HIF-1α/BNIP3 Signaling Pathway-induced-autophagy Plays Protective Role During Myocardial Ischemia-reperfusion Injury

Coronary heart disease(CHD)is known as the "first killer" of human health and life.The incidence and mortality of CHD are increasing year by year in the world.In addition to traditional drug therapy,coronary thrombolysis,percutaneous coronary intervention and coronary artery bypass grafting are also widely used in the treatment of coronary heart disease.Their advantages are that they can open occlusive and narrow vessels and restore coronary blood flow,but they also inevitably lead to myocardial ischemiareperfusion injury after vascular opening.This has become a hot topic in the study of coronary heart disease.Coronary artery occlusion will lead to ischemia of the heart,which will obviously affect the blood supply of myocardial cells and produce certain damage.The recovery of blood supply after myocardial ischemia will lead to further damage of ROS and affect the function of myocardial cells,that is,myocardial ischemia-reperfusion(I / R)injury.In the process of I / R injury,there are many adverse reactions,such as serious hypoxia,acidosis,calcium overload,energy depletion and ion dynamic balance disorder.Under the influence of these factors,the heart muscle is damaged and finally leads to the death of myocardial cells.Mitochondria can provide support for the activities of cardiomyocytes,and play an important role in I / R injury.The active oxygen produced by mitochondria in the damaged condition is significantly higher than that in the normal condition,and the active oxygen will have a strong damage effect on mitochondrial protein and DNA,which also leads to the destruction of mitochondria,and then damage cells.Moderate mitochondrial autophagy can clear some damaged mitochondria and reduce this damage.Therefore,proper regulation of mitochondrial autophagy can reduce myocardial ischemia-reperfusion injury.At present,it has been confirmed that there are several ways for cells to perform autophagy related research,and many kinds of autophagy regulatory pathways have been found.Including the PINK1(PTEN induced putative kinase 1)/ parkin(Parkinson juvenile release protein 2)pathway,which regulates mitochondrial depolarization,the mitochondrial autophagy pathway mediated by the mitochondrial autophagy receptor molecule fundc1(fun14 domain containing 1),and the other pathway,which is the HIF-1 α mediated autophagy pathway of BNIP3 under the condition of ischemia and hypoxia.BNIP3(Bcl-2 nineteen kilodalton interacting protein 3)is a member of the BH3 only subfamily of Bcl-2 family proteins.It is mainly distributed in the endoplasmic reticulum(ER)and loosely bound to the outer membrane of mitochondria.Its position in the cell determines the way of cell death.BNIP3,through its BH3 domain,opens mitochondrial permeability transition pore(MPTP)or / and activates mitochondrial membrane,thus affecting Bax / bak to cause mitochondrial dysfunction and promote apoptosis.On the other hand,experimental research also found that its BH3 domain destroyed Bcl-2 / beclin-1 in normal cells The increased expression of beclin-1 can be used as an important indicator of the increased level of autophagy.BNIP3 can activate mitochondrial autophagy when the nutrition and oxygen of cells are reduced.BNIP3 can mediate mitochondrial autophagy without changing mitochondrial permeability and apoptosis.Moderate mitochondrial autophagy can eliminate the damaged mitochondria and maintain the stability of the internal environment.Initially,Hypoxia inducible factor-1(HIF-1)is a heterodimer,which is composed of unstable functional subunits,HIF-lα and stable structural subunits,HIF-1β.Under the condition of constant oxygen,HIF-lα is very unstable,It has poor stability under normal oxygen condition and is catalysed and hydroxylated by prolyl hydroxylase.After being hydroxylated with HIF-l α,it is easy to bind tumor suppressor protein von Hippel Lindau(VHL)binds and is recognized as a ubiquitinated site.After that,it is recognized as a ubiquitinated site.After that,it is rapidly degraded through the ubiquitin proteasome pathway.After being recognized,it is rapidly degraded based on the ubiquitin proteasome pathway.Under the condition of hypoxia,this degradation process was inhibited and blocked under the condition of hypoxia;HIF-lα transferred to the nucleus and combined with HIF-1β and CBP / P300 to form a complex,which combined with the resultant product combined with the hypoxia response element(HRE),and mediated the transcription of over 60 target genes.The results of previous studies on the ischemia-reperfusion injury of tumor cells,liver cells and kidney cells show that mitochondrial autophagy mediated selective autophagy can lead to mitochondrial destruction and release of selective autophagy.Studies show that HIF-1 α in mitochondrial autophagy is an endogenous HIF-1α dependent hypoxia response.Sexual process plays an important role in regulation.Many studies have shown that the up regulation of BNIP3 and BNIP3 L caused by HIF-1α and HIF-1α is closely related to the up regulation of BNIP3 and BNIP3 L in hypoxia mediated autophagy.In this study,peripheral blood of patients with acute myocardial infarction,SD rats and H9C2 myocardial cells were studied.Enzyme linked immunosorbent assay(ELISA),Western Blot,Real-time fluorescence quantitative polymerase chain reaction(RT-PCR),MTS,LDH,flow cytometry and electron microscopy were used to study the expression of BNIP3 in myocardial ischemia-reperfusion injury,and then the viