| With the development of China’s economy and the improvement of people’s living standards,dietary structure and environmental factors have changed.The incidence of cardiovascular disease as the representative of major chronic diseases were increasing.The incidence of hypertension,atherosclerosis and other diseases has increased every year.Accompanied by acute myocardial infarction(AMI)incidence increased every year,accompanied by younger trend.Percutaneous coronary intervention(PCI)and other reperfusion treatment schemes can restore blood flow in time,greatly reducing the degree of heart damage and disease mortality.However,the occurrence of reperfusion-induced myocardial stunning,microvascular dysfunction,no reflow phenomenon,and reperfusion arrhythmia have weakened the patients’ myocardial benefits.This myocardial damage caused by ischemia and reperfusion is called Ischemia/Reperfusion Injury(I/R Injury).Mainly manifested as myocardial cell death and impaired cardiac function.The mechanism of is very complicated,including calcium overload,oxidative stress,inflammatory response,etc.How to find the key links and key molecules of myocardial injury after reperfusion and develop new drugs or treatments on this basis has been a thorny problem that has plagued basic research and clinical treatment.Studies have shown that changes in gut microbes is closely related with a variety of cardiovascular diseases.Including atherosclerosis,hypertension,hyperlipidemia,and myocardial infarction.Recently many papers reported that acute ST-segment elevation myocardial infarction due to insufficient blood supply to the intestinal permeability changes in patients with myocardial infarction lead to intestinal bacterial translocation into the blood,resulting in "class sepsis-like response",and after myocardial infarction without conscience closely related vascular events,myocardial infarction antibiotic treatment can significantly reduce infarct size in mice and expression of inflammatory cytokines.However,the mechanism of gut microbes’participation in I/R Injury remains to be further elucidated.As the first line of defense of the human body’s natural immunity,neutrophils have a very fast response to microbial invasion.It is generally believed that neutrophils resist microbial invasion by phagocytosing and releasing antimicrobial peptides.In recent years,researchers have discovered a new mechanism of neutrophil antibacterial-Neutrophils Extracellular Traps(NETs).The production of NETs can help capture and kill bacteria.However,there is increasing evidence that NETs also play a role in non-communicable diseases such as autoimmune diseases,arteriovenous thrombosis,malignancies,atherosclerosis and I/R Injury It plays an important role,and a large number of articles have reported that NETs can be a new therapeutic target in I/R Injury.We hypothesis that the intestinal flora is translocated into the blood after myocardial ischemia to form a "septicemia-like reaction",which causes a large number of NETs and NETs-related micro thrombosis,and further causes myocardial tissue microcirculation dysfunction to further increase myocardial damage.In this study,we used intestinal microorganism clearance mice and neutrophil clearance mice to construct a model of myocardial ischemia-reperfusion to observe the relationship between intestinal microorganisms and NETs and I/R Injury.The neutrophils were isolated by in vitro experiments to explore the role and molecular mechanism of intestinal flora translocation into the blood to aggravate myocardial cell injury.The mouse myocardial ischemia/reperfusion model was successfully constructed,and the mouse myocardial tissue was observed by immunofluorescence,and there were a large number of NETs in the tissue.In order to observe the effect of NETs on ischemia/reperfusion injury,we cleared mouse neutrophils by Ly6g neutralizing antibody,and confirmed the effect of neutrophil clearance by flow cytometry.Further using neutrophil-clearing mice to construct ischemia-reperfusion models,the results of cardiac TTC staining showed that the removal of neutrophils can reduce the area of myocardial infarction.In order to confirm whether there is a link between intestinal microbes and myocardial ischemia/reperfusion injury,we fed mice with quadruple antibiotic solution to eliminate intestinal microbes.Mice with significant clearance effect were used to construct a model of cardiac ischemia-reperfusion.The results of TTC staining of the heart showed that the removal of intestinal microorganisms can also reduce the area of myocardial infarction.In experiments that confirmed the association between intestinal microbial clearance and the formation of NETs after cardiac ischemial reperfusion,we first used mice with double clearance of intestinal microbes and neutrophils to construct a model of cardiac ischemia-reperfusion.The results of cardiac TTC showed that the area of myocardial infarction did not decrease further.Immunohistochemical staining was used to detect the infiltration of neutrophils and the formation of NETs.The results showed that the number of neutrophil infiltration in the myocardial tissue in mice with I/R after the removal of intestinal microorganisms decreased,and the number of NETs formed decreased.The content of MPO and citH3-DNA complexes in the serum of mice in the gut microbial clearance group was detected by ELISA,and it was found that the content of the above two molecules decreased after the gut microbial clearance,and there was a significant difference.To investigate the time points of NETs formation in tissues and blood after cardiac ischemia/reperfusion injury,we used WB to detect the content of citH3 in tissues at different time points.The content of MPO and citH3 in serum at different time points were detected by ELISA.The results showed that after cardiac I/R occurred.Tissue citH3 expression increased.Gray-scale analysis showed that the expression level reached the highest level after 3 hours of reperfusion.ELISA results showed that the content ofNETs in serum reached the highest after 12 hours of reperfusion.The MPO content reached its highest level 3 hours after re-irrigation.This difference in time may be due to the inconsistent release order of the different components of NETs.In experiments to study how NETs damage tissues,we successfully isolated neutrophils with higher purity and induced NETs in vitro.NETs were used to stimulate NRVM and CMEC cells,WB was used to detect apoptosis-related proteins,and TUNEL was used to detect apoptosis.The results show that NETs can induce apoptosis in NRVM and CMEC.In Conclusion,we find that intestinal microbes participate in cardiac ischemia-reperfusion injury by regulating the formation of NETs,and NETs triggered cardiomyocyte and microvascular endothelial cell apoptosis... |
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