Vitexin Attenuates Myocardial Ischemia/Reperfusion Injury By Downregulating IRhom2 To Mediate Vascular Endothelial Cell Pyroptosis

Objective:To investigate the protective effect of peuromycin-regulated iRhom2signaling-mediated vascular endothelial cell pyroptosis on acute myocardial ischemia-reperfusion injury and its potential mechanism.Methods:1.Human coronary artery endothelial cells（HCAEC）cells were divided into control group,H/R group,H/R + VT-5 μM group,H/R + VT-10 μM group and H/R +VT-20 μM group.LDH kit and CCK-8 were used to detect cell injury and cell viability.Annexin V-FITC/PI kit to detect apoptosis;immunofluorescence staining and Western blot to detect the expression of iRhom2 and GSDMD proteins.2.HCAEC cells were divided into control group,H/R group,H/R+VT-10 μM group and H/R+iRhom2-si RNA group.CCK-8 and LDH kits detected cell viability and cell damage.Annexin V-FITC/PI kits detected apoptosis.ELISA kits for TNF-α,IL-1β and IL-18 release.Real-time PCR for iRhom2,ADAM17,NLRP3,GSDMD and NF-k B m RNA expression.Immunofluorescence for iRhom2 and GSDMD protein expression.The expression levels of iRhom2,ADAM17,TNF-α,NLRP3,cleaved Caspase-1,ASC,GSDMD-N,IL-1β and IL-18 proteins were detected by Western blot.3.HCAEC cells were divided into control group,H/R group and H/R+VT-10 μM group.AC16 cells were divided into control group and H/R group.Supernatants from HCAEC cells were added to AC16 cells.The CCK-8 kit was used to detect AC16 cell viability.Western blot was performed to detect the expression levels of iRhom2,NLRP3,cleaved Caspase-1 and GSDMD-N proteins in AC16 cells.4.Fifty male C57/BL mice were randomly divided into sham-operated group,IR group,IR+VT-3 mg/kg group,IR+VT-6 mg/kg group,and IR+VT-12 mg/kg group.VT was administered intraperitoneally with oxytocin 2 h before the staging.I/R mice were modeled by ligation of the left anterior descending coronary artery of the heart for 30 minutes and reperfusion for 2 h.Electrocardiographic changes were measured by biofunctional system;ultrasound to detect changes in cardiac function.H＆E staining to observe myocardial histopathological damage.Immunohistochemistry to detect NLRP3 expression in myocardial tissue.The expression levels of iRhom2,ADAM17,TNF-α,NLRP3,cleaved Caspase-1,ASC,GSDMD-N,IL-1β and IL-18 proteins were detected by Western blot.Results:1.CCK-8 kit and LDH kit assay results showed that compared with the control group,HCAEC cells in the H/R group were damage significantly,LDH release and apoptosis leave increased,and the expression of iRhom2 and GSDMD-N protein were significantly increased.Compared with the H/R group,the H/R+VT（5,10,20 μM）group reduced cell injury and inhibited apoptosis,as well as inhibited the protein expression of iRhom2 and GSDMD-N in HCAEC cells.2.The results of iRhom2-si RNA experiments showed that compared with the control group,HCAEC cells in the H/R group were damaged,with increased LDH release and apoptosis leave,as well as increased levels of inflammatory factors TNF-α,IL-1β and IL-18 in the supernatant.In addition,iRhom2 m RNA expression was upregulated in cells,causing the activation of ADAM17,leading to its downstream NLRP3,NF-κB,GSDMD m RNA expression was upregulated.While iRhom2 protein expression was increased in cells,downstream ADAM17 protein was actived,and TNF-α protein expression was increased,that leading to NLRP3 inflammatory vesicle signaling activation in cells,and its downstream ASC,cleaved Caspase-1,GSDMD-N,IL-1β and IL-18 protein expression was increased.Compared with the H/R group,the H/R+iRhom2-si RNA group attenuated cell injury,inhibited apoptosis,and decreased TNF-α,IL-1β and IL-18 release.The iRhom2 m RNA expression was down-regulated after silencing,ADAM17 m RNA expression was inhibited,and its downstream m RNA expression of NLRP3,NF-κB,and GSDMD was significantly down-regulated.At the same time,iRhom2 protein expression was reduced,inhibited ADAM17 protein activation,decreased TNF-α protein expression,and suppressed its downstream NLRP3,ASC,cleaved Caspase-1,GSDMD-N,IL-1β,and IL-18 protein expression.The H/R+VT-10 μM group produced the same effect as the H/R+iRhom2-si RNA group with similar effects,both inhibiting the H/R-induced inflammation and cell pyroptosis pathways.3.AC16 cells were pretreated with H/R（with or without VT-10 μM pretreatment）followed by HCAEC cells supernatant.The results showed that normal AC16 cells,incubated with HCAEC cells supernatant after H/R,decreased AC16 cells viability,upregulated cellular iRhom2 protein expression,and activated its downstream NLRP3 inflammatory vesicle signaling,causing caspase-1 activation and increased GSDMD-N shear;normal AC16 cells incubated with VT-10 μM pretreated H/ R followed by incubation with HCAEC cells supernatant inhibited the decrease in AC16 cells viability and decreased expression of the above-mentioned related proteins.In addition,incubation of hypoxia-reoxygenated AC16 cells with the supernatant of HCAEC cells after H/R revealed a significant decrease in cardiomyocyte viability,increased cell injury,significant upregulation of intracellular iRhom2 protein expression,and activation of downstream NLRP3 inflammatory vesicle signaling,causing increased caspase-1 activation and GSDMD-N shear,and incubation with VT-10 μM pretreated AC16 cells after H/R incubation with HCAEC cell supernatant showed that AC16 cells showed increased viability and reduced injury,and inhibited iRhom2 protein upregulation and the cardiomyocyte pyroptosis pathway.4.Compared with the Sham group,mice in the model group had significantly elevated ST segment of ECG.The serum LDH activity and MDA content increased significantly,SOD activity decreased,and the contractile function of the left anterior wall of mouse heart decreased significantly;compared with the I/R group,the VT treatment group could inhibit ST-segment elevation,inhibit LDH leakage,reduce MDA content and increase SOD activity.In addition,VT could significantly improve the contractile function of the left anterior wall of mouse heart.The H＆E results showed that VT reduced histopathological damage in the myocardium of mice compared with the I/R group.Immunohistochemical results showed that NLRP3 protein expression level in myocardial tissues of mice in the I/R group was significantly higher than that in the Sham group,and NLRP3 expression level in myocardial tissues of mice in the VT-treated group was decreased.Westren Blot results showed that iRhom2 protein expression was upregulated,ADAM17 activation,TNF-α protein expression was increased in myocardial tissues of mice in the I/R group compared with the Sham group.The activation of NLRP3 inflammatory vesicle signaling downstream caused a significant increase in ASC,cleaved Caspase-1 and cell pyroptosis executive protein GSDMD-N,IL-1β and IL-18 protein expression.VT inhibited the upregulation of iRhom2 protein expression,suppressed ADAM17 activation and decreased TNF-α protein expression in myocardial tissue of mice and inhibited cardiac myocyte pyroptosis pathway.Conclusion:The results of this study suggest that VT has a protective effect on vascular endothelial cells after H/R and attenuates acute myocardial ischemia-reperfusion injury,and the underlying mechanism may be through the regulation of iRhom2 signaling,which inhibits vascular endothelial cells and myocardial pyroptosis,thus attenuating myocardial ischemia-reperfusion injury.