| Research background: Myocardial ischemia reperfusion injury（MIRI）is generally caused by partial or complete occlusion of coronary arteries caused by the shedding or accumulation of atherosclerotic plaques,and then regains recanalization within a certain period of time.and related tissue damage.Its specific mechanism is not very clear,but recent studies have found that inflammatory response plays a very important role in myocardial ischemiareperfusion injury."Endothelial insufficiency or dysfunction" is the pathological basis for the occurrence and development of various cardiovascular diseases,including ischemic heart disease.Protecting endothelial cell function is an important way to prevent and treat cardiovascular diseases.Vitexin（VT）is a natural flavonoid drug with strong anti-MIRI effect and can be used for the treatment of cardiovascular diseases such as angina pectoris and hyperlipidemia,but the detailed mechanism is still unclear.So far,there is no research report on the interaction of VT on coronary endothelial cells and cardiomyocytes.Therefore,this project aims to observe the changes and regulation of inflammation-related factors in vascular endothelial cells and cardiomyocytes and their relationship with cardiomyocyte pyroptosis by establishing ischemia,hypoxia and reperfusion models in cells and animals.Part 1: The role of HIF-1α/i RHOM2 signaling in the inflammatory response induced by hypoxia and reoxygenation of vascular endothelial cells and the molecular mechanism that mediates the regulation of cardiomyocyte pyroptosis after myocardial ischemiareperfusion in miceObjective:1.To study the role of HIF-1α/i RHOM2 signaling in the inflammatory response of vascular endothelial cells induced by hypoxia and reoxygenation.2.To clarify the molecular mechanism of vascular endothelial cell HIF-1α/i RHOM2 signaling mediating the regulation of cardiomyocyte pyroptosis after myocardial ischemiareperfusion in mice.Method:The differential expression of different genes and the relationship between proteins were predicted by bioinformatics analysis.The primary mouse CECs were isolated by collagenase digestion and time gradient method,and the H/R model of primary CECs was established.WB,RT-PCR,and immunofluorescence were used to detect the effect of BAY,i Rhom2-si RNA and IOX2 on H/R under different conditions.Effects of HIF-1α,NFκB（P65）,i Rhom2 and TNF-α protein and m RNA expression in CECs after R;the interaction between i Rhom2 and NFκB（P65）and HIF-1α in CECs after H/R was detected by co-IP;ELISA detected the changes of TNF-α,IL-1α and IL-1β in the supernatant of CECs after H/R with different treatments;TUNEL and flow cytometry were used to detect the effects of different treatments on the apoptosis of CECs after H/R;The effect of different treatments on the viability of CECs after H/R was detected by CCK-8 method;the effect of different treatments on the barrier function of CECs after H/R was detected by FITC-fluorescence permeation assay;the TNF-α receptor inhibitor Infliximab（IN）was detected by WB method The effects of HIF-1α,i Rhom2,TNF-α and i Rhom2 in CECs after H/R;the expression of HIF-1α,NFκB（P65）,ADAM17 and i Rhom2 and ADAM17 in CECs after H/R were detected by immunofluorescence assay.Interaction with i Rhom2 protein.After the co-culture model was established by culturing HCAEC and AC16 cell lines by passage method,the effect of TNF-α receptor inhibitor IN on the expression of GSDME-NT after H/R in AC16 cells cultured alone and co-cultured with HCAECs was detected by immunofluorescence assay;ELISA The effect of IN on the contents of TNF-α,IL-1α and IL-1β in the supernatant after H/R of HCAECs cells and AC16 cells cultured alone or co-cultured,respectively,and the effect of i Rhom2-si RNA transfection on the expression of HCAECs cells and AC16 cells were detected by the method.The effect of TNF-α content in cell supernatant after H/R;the changes of AC16 cell morphology and the formation of pyroptotic pores after co-culture were observed by electron microscope.After the myocardial I/R injury model of C57 mice was established by ligation of left anterior descending artery,the changes of myocardial pNFκB（P65）,activated Caspase3 and GSDME-NT of I/R mice were detected by immunohistochemistry;Changes in the expression of GSDME-NT,activated Caspase-3 and IL-1β proteins in the myocardium of I/R mice;the changes of cardiac function in I/R mice induced by BAY were detected by echocardiography;the effects of BAY on LDH in I/R mice were detected by ELISA,NO,i NOS and ET-1 serum levels;HE staining was used to detect the cardiac pathological changes in mice after I/R;immunohistochemistry was used to detect the effect of BAY and i Rhom2 knockdown on myocardial HIF-1α in I/R mice and i Rhom2 protein expression;the effects of BAY and i Rhom2 knockdown on myocardial IL-6,TNF-α and NFκB（P65）protein expression in I/R mice were detected by immunofluorescence.Result:1.Gene expression