Study On Antithrombotic Mechanism Of Qishen Yiqi Decoction Based On Platelet Mitochondrial Autophagy

Background:Cardiovascular disease is a serious threat to human health,with high morbidity and mortality worldwide,and the death toll exceeds one quarter of the total^[1].Thrombosis is a key pathogenic factor in many cardiovascular diseases?myocardial infarction,venous thromboembolism,etc.?and is one of the important causes of death in patients with cardiovascular disease^[2].Intravascular arterial thrombosis is a chronic inflammatory disease,mainly due to the rupture of atherosclerotic plaque,which in turn leads to the occurrence of ischemic injury diseases^[3,4].Acute arterial thrombosis is the main cause of most cardiovascular diseases and eventually death,often associated with platelet activation^[5].Existing clinical studies have made great achievements in the treatment of thrombosis,such as the combined use of dual antiplatelet drugs and the use of surgical therapy for thrombolytic treatment of thrombosis sites.The efficacy is significant,but it still has certain risks,such as the gastrointestinal tract Bleeding,surgical thrombolysis have a higher risk and cost.Therefore,we need to find safe and effective drugs for treatment.In recent years,the advantages of traditional Chinese medicine in the prevention and treatment of cardiovascular diseases have also become increasingly prominent.Due to the multi-component,multi-target and multi-path characteristics of traditional Chinese medicine,the safety of medicine has been improved and there are fewer adverse reactions.Chinese medicine believes that heart failure is caused by heart and Qi deficiency,and thrombosis is caused by poor blood flow,so it is treated with Yiqi Huoxue Chinese medicine.Therefore,we have conducted research on Yiqi Huoxue drugs already on the market,looking for qualified prescriptions,and further exploring its mechanism of action.Objective:This study uses the method of network pharmacology to integrate and analyze Yiqihuoxue traditional Chinese medicines and heart failure and the target of thrombosis in key pathological links,to predict the main mechanism of action of the drug on the disease and the target of possible intervention.On the key pathological link of thrombus,research was carried out,using 10%Fe Cl3 to damage the rat carotid artery to prepare a thrombus model;using a small animal ultrasound imaging system,a hemodynamic system;scanning electron microscopy,serum pharmacology,Determination of active oxygen in platelets,Mito-Tracker mitochondrial activity measurement,Western blotting and other methods to investigate the effect of Qishen Yiqi Recipe on platelet function and thrombosis,and further explore and verify its mechanism of action.Methods:1.Classify and sort out the existing traditional Chinese medicines for benefiting qi and activating blood in the market,analyze them with the aid platform of traditional Chinese medicine,find out the commonly used compatible drug pairs,and then analyze the active ingredients and action targets of single medicines;Correlation analysis of the active ingredients and the target,looking for its key target and signal pathway.2.Establish Fe Cl3 induced rat carotid artery thrombosis model,this experiment is divided into five groups:sham operation group,model group,Qishen Yiqi 150 mg/kg group,Qishen Yiqi300 mg/kg group,aspirin positive drug group;Seven days after pre-administration,the small animal ultrasound Doppler system was used to observe the blood flow occlusion after modeling,and the scanning electron microscope was used to observe the changes between the shape and structure of the carotid vascular endothelium,thrombosis and vascular damage in rats.ELISA was used to detect the expression of thromboxane?TXB2?B2 and6-keto-prostaglandin?6-keto-PGF1??in plasma,and the ratio of TXB2/6-keto-PGF1?was also determined.Using serum pharmacology method,the drug-containing serum was given to human platelets to determine the effect of Qishen Yiqi Formula on human platelet adhesion and aggregation.3.Seven days after pre-administration of rats,the rat carotid artery thrombosis model was established by Fe Cl₃,and the platelets of each group were extracted for active oxygen ROS and mitochondrial activity determination,and the autophagy-related proteins ATG7,LC3?/?,Beclin-1,p62,ATG3,ATG12,PINK,were detected by Western blot.And apoptosis-related proteins Bcl-2,Bax,Caspase-3 and PI3K/Akt/m TOR pathway related proteins were measured.4.Platelets of normal rats administered for 7 days were taken,and the platelet injury model induced by H₂O₂ was divided into Contrl,Model,QSYQ300 mg/kg,QSYQ300 mg/kg+autophagy agonist rapamycin?RAPA?,QSYQ300 mg/kg+Inhibitors chloroquine?CQ?,QSYQ 300 mg/kg+Akt inhibitor?KM2006?;Western blot method was used to express autophagy-related proteins ATG7,ATG3,ATG12,LC3?/?,Beclin-1,p62,PINK;Apoptosis-related proteins Bcl-2,Bax,Caspase-3 and PI3K/Akt/m TOR pathway related proteins were verified.Finally,the mitochondrial activity and ROS were measured again.Results:1.The traditional Chinese medicines obtained through the auxiliary platform of traditional Chinese medicine are Huangqi,Danshen,Chuanxiong,Angelica,Ginseng,Panax notoginseng,etc.,which are compatible with Qishen Yiqi Recipe,so we choose Qishen Yiqi Recipe as the research object.According to IPA analysis,Qishen Yiqi Compound has multiple targets and pathways for the treatment of heart failure and thrombosis.It has predicted 20 key upstream factors and found that it is closely related to the PI3K/Akt/m TOR signaling pathway.2.After evaluating the blood flow of the rat carotid artery thrombosis model induced by Fe Cl3,the blood flow of each group was significantly reduced after the model was established,and there was significant thrombosis in the carotid artery of each group.The thrombosis of the administration group was significantly improved;the results of scanning electron microscopy showed that the thrombosis of the Qishen Yiqi Formula 300 mg/kg administration group was significantly better than that of the other groups.Elisa test results showed that Qishen Yiqi significantly inhibited TXB2,TXB2/6-keto-PGF1?and significantly increased6-keto-PGF1?compared with the model group.Further serum pharmacological results show that Qishen Yiqi can significantly inhibit the effects of thrombin and U46619 agonists on platelet adhesion and aggregation.3.In the evaluation of platelet function,it was found that compared with the model group,the ROS content in the platelet of rats in the Qishen Yiqi administration group was significantly reduced,and the mitochondrial activity was significantly increased.The autophagy-related proteins ATG7,ATG3,ATG12,LC3?/?,Beclin-1.The expression of mitochondrial autophagy-related protein PINK is significantly increased,and the expression of p62 is significantly reduced;it can significantly increase the content of Bcl-2,reduce the content of Bax and Caspese-3;significantly reduce the phosphorylation level of PI3K,Akt and m TOR protein.4.In the evaluation of the effect of Qishen Yiqi Recipe on platelet autophagy,it was found that the expression of autophagy-related proteins ATG7,ATG3,ATG12,LC3?/?,Beclin-1,PINK in the autophagy agonist group was significantly increased compared with the model group,The expression of p62 was significantly reduced;the expression of autophagy-related proteins ATG7,ATG3,ATG12,LC3?/?,Beclin-1,PINK in the autophagy inhibitor group was significantly reduced,and the expression of p62 was significantly increased;Qishenyiqifang mainly stimulated platelets Mitochondrial autophagy exerts a protective effect on platelets.5.Qishen Yiqi Recipe's regulation of the classic autophagy pathway PI3K/Akt/m TOR found that:compared with the model group,the Akt inhibitor group can significantly reduce the expression of p-PI3K,p-Akt and p-m TOR protein;it can significantly Increase the expression of autophagy-related protein and decrease the expression of p62 protein.This indicates that Qishen Yiqi Recipe further inhibits the activation of the pathway by inhibiting the phosphorylation of PI3K/Akt/m TOR,thereby stimulating platelet autophagy.Conclusion:In summary,this study found that Qishen Yiqi Recipe mainly inhibits the activation of PI3K/Akt/m TOR pathway and regulates mitochondrial autophagy in platelets,thereby inhibiting platelet apoptosis and has an inhibitory effect on thrombosis.The results of network pharmacology predictions are consistent.