4 °C,and the supernatant was enriched.The plasma of platelets was centrifuged at 300 °C for 10 min at 4 °C,and the supernatant was discarded.The obtained pellet was platelets.Platelets were resuspended by adding an equal volume of normal saline to the plasma,and stored at room temperature for shaking.4.Extraction of mitochondria: Take appropriate amount of platelet suspension and operate according to the instructions of the cell mitochondrial extraction kit.5.Detection of mitochondrial SOD,MDA,GSH-Px,SDH and ATPase levels in platelets: Take appropriate platelet suspension and follow the kit instructions to detect SOD,MDA,GSH-Px,SDH and ATPase levels in platelets.Platelet mitochondrial swelling was measured.Result: 1.Changes in SOD,GSH-Px and MDA levels in platelets of AP patients Compared with the control group,MDA levels in platelets,MAP group,MSAP and SAP group increased,SOD and GSH-Px levels decreased.Compared with the MAP group,the levels of platelet MDA and the decrease of SOD and GSH-Px levels were more obvious in the MSAP group and the SAP group.2.Changes in platelet ATPase and SDH levels in AP patients Compared with the control group,the levels of ATPase and SDH in platelets of MAP group and SAP group were decreased.Compared with the MAP group,the levels of SDH and ATPase in the MSAP group and the SAP group were significantly lower.3.Changes in platelet mitochondrial swelling in AP patients After adding p H 7.2 reaction buffer,the absorbance of mitochondria at 520 nm decreased slowly in the control group,suggesting that mitochondria appeared swelling due to abnormal osmotic pressure.The decrease of absorbance in MAP group was lower than that in the control group,suggesting that it was not sensitive to changes in environmental p H,indicating that the function of mitochondria was impaired.The decrease of absorbance in MSAP group was lower than that in MAP group,suggesting that mitochondrial function was more obvious;the decrease of absorbance in SAP group It is lower than the MSAP group,suggesting that mitochondrial function is most impaired.See Figure 1A.After adding 0.3 mmol/L Ca Cl 2 reaction buffer,the mitochondrial absorbance of the control group decreased significantly,suggesting that the mitochondria showed obvious swelling.The decrease of absorbance in MAP group was lower than that in the control group,suggesting that mitochondrial function was impaired.The decrease of absorbance in MSAP group was lower than that in MAP group,suggesting that mitochondrial function was more obvious.The decrease of absorbance in SAP group was lower than that in MSAP group,suggesting that mitochondrial function was impaired.Most obvious.Conclusion: 1.Compared with the control group,the levels of MDA in the platelets of the mild acute pancreatitis(MAP)group,moderate severe acute pancreatitis(MSAP)group and severe acute pancreatitis(SAP)group increased,SOD and GSH-Px levels decreased,SDH and ATPase activity is inhibited and mitochondrial swelling is reduced.2.Compared with MAP group and MSAP,the levels of platelet MDA and the decrease of SOD and GSH-Px levels were more obvious in SAP group,and the inhibition of SDH and ATPase activity was more obvious.At the same time,the decrease of mitochondrial swelling degree was significantly reduced.3.In patients with acute pancreatitis,mitochondrial oxidative damage is evident in platelets,and energy synthesis is inhibited,which is associated with the severity of acute pancreatitis.