comprehensive（GEO）analysis showed that the gene expression of endothelial cells HIF-1α,i Rhom2 and inflammation-related factors were significantly changed in the I/R model.2.In the mouse primary CECs model,WB,RT-PCR,and immunofluorescence assays showed that compared with the control group,the m RNA and protein expressions of TNF-α,NFκB（P65）in CECs cells in the H/R group were up-regulated,and the nuclear translocation of NFκB（P65）was significantly increased.,HIF-1α inhibitor BAY and i Rhom2 knockdown can significantly reverse the up-regulation of TNF-α,NFκB（P65）m RNA and protein expression,and inhibit NFκB（P65）nuclear translocation;The co-IP experiment results showed that i Rhom2 did not aggregate with HIF-1α,but interacted with NFκB（P65）;CCk-8 detection results showed that BAY and i Rhom2-si RNA transfection could significantly enhance the cell viability of CECs after H/R,and the apoptosis results showed that BAY or i Rhom2-si RNA transfection could significantly inhibit the apoptosis of CECs after H/R;2.4 Fluorescein endothelial barrier assay and ELISA results showed that BAY or i Rhom2-si RNA transfection could enhance the cell barrier function of CECs after H/R and inhibit IL-1α,IL-1β and TNF-α in the supernatant of CECs after H/R level;2.5WB and immunofluorescence results showed that compared with the control group,IN could significantly inhibit the protein expressions of NFκB（P65）,TNF-α,ADAM17 and i Rhom2 in CECs after H/R;3.In the AC16 s and HCAECs co-culture model,ELISA results showed that compared with the H/R control group,IN could significantly reduce the levels of TNF-α,IL-1α,and IL-1β in the supernatant of AC16 s after H/R;After R,the levels of TNF-α,IL-1α,and IL-1β in the supernatant were further increased,and IN could reverse the increased levels of inflammatory factors in co-cultured AC16 s.The levels of inflammatory factors released in H/R A16 s cells were significantly lower than those in HCAECs cells;The results of immunofluorescence detection showed that AC16 s co-cultured with HCAECs could further promote the expression of GSDME-NT after H/R,and IN could also reverse the upregulation of GSDME-NT expression of co-cultured AC16 s after H/R;Electron microscopy results showed that the cardiomyocytes had more pyroptotic pores after H/R,and the cell volume was relatively enlarged and rounded.In addition to the increased pyroptotic pores,the cardiomyocytes co-cultured with HCAECs also burst.improve this phenomenon.4.In the mouse MIRI model,The results of immunohistochemistry showed that compared with the sham operation group,the cardiomyocytes of the I/R group were activated by caspase 3,and the expressions of GSDME-NT and NFκB（P65）were increased;WB experiment showed that compared with the sham operation group,the expressions of cleaved-caspase-3,IL-1β and GSDME-NT in the cardiomyocytes of the I/R group were significantly increased;The results of immunohistochemical experiments showed that compared with the I/R group,BAY could inhibit the up-regulation of HIF-1α and i RHOM2 in mouse cardiomyocytes after I/R;HE staining results showed that BAY could improve the obvious vacuolization and mild infiltration of inflammatory cells in the cardiomyocytes of I/R mice;Immunofluorescence experiments showed that BAY could inhibit the protein expression of IL-6,NFκB（P65）and TNF-α in cardiomyocytes of I/R mice;Ultrasound results showed that compared with the I/R group,the cardiac function of the mice after i Rhom2-sh RNA-AAV knockdown was significantly improved,and the LVEF and LVSF were significantly increased;Immunohistochemical results showed that i Rhom2 in the mouse heart could not be up-regulated by I/R after knockdown;and compared with the I/R group,the protein expressions of Cleaved-caspase3,p-NFκB and GSDME-NT were significantly higher after i RHOM2 knockdown.reduce.Conclusion:1.After H/R of vascular endothelial cells,the HIF-1α/i Rhom2 signaling pathway is activated,which promotes the maturation of ADAM17,leads to increased release of pro-inflammatory factors and inflammatory response,and initiates the occurrence of pyroptosis;2.After myocardial ischemia-reperfusion in mice,the activation of HIF-1α/i RHOM-2 signaling pathway in CECs is involved in I/R-induced cardiomyocyte injury,and inhibition of HIF-1α/i RHOM-2 signaling can reduce the release of CECs and cause inflammation factor,reduce the inflammatory response,thereby inhibiting the occurrence of cardiomyocyte pyroptosis.Part 2 Vitexin regulates HIF-1α/i RHOM2 pathway to protect vascular endothelial cells from H/R injury and its molecular mechanism of mediating the inhibition of cardiomyocyte pyroptosis after MIRI in miceObjective:1.To clarify the protective effect of vitexin on vascular endothelial cells after H/R and the regulation effect of inhibiting HIF-1α/i RHOM2 pathway on inflammatory response;2.To explore the molecular mechanism of vitexin mediating cardiomyocyte pyroptosis after myocardial