SDR5-Fc Inhibits Neutrophil Infiltration And Activation Reduces Myocardial Ischemia-reperfusion Injury

Background:To avoid long-term ischemia leading to increased myocardial damage,use of thrombolysis,percutaneous coronary intervention,coronary artery bypass grafting,etc.,to restore myocardial ischemia.However,blood reperfusion can cause myocardial damage,this phenomenon is called myocardial ischemia-reperfusion injury(I/R).Multiple factors are involved in myocardial I/R injury,and neutrophils from the peripheral blood are one of the factors that cause myocardial damage.Neutrophils are a hallmark of myocardial I/R injury.When I/R occurs in the myocardium,a large number of neutrophils accumulate in the myocardial injury area.A fraction of neutrophils accumulate at the capillary of the myocardium and block the capillaries,causing a "no-reflow" phenomenon in the reperfused myocardium;the other part of the neutrophils chemotizes to the damaged myocardial tissue,which intensifies the inflammatory response of the myocardium...Our previous results showed that s DR5-Fc can reduce myocardial I/R injury,but the underlying mechanism of its protective effect is not completely clear.Infiltration and activation of neutrophils can aggravate myocardial I/R injury.Therefore,we hypothesize whether s DR5-Fc can exert its protective effect on myocardial tissue by regulating neutrophils.Purpose:By constructing an animal model of acute myocardial ischemia/reperfusion injury and Hypoxia/Reoxygenation(H/R)model of H9c2 cells,we verified whether s DR5-Fc can reduce myocardial I/R injury by inhibiting neutrophil infiltration and activation.Methods:To verify that s DR5-Fc inhibits neutrophil infiltration into damaged myocardial tissue.We established a rat model of acute myocardial infarction by ligation of rat coronary arteries.We used a small animal electrocardiograph to detect whether an acute myocardial infarction model was successfully constructed.TTC staining was used to detect whether the rat myocardial I1R24 h model was successfully constructed;by Evans blue-TTC staining,ELISA was used to detect the content of cardiac troponin I(c Tn I)to assess whether s DR5-Fc can reduce myocardial I/R injuryin rats.The level of neutrophils in myocardium was detected by MPO immunohistochemical staining to evaluate the inhibitory effect of s DR5-Fc on neutrophil infiltration;use of flow cytometry,MPO immunohistochemical staining to detect neutrophil depletion by anti-Rat PMN;by TTC staining,HE staining,TUNEL,Compare the protective effect of s DR5-Fc and Anti-Rat PMN on myocardial tissue.To verify the inhibitory effect of s DR5-Fc on neutrophil activation,we used neutrophil separation fluid to isolate rat peripheral blood neutrophils;use hypoxia workstation to establish H2R3 h model of H9c2 cells;using Hoechst,MTS to detect whether s DR5-Fc can reduce H9c2 cell damage by inhibiting neutrophil activation in co-culture system;s DR5-Fc was used to verify the activation of TRAIL-induced neutrophils by q PCR and ELISA.Results:After coronary artery ligation in rats,the ST segment of the rat’s electrocardiogram was significantly elevated,and the rat model of acute myocardial infarction was successfully constructed.TTC staining showed that the weight of the infarcted region of the rat in the I1R24 h group accounted for about 50% of the left ventricular mass,and the rat myocardial I1R24 h.Evaluate whether s DR5-Fc can reduce myocardial I/R injury in rats by Evans blue-TTC staining and ELISA;MPO immunohistochemical staining showed that s DR5-Fc could suppression of neutrophil infiltration of damaged myocardial tissue;TTC staining,HE staining,TUNEL results showed that: s DR5-Fc can inhibit the infiltration of neutrophils to reduce myocardial I/R injury in rats.These results indicate that s DR5-Fc can reduce myocardial I/R injury in rats by inhibiting the infiltration of damaged myocardium by neutrophils.In cell experiments,blood cell counters,flow cytometry,and Wright-Giemsa staining showed that the purity of isolated neutrophils accounted for more than 89%,and neutrophils were successfully isolated;the MTS and LDH results showed: In H2R3 h,the activity of H9c2 cells decreased and the release of LDH increased.Therefore,H2R3 h was selected as the best time point for H/R of H9c2 cells;Transwell assay showed that H9c2 cells had a large number of neutrophils passing from the upper chamber during H2R3 h.The lower chamber shows that H9c2 cells after H/R have a strong chemotactic effect on neutrophils;Hoechst results show that neutrophils can increase the apoptosis of H9c2 cells,s DR5-Fc can inhibit the apoptosis of H9c2 cells caused by neutrophils;MTS results showed that s DR5-Fc could reduce the damage of neutrophils to H9c2cells,indicating that s DR5-Fc can protect H9c2 cells by inhibiting the activation of neutrophils;q PCR,ELISA results show that s DR5-Fc can block TRAIL-induced neutrophil expression of TNF-α、IL-1β.The above results indicate that s DR5-Fc can reduce the damage of H9c2 cells in co-culture system by inhibiting the activation of neutrophils.Conclusion:1.s DR5-Fc reduces myocardial I/R injury by inhibiting peripheral blood neutrophil infiltration.2.s DR5-Fc reduced the damage of H9c2 cells in the co-culture system by inhibiting the activation of neutrophils.