| Myocardial ischemia/reperfusion injury(MI/RI)is an additional injury to the myocardium and coronary circulation induced by myocardial reperfusion after acute myocardial infarction(AMI).MI/RI is the main reason affecting the prognosis of AMI,and it is also an important pathological factor leading to heart failure(HF)in the later stage of AMI.Myocardial cell injury caused by MI/RI mostly occurs in the early stage of reperfusion.Therefore,it is of great significance to explore the myocardial protection mechanism and prevention strategies in the acute phase of MI/RI.Pyroptosis is a key mechanism involved in the MI/RI process.Cardiomyocyte pyroptosis and its secondary inflammatory cascade can expand the infarct size(IS)and aggravate the MI/RI process.Therefore,improving cardiomyocyte pyroptosis is an important means to prevent MI/RI.As a new type of high-performance communication carrier,exosomes can not only promote cell-to-cell communication but also play an important role in MI/RI cardiac injury repair,immune response,pyroptosis,and its mediated inflammatory waterfall.It has broad application prospects to study the prevention and treatment mechanism of MI/RI from a new perspective of exosomes.Qingxin Jieyu Formula is modified from the empirical formula of Academician Chen Keji in the treatment of AMI.It is a representative prescription for the etiology and pathogenesis of "stasis and toxin",and has the effects of replenishing qi and resolving turbidity,activating blood,and detoxification.The team’s previous clinical studies have shown that Qingxin Jieyu Formula can reduce the occurrence of long-term clinical combined endpoint events in patients with stable coronary heart disease,reduce the readmission rate of patients after AMI,improve clinical symptoms and anti-inflammatory immunity;the basic research results show that Qingxin Jieyu Formula has the effects of inhibiting macrophage pyroptosis,regulating intestinal flora,improving lipid metabolism,inhibiting the inflammatory response and improving ventricular remodeling after AMI.Therefore,Qingxin Jieyu Formula is an effective compound for the prevention and treatment of acute coronary events.Pyroptosis and its mediated inflammatory cascade are one of the important pathological mechanisms of MI/RI,and exosomes play an important role in cardiac repair and pyroptosis of MI/RI.The preliminary experimental results of this subject suggest that Qingxin Jieyu Formula can down-regulate the expression of NLRP3 inflammasome and GSDMD in myocardial tissue,indicating that the effect of Qingxin Jieyu Formula on improving MI/RI in rats may be related to pyroptosis.However,the specific mechanism of action and the type of pyroptosis involved are still unknown,and further research is needed.Therefore,we consider whether Qingxin Jieyu Formula and its induced exosomes can inhibit the occurrence of myocardial pyroptosis,thereby improving MI/RI and exerting myocardial protection?Is this effect achieved through the NLRP3/Caspase-1/GSDMD pathway?Therefore,we propose the hypothesis:1.Qingxin Jieyu Formula may inhibit the pyroptosis of cardiomyocytes by regulating circulating exosomes,thereby improving MI/RI myocardial injury,the above effects may be achieved by affecting the classical pyroptosis pathway NLRP3/Caspase-1/GSDMD pathway.In order to verify the above hypothesis,three parts of literature metrology research,in vivo experiment,and in vitro experiment were carried out.Based on bibliometrics,the research potential of exosomes in the cardiovascular field was explored to provide literature evidence and theoretical support for subsequent in vitro and in vivo experiments from the new perspective of exosomes.In vivo and in vitro experiments explored the relationship between traditional Chinese medicine compound effect,exosomes,and myocardial cell pyroptosis from the perspective of exosomes.The purpose of this study is to verify the effect characteristics and mechanism of Qingxin Jieyu Formula and its induced circulating exosomes on myocardial cell pyroptosis