Study Of The Effect Of CGRP Released From Nitroglycerin On Myocardial Ischemia-reperfusion Via Ferroptosis

Background:Myocardial ischaemia-reperfusion injury（MIRI）is the damage caused to myocardial cells or tissues when reperfusion is restored after being subjected to ischaemia and hypoxia.It is now believed that MIRI is one of the main reasons for limiting the effectiveness of various treatments to save acute myocardial infarction.Nitroglycerin is the classic drug for myocardial infarction,but its rapid tolerability and the potential for MIRI are limiting factors,meanwhile,the molecular mechanisms involved have not been investigated;MIRI occurs when myocardial cells and tissues produce large amounts of ROS and leads to structural changes in mitochondrial function,which are closely linked to the development of ferroptosis.Calcitonin gene-related peptide（CGRP）is the most potent endogenous vasodilator in humans and has been shown to be associated with the action of nitroglycerin.The present study is intended to demonstrate at the animal and cellular levels that CGRP release facilitated by nitroglycerin is associated with MIRI and that CGRP exerts a protective effect by improving mitochondrial function at the onset of MIRI and thus influencing the onset of ferroptosis,providing new insights into the mechanism of action of nitroglycerin and the search for alternative drugs to nitroglycerin.Objectives:（1）To investigate the protective role of CGRP released by nitroglycerin in MIRI;（2）To determine the structural changes in mitochondrial function and the development of ferroptosis during MIRI;（3）To explore the mechanism that CGRP regulates the development of ferroptosis through the improvement of mitochondrial function.Methods:（1）Ischemia-reperfusion models were established using SD rat and rat cardiomyocyte lines（H9C2）using anterior coronary descending artery ligation and hypoxia/reoxygenation,respectively.And TTC staining was performed to detect the effects of nitroglycerin,cinnamaldehyde,CGRP8-37,and HC030031 on infarct size,as well as changes in serum CK,CGRP,and LDH;（2）After experiencing I/R or H/R model injury,the addition of CGRP as well as the ferroptosis inhibitor Ferrostatin-1,JC-1 probe and DCFH-DA detected the changes in mitochondrial membrane potential and ROS production after H/R;transmission electron microscopy directly observed the structural changes of mitochondria,and Western blot detected the expressions of mitochondrial split fusion proteins DRP-1and ferroptosis-related proteins:ACSL4、GPX4、Fsp1 and DHODH;also measuring the concentration of Fe³⁺and Fe²⁺and the change of LPO.Results:（1）Compared with the I/R model group,NTG significantly reduced the infarct area and serum CK values in myocardial ischemia-reperfusion.When NTG was administered together with the CGRP-specific antagonist CGRP8-37,myocardial infarct area increased significantly and CK values increased significantly in rats.（2）The expression of DRP-1 was significantly reduced;JC-1 and ROS were detected:compared with the control group,the decrease in cell number in the model group was accompanied by a significant decrease in red fluorescence,a significant increase in green fluorescence and a significant increase in ROS levels.At the same time,after the addition of Ferrostatin-1,an ferroptosis inhibitor,the red fluorescence was increased and the green fluorescence and ROS levels were decreased compared with the model group;both Fe²⁺and total iron content were significantly increased in the model group compared with the control group,and the increase in Fe²⁺was predominant;compared with the control group,both intra-tissue and intracellular LPO were significantly increased in the model group,and the expression of GPX4 and ACSL4;（3）Compared with the model group,400n M CGRP and 5μM Ferrostatin-1 significantly modulated the expression of GPX4、FSP1 and DHODH in H9C2cells,and this effect was counteracted by CGRP8-37;5m LFerrostatin-1significantly reduced the damaging effect of H/R on H9C2 cells,while the addition of CGRP8-37 re-induced such damage.Compared with the H/R model group,400n M CGRP and 5μM Ferrostatin-1 maintained normal mitochondrial membrane potential and ROS levels in H9C2 cells during H/R injury,whereas the addition of CGRP8-37 disrupted this balance,leading to a decrease in mitochondrial membrane potential and a dramatic increase in ROS in H9C2 cells.Conclusions:In an SD rat ischemia-reperfusion model,nitroglycerin promotes the release of CGRP by activating TRPA1,which reduces the occurrence of ferroptosis in cardiomyocytes by improving mitochondrial function and thereby reducing MIRI injury.