Mutual Regulation Between CMECs And Myocardial Cells In Co-culture System

Ischemic heart disease?IHD?,such as myocardial infarction,stroke and vascular disease are among the most common causes of death and disability in the world.The degree of tissue damage is directly related to the degree of blood flow and the time of ischemia^[1].Drug thrombolysis,percutaneous coronary intervention,and coronary artery bypass grafting are currently important treatment options for IHD and are important for reducing the area of myocardial infarction and improving cardiac function.However,even if the coronary blood flow is improved,there are clinically significant reductions in microvascular perfusion,chronic heart function impairment,and dilated cardiomyopathy caused by microvascular damage.Ischemia/reperfusion injury?I/RI?is a pHenomenon that causes the microvascular endothelial dysfunction and myocardial cell damage to be aggravated by the restoration of coronary blood flow.About 30%of patients with acute myocardial infarction undergoing reperfusion therapy have this pHenomenon^[2].At the same time,the sudden restoration of blood flow can also cause some micro-vascular obstruction.This pHenomenon is called no-reflow pHenomenon^[3].No reflow results in poor prognosis and is associated with increased infarct size,congestive heart failure,and increased mortality^[4].The key to the pathogenesis of myocardial I/R injury is endothelial cell microvascular dysfunction,in which cardiac microvascular endothelial cells?CMECs?play an important role^[5].Therefore,understanding the mechanism of action of CMECs in the I/R process may provide new therapeutic directions for IHD.CMECs are key interfaces between blood vessels and vessel walls.They have an important function of regulating the balance of tissue fluids and providing the necessary nutrients for the survival of organisms.They are the main noncardiomyocytes in the heart.Previous studies have demonstrated that reperfusion induced CMECs to release soluble pro-apoptotic mediators to promote cardiomyocyte apoptosis^[5],suggesting that CMECs play a key role in triggering myocardial cell injury during myocardial I/R injury.In addition,CMECs apoptosis precedes cardiomyocytes during I/R,and apoptotic cardiomyocytes surround the apoptotic CMECs^[6],and in vitro CMECs can secrete IL,TNF?,MCP,ET,and Ang II to exacerbate myocardial injury,and also secrete some cytokines to protect cardiomyocytes,such as NO.In summary,the interaction of CMECs-cardiomyocytes plays an important role in cardiac MI/RI,but the specific mechanism of interaction is not yet clear.In this experiment,a co-culture model of CMECs-H9c2 was established to simulate hypoxia/reoxygenation?H/R?model by simulating ischemia/reperfusion injury.Observe whether CMECs and H9c2 interact with each other under H/R conditions.Initially explore its mechanism of action.Method?1?Construction of rat I/R model:Rats were ligated for 1 hour and left for 24 hours along the left anterior descending coronary artery,and blood samples were t-aken before and after operation;staining of HE was used to observe the changes of cardiac morpHology;staining of TUNEL was used to observe the damage of heart tiss ue in rats.?2?Compare the expression of NO and ET-1 in rat I/R group and sham group:the expression of NO and ET-1 in rat serum was detected by kit.?3?Isolation and identification of CMECs:CMECs were isolated by enzymati -c digestion,and CMECs were identified by immunofluorescence and Matrigel Matrige l-induced angiogenesis experiments.?4?Construction of CMECs/H9c2 hypoxia/reoxygenation model:Hypoxia/reoxyg enation models of CMECs/H9c2 were established using hypoxia workstations,and their corresponding indicators were tested.?5?Effect of CMECs on H9c2:Collect H/R supernatants from CMECs and apply them to H9c2,LDH,MTS and RT-PCR to detect the effect of CMECs on H9c2;construct H9c2 and CMECs to co-culture H/R system,WB detected H9c2 apoptosis-related protein expression.?6?Effect of H9c2 on CMECs:H9c2 cell culture supernatant was collected after H/R,and it was applied to H/R-received CMECs.LDH,MTS,Transwell,and RT-PCR were used to detect the effect of H9c2 on CMECs.The H/R system was co-cultured with H9c2 and CMECs,and the expression of inflammatory cytokines in CMECs was detected by RT-PCR.?7?The mechanism of the interaction between H9c2 and CMECs was preliminarily explored:The content of NO and ET-1 in the culture medium of CMECs before and after H/R was detected by the kit.Result?1?HE staining:In the rat I/R group,the myocardial tissue was disordered,the blood vessel was damaged,and there was inflammatory cell infiltration;TUNEL staining:Compared with the sham group,the TUNEL positive signal of the myocardial and vascular cells in the infarcted area of the model group was significantly increased.?3?Serum NO and ET-1 results showed that compared with sham group,NO and ET-1 expression in model group increased,but NO/ET-1 decreased.?4?Successful establishment of a stable platform for isolation,identification and in vitro expansion of adult rat CMECs.?5?After H/R CMECs cell culture supernatants were treated with H9c2,MTS and LDH assays showed that the supernatants of CMECs after H/R promoted the growth of H9c2and reduced the H9c2 hypoxia/reoxygenation injury;RT-PCR The results showed that the supernatant of culture medium after CMECs H/R inhibited the expression of inflammatory factors after H9c2 H/R;WB assay results showed that the expression of apoptosis-related proteins decreased after H9R2 co-cultured with CMECs.?6?After H/C/H9c2 cell culture supernatants act on CMECs after H/R,MTS and LDH assay results show that H9c2 H/R culture supernatant aggravates hypoxia/reoxygenation injury of CMECs;RT-PCR results show H9c2 H/R After culture supernatants promoted the expression of inflammatory factors after hypoxia/reoxygenation of CMECs;Transwell assay showed that the culture medium after H9c2 H/R inhibited the migration of CMECs.Conclusion?1?Injury of myocardial cells and vascular endothelial cells in the infarcted region of rat I/R heart was intensified,and the expression of NO and ET-1 were abnormal;?2?After anoxia/reoxygenation of CMECs,the supernatant was protected agai nst H9c2,and H9c2 was co-cultured with CMECs to reduce H9c2 injury after hypoxi a/reoxygenation.?3?After hypoxia/reoxygenation of H9c2,the supernatants aggravated hypoxia/reoxygenation injury of CMECs,and CMECs and H9c2 co-cultured damaged CMECs after hypoxia/reoxygenation.