from three aspects:bibliometrics,in vivo experiments and in vitro experiments.Part 1 Bibliometric study of exosomes in the cardiovascular fieldObjective:To explore the distribution of scientific cooperation network,research hotspots,and frontier trends of exosomes in the cardiovascular field,reveal the contribution and development context of exosomes in the cardiovascular field,provide a new perspective for clinical and scientific researchers,and provide literature evidence and theoretical basis for subsequent in vitro and in vivo experiments.Methods:Visual analysis of the literature on exosomes in the cardiovascular field from the Web of Science Core Collection from 2002 to 2022 in the past 20 years.CiteSpace 5.8.R3 software was applied to the quantitative analysis,burst analysis,co-occurrence analysis,cluster analysis,and other aspects of this study to construct a knowledge map.Results:The analysis of 1919 articles showed that the number of articles published on exosomes in the cardiovascular field continued to rise in the past 20 years,especially in China and the United States.The research results of France,England and Germany played key role in global network cooperation.Nanjing Medical University,Shanghai Jiao Tong University,and Fudan University are the top research institutions in the number of publications.Eduardo Marbán has published the most papers in this field.Circulation Research is not only the most published journal in related fields but also the most co-cited journal.Through keyword co-occurrence analysis,myocardial infarction,mesenchymal stem cell,micro RNA,heart failure,mechanism,therapy,angiogenesis,inflammation,and biomarker are the top 20 keywords.Conclusion:Although the literature on exosomes in the cardiovascular field continues to rise,the breadth and intensity of scientific cooperation networks are not ideal,and transnational cooperation and exchanges are significantly insufficient.In the future,it is necessary to break academic barriers and further strengthen international cooperation.Exosomes have great potential for diagnosis and treatment in the fields of AMI,MI/RI and HF.The mechanism of exosomes in immune inflammation,angiogenesis,cell death and cardiac repair has attracted much attention and has broad development prospects.Part 2 Effects of Qingxin Jieyu Formula on pyroptosis and exosomes in myocardial tissue of Wistar ratsObjective:To observe the effects of Qingxin Jieyu Formula on myocardial IS,pathological morphology,myocardial pyroptosis level(TEM,TUNEL,and immunohistochemical method),serum inflammatory factors,mRNA and protein expression of core molecules in classical pyroptosis pathway and plasma exosomes in MI/RI Wistar rats.Methods:Seventy-five Wistar rats were randomly divided into sham operation group(Sham),model group(Model),low-dose Qingxin Jieyu Formula(L-QXJYF)group,medium-dose Qingxin Jieyu Formula(M-QXJYF)group,and high-dose Qingxin Jieyu Formula(H-QXJYF)group by random number table method,with 15 rats in each group.The rats were given intragastric administration for 28 days,twice a day,and the model was prepared 1 hour after the last administration.MI/RI rat model was established by ligation of LAD for 30 min and reperfusion for 3h.The area of myocardial ischemia risk area and myocardial IS in Wistar rats were observed by the Evans blue-TTC method.The pathological changes of myocardial tissue in Wistar rats were observed by HE staining.The morphological characteristics of pyroptosis in the myocardial tissue of Wistar rats were observed by TEM.DNA damage in myocardial tissue of Wistar rats was determined by TUNEL staining.The expression of GSDMD protein in the myocardial tissue of Wistar rats was detected by immunohistochemistry.The levels of serum IL-1β and IL-18 in Wistar rats were detected by ELISA.The expression of NLRP3,Caspase-1 and GSDMD mRNA and protein in the myocardial tissue of Wistar rats was detected by qPCR and WB.The morphological characteristics,marker proteins,particle size distribution,and concentration changes