ischemia-reperfusion in mice by regulating HIF-1α/i RHOM2 signaling in vascular endothelial cells.Method:After culturing HCAEC cell lines by passage method and establishing HCAECs model,CCK-8 method was used to detect the optimal hypoxia time of HCAECs,and the optimal concentration of VT and BAY to resist H/R injury;Elisa method was used to detect the effect of VT on H/R cells.The effects of TNFα,IL-1α and IL-1β levels in the supernatant;the effect of VT on the expression of i Rhom2 in cells after H/R was detected by WB method.After the primary CECs were separated by collagenase digestion and time gradient method,the primary CECs model was established,and the effects of different concentrations of VT and i Rhom2-si RNA on the apoptosis of CECs after H/R were detected by flow cytometry;The effects of concentrations of VT and i Rhom2-si RNA on the expression of i Rhom2 in cells after H/R;the effects of different concentrations of VT on the expression of i Rhom2 in cells after H/R and the inhibition of HIF-1α,VT 10 μmol/L and Rhom2-si RNA were detected by WB Effects on the expression of i Rhom2 and HIF-1α in cells after H/R;the effects of HIF-1α inhibition,VT 10 μmol/L and Rhom2-si RNA on the m RNA expression levels of i Rhom2 and ADAM17 in cells after H/R were detected by q PCR.After the primary cardiomyocytes and CECs were separated by collagenase digestion and time gradient method,the H/R model of primary cardiomyocytes and CECs was established.Effects of IL-6 protein expression.The myocardial I/R injury model in mice was established by ligation of the left anterior descending artery,and the effects of different concentrations of VT on the pathological changes of myocardial cells after I/R surgery were detected by H＆E staining.The effects of 6 and 12 mg/kg VT on the electrocardiogram and cardiac function of I/R mice;Elisa method was used to detect the effects of 6 and 12 mg/kg VT on the changes of LDH,MDA and SOD in plasma of mice induced by I/R;The effects of 3,6 and 12 mg/kg VT on the expressions of NFκB（P65）,Caspase3 and GSDM-E in I/R-induced hearts were detected by immunohistochemistry.Result:1.In the HCAECs model,Elisa results showed that 10 μmol/L VT could significantly reduce the H/R-induced increases in the levels of TNFα,IL-1α and IL-1β in the supernatant of HCAECs;WB results showed that VT could inhibit H/R-induced increase of i Rhom2 expression in HCAECs;2.In the CECs model,Flow cytometry showed that VT and i Rhom2-si RNA significantly down-regulated H/R-induced apoptosis of CECs;Immunofluorescence results showed that VT and i Rhom2-si RNA significantly reduced the increase of i Rhom2 fluorescence intensity and protein expression in H/R-induced CECs;WB results showed that VT significantly reduced the expression of i Rhom2 protein in H/R-induced CECs;WB results also showed that the HIF-1α inhibitor BAY not only significantly inhibited H/R-increased HIF-1α expression,but also significantly inhibited H/R-increased i Rhom2 expression;VT 10 μmol/L increased H/R The expression of HIF-1α and i Rhom2 were also significantly inhibited;q PCR results showed that VT 10 μmol/L significantly inhibited the m RNA expression levels of i Rhom2 and ADAM17 increased by H/R,and the m RNA expression levels of i Rhom2 and ADAM17 induced by H/R were also significantly decreased after HIF-1α inhibitor or i Rhom2 knockdown;3.In the primary cardiomyocyte model,WB results showed that the supernatant of CECs after H/R could further up-regulate the protein expressions of NLRP3 and IL-6 in cardiomyocytes after H/R;4.In the mouse MIRI model,The results of H＆E staining showed that 3,6 and 12 mg/kg VT significantly improved myocardial cell arrangement and nuclear staining caused by MIRI;The ECG results showed that,compared with the Sham group,the ECG of the mice in the I/R group had obvious "J point" elevation;echocardiography showed that VT could significantly improve the left ventricular ejection fraction of the mice caused by I/R.（LVEF）and decreased left ventricular systolic function（LVSF）;Elisa results showed that 6 and 12 mg/kg VT could significantly improve I/R,which caused the increase of LDH and MDA and the decrease of SOD in mouse plasma;The results of immunohistochemistry showed that 3,6 and 12 mg/kg VT could significantly inhibit MIRI-induced increases in the expression of cardiac NFκB（P65）,Caspase3 and GSDM-E.Conclusion:1.After H/R injury of vascular endothelial cells,the HIF-1α/i Rhom2 signaling pathway is up-regulated and promotes the increase of ADAM17 expression;2.VT ameliorated H/R-induced CECs damage and reduced the release of inflammatory factors,which was related to the inhibition of up-regulated HIF-1α/i Rhom2 signaling pathway and the increased expression of ADAM17;3.The protective effect of VT on MIRI in mice may be produced by reducing cardiomyocyte pyroptosis and inhibiting the up-regulated HIF-1α/i Rhom2 signaling pathway. |
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