Piceatannol Protects Against Myocardial Ischemia/Reperfusion Injury By Inhibiting Ferroptosis Via Nrf-2 Signaling-Mediated Iron Metabolism

Objective:To study the protective effect of picatanol on myocardial ischemia-reperfusion injury in mice,and to further explore whether picatanol regulates iron metabolism in cardiomyocytes and reduces the level of intracellular free ferrous ions through the Nrf-2 pathway to inhibit ferroptosis in cardiomyocytes The protective effect and mechanism of mediating myocardial ischemia-reperfusion injury in mice This study tried to explore the protective effect of piceatanol in inhibiting cardiomyocyte ferroptosis mediated cardiac ischemia-reperfusion injury from a new perspective,and has a clinical effect on the prevention and treatment of myocardial ischemia-reperfusion injury.Blood reperfusion injury provides theoretical and experimental basis.Methods:1.Male C57B/L6 mice were randomly divided into Sham operation group,I/R group,I/R+PCT10（mg/kg）group,I/R+PCT20（mg/kg）group,I/R+PCT40（mg/kg）group,PCT was administered by intraperitoneal injection 3 hours before the operation.Mice were anesthetized by intraperitoneal injection of 1.2%tribromoethanol（0.2ml/10 g）,and ventilated with an animal ventilator after tracheal intubation.Connect lead II electrocardiogram（ECG）to observe ST segment elevation.The chest cavity was opened and the heart was exposed at the fourth intercostal space in the midline of the left subclavian.The left anterior descending coronary artery（LAD）was ligated for30 minutes and reperfused for 2 hours.The Sham operation group underwent the same operation without ligation of the LAD.Echocardiography was used to detect cardiac function changes;HE and Prussian blue staining were used to detect myocardial histopathological damage and iron ion levels;immunohistochemistry was used to detect the expression of related proteins;Western blot was used to detect Nrf-2,ACSL4,HO-1,and Tf R-1,FPN1,GPX4 and other protein expression levels.2.The cells were randomly divided into the following experimental groups:（1）Control,（2）H/R group,（3）H/R+PCT-10μM group,（4）H/R+PCT-20μM group,（5）H/R+PCT-40μM group.To explore the role of Nrf-2 in H/R and compare the role of PCT,we used the Nrf-2 inhibitor ML385 and performed another experiment with the following groups:（1）Control,（2）H/R group,（3）H/R+PCT-40μM group,（4）H/R+ML385（1μM）group,（5）H/R+ML385+PCT group.Control group AC16 cells were cultured in normal medium under normal atmospheric conditions（37°C,21%O₂,5%CO₂）.In the H/R group,AC16 cell culture medium was replaced with Krebs-Ringer bicarbonate buffer（KRB）,and cultured in an anoxic incubator（37°C,1%O₂,5%CO₂）for 5 hours,Afterwards,the KRB buffer solution was replaced with ordinary medium,and incubated for another 2 hours under normoxic conditions.Cell viability and cell damage were detected by CCK-8 and LDH;lipid peroxidation products and ROS levels were detected by flow cytometry;expression levels of key proteins were detected by immunofluorescence;mitochondrial changes and non-dissociated iron lattices were observed by transmission electron microscopy,The expression levels of Nrf-2,ACSL4,HO-1,Tf R-1,FPN1,GPX4 and other proteins were detected by Western blot.Results:1.Compared with the sham operation group,after I/R treatment,the ST segment of the electrocardiogram of the mice was significantly elevated,and the LVEF and LVFS values of the mouse heart were significantly decreased.The PCT treatment group could inhibit the ST segment elevation of the electrocardiogram of the mice after I/R,and the mice Cardiac LVEF and LVFS values were higher than I/R group.The detection results of serum LDH and MDA in mice showed that compared with the Sham group,the levels of LDH and MDA in the serum of mice in the I/R group were significantly increased,and the activity of SOD was significantly decreased.Compared with the I/R group,serum levels of mice in the PCT treatment group The release of LDH was significantly reduced,the level of MDA was reduced,and the activity of SOD was significantly increased.The results of HE staining of mouse heart tissue showed that vacuoles and a large number of inflammatory cell infiltration appeared in the cardiomyocytes of the mice in the I/R group.Improve myocardial pathological injury after I/R.The results of Prussian blue staining showed that PCT could significantly reduce the iron ion in myocardial tissue after I/R.The results of immunohistochemistry showed that the expression of Tf R-1 protein in the myocardial tissue of the mice in the I/R group was significantly increased,and the expression of FPN1 was significantly decreased.The results of Westren Blot showed that the expression of Nrf-2,HO-1 and FPN1 in the heart tissue of mice in the I/R group were significantly down-regulated,and the expressions of Tf R-1 and ACSL4 were increased,and the expression of Nrf-2,HO-1.The expression of FPN1 protein was significantly up-regulated,while the expression of Tf R-1 and ACSL4 was down-regulated.2.The results of CCK-8 and LDH detection showed that PCT could significantly increase the viability of AC16 cardiomyocytes after H/R and inhibit the release of LDH,suggesting that PCT had a protective effect on AC16 cardiomyocytes after H/R.The results of fluorescence photography and flow cytometry detection of ROS and lipid peroxidation products showed that PCT could significantly reduce the peroxidation products of AC16 cells after H/R.The detection results of Fe²⁺fluorescent kit showed that the free Fe²⁺level in AC16 cells decreased significantly after PCT treatment.The results of absorbance,magnetic detection and lattice diffraction fringes of PCT and Fe²⁺reaction products show that PCT can convert free Fe²⁺into non-free Fe₃O₄.Immunofluorescence results showed that,compared with the H/R group,PCT could up-regulate the expression of Nrf-2 and FPN1 and down-regulate the expression of Tf R-1 in AC16 cells after H/R.The results of Westren Blot showed that,compared with the H/R group,the expression levels of Nrf-2,FPN1,and HO-1 in AC16 cells in the PCT treatment group were significantly up-regulated,and the expression levels of Tf R-1 and ACSL4 were significantly down-regulated.Conclusion:The results of the study show that PCT can regulate iron metabolism through Nrf-2 and convert free iron into non-free iron,thereby reducing the level of free iron in cells and alleviating MIRI-induced ferroptosis in cardiomyocytes.