of plasma exosomes of Wistar rats were identified by nano TEM,WB,and NTA techniques.Results:Evans blue-TTC staining showed that the AAR/LV ratio of Wistar rats in H-QXJYF group was lower than that in Model group(P<0.05).The IS/AAR ratio of Wistar rats in L-QXJYF group,M-QXJYF group and H-QXJYF group was lower than that in Model group(P<0.05).The results of HE staining showed that the pathomorphological characteristics of myocardial tissue in H-QXJYF group were significantly improved compared with Model group.TEM results showed that compared with the Sham group,the myocardial tissue cells of Wistar rats in the Model group were significantly swollen,and a large number of bubble-like protrusions were seen outside the cells.Compared with the Model group,the myocardial tissue cells of Wistar rats in the H-QXJYF group were more complete,no obvious vesicle structure was observed,and the surrounding mitochondrial structure was normal.The results of TUNEL assay showed that the rate of TUNEL positive cells in myocardial tissue of Wistar rats in L-QXJYF group,M-QXJYF group and H-QXJYF group was lower than that in Model group(P<0.05).The results of immunohistochemistry showed that the expression of GSDMD protein in the myocardial tissue of Wistar rats in the M-QXJYF group and H-QXJYF group was lower than that in the Model group(P<0.05).ELISA showed that the levels of serum IL-1β in the L-QXJYF group,M-QXJYF group,and H-QXJYF group were lower than those in the Model group(P<0.05).The serum IL-18 level of Wistar rats in the M-QXJYF group and H-QXJYF group was lower than that in the Model group(P<0.05).QPCR showed that the relative expression of NLRP3 mRNA in myocardial tissue of Wistar rats decreased in H-QXJYF group compared with Model group(P<0.05).The relative expression of Caspase-1 mRNA in myocardial tissue of Wistar rats decreased in H-QXJYF group compared with Model group(P<0.05).The relative expression of GSDMD the mRNA in the myocardial tissue of Wistar rats in the M-QXJYF group and H-QXJYF group was lower than the that in Model group(P<0.05).The results of WB showed that the relative expression of NLRP3 protein in myocardial tissue of Wistar rats was lower in H-QXJYF group than in Model group(P<0.05).The relative expression of Caspase-1 protein in myocardial tissue of Wistar rats in L-QXJYF group,M-QXJYF group and H-QXJYF group was lower than that in Model group(P<0.05).The relative expression of GSDMD protein in myocardial tissue of Wistar rats was decreased in L-QXJYF group,M-QXJYF group and H-QXJYF group compared with Model group(P<0.05).Nano TEM identification showed that the plasma exosomes of Wistar rats showed a very significant membrane structure,which was saucer-like or concave hemisphere-like;the results of WB showed that the marker proteins CD81 and TSG101 of plasma exosomes of Wistar rats in Sham group,Model group,L-QXJYF group,M-QXJYF group,and H-QXJYF group were positively expressed.The results of NTA showed that the peak diameter of plasma exosomes in Wistar rats in the Model group was 105nm,and the peak diameter of plasma exosomes in Wistar rats in the other groups was 135nm.There was no significant difference in the concentration of plasma exosomes in each group(P>0.05).Conclusion:Qingxin Jieyu Formula can inhibit the pyroptosis level of myocardial tissue cells in MI/RI Wistar rats,reduce myocardial AAR and IS,inhibit inflammatory response,improve the survival state of myocardial cells,and promote the repair of damaged myocardium to exert myocardial protection.The mechanism may be related to the regulation of NLRP3/Caspase-1/GSDMD signaling pathway.Part 3 Effect of Qingxin Jieyu Formula and its induced circulating exosomes on pyroptosis of H9C2 cardiomyocytesObjective:To observe the effects of Qingxin Jieyu Formula and its induced circulating exosomes on pyroptosis level,inflammatory factor level in cell supernatant,and mRNA and protein expression of core molecules in classical pyroptosis pathway in H9C2 cardiomyocytes after oxygen-glucose deprivation/reperfusion(OGD/R).Methods:According to the administration and modeling,the rats were divided into the Normal group,Model group,QXJYF group,MCC950 group,circulating exosomes induced by M-QXJYF group in Wistar rats(Exo-medium)group,and circulating exosomes induced by Model group in Wistar rats(Exo-model)group.After pretreatment with corresponding drugs for 48 h,each group was replaced with a sugar-free medium and placed in a three-gas incubator.After 4h of hypoxia and glucose deprivation,it was replaced with a complete medium and placed in a conventional incubator for 2h of reoxygenation to construct an OGD/R H9C2 cardiomyocyte model.The CCK8 method was used to determine the relative survival rate of H9C2 cardiomyocytes to screen the optimal treatment concentration of Qingxin Jieyu Formula-containing serum.Dil and Cell Tracker Green stains were used to stain exosomes and H9C2 cardiomyocytes,and the uptake of exosomes was observed by co-culture of exosomes and H9C2 cardiomyocytes.The LDH efflux of H9C2 cardiomyocytes was detected by CytoTox96?.The morphological characteristics of pyroptosis in H9C2 cardiomyocytes were identified by TEM.The pyroptosis of H9C2 cardiomyocytes was detected by AO/EB staining,7-AAD,and PI staining flow cytometry.The levels of IL-1β and IL-18 in the supernatant of H9C2 cells were determined by ELISA.The expressions of NLRP3,Caspase-1,and GSDMD mRNA and protein in H9C2 cardiomyocytes were detected by WB and qPCR.Results:CCK8 method showed that the optimal intervention concentration of serum containing Qingxin Jieyu Formula was 3%.It was found that exosomes could be successfully taken up by H9C2 cardiomyocytes by co-culture of labeled exosomes and H9C2 cardiomyocytes.The results of TEM showed that H9C2 cardiomyocytes were swollen and swollen,and a large number of vesicles were formed in the cells,and the cell membrane was ruptured,accompanied by vesicles outflow.CytoTox96 ?test results showed that the LDH efflux rate of H9C2 cardiomyocytes was lower in QXJYF group,MCC950 group and Exo-medium group than in Model group(P<0.05).AO/EB staining showed that the positive rate of EB staining in H9C2 cardiomyocytes was lower in QXJYF group,MCC950 group and Exo-medium group than in Model group(P<0.05).The results of flow cytometry(PI staining)showed that the positive rate of PI staining in H9C2 cardiomyocytes in the QXJYF group,MCC950 group,and Exo-medium group was significantly lower than that in the Model group(P<0.05).Flow cytometry(7-AAD staining)showed that the positive rate of 7-AAD staining in H9C2 cardiomyocytes was lower in QXJYF group,MCC950 group and Exo-medium group than in Model group(P<0.05).ELISA showed that the contents of IL-1β and IL-18 in the supernatant of H9C2 cardiomyocytes in the QXJYF group,MCC950 group,and Exo-medium group were lower than those in the Model group(P<0.05).QPCR showed that the relative expression of NLRP3 mRNA in H9C2 cardiomyocytes was lower in QXJYF group,MCC950 group and Exo-medium group than in Model group(P<0.05).The relative expression of Caspase-1 mRNA in H9C2 cardiomyocytes was lower in QXJYF group,MCC950 group,Exo-medium group and Exo-model group than in Model group(P<0.05).The relative expression of GSDMD mRNA in H9C2 cardiomyocytes in QXJYF group,MCC950 group and Exo-medium group was lower than that in Model group(P<0.05).WB showed that the relative expression of NLRP3 protein in H9C2 cardiomyocytes was lower in QXJYF group,MCC950 group and Exo-medium group than in Model group(P<0.05).The relative expression of Caspase-1 protein in H9C2 cardiomyocytes was lower in QXJYF group,MCC950 group and Exo-medium group than in Model group(P<0.05).The relative expression of GSDMD protein in H9C2 cardiomyocytes was lower in QXJYF group,MCC950 group and Exo-medium group than in Model group(P<0.05).Conclusion:Qingxin Jieyu Formula and its induced circulating exosomes may inhibit pyroptosis of H9C2 cardiomyocytes by regulating NLRP3/Caspase-1/GSDMD signaling pathway to alleviate OGD/R injury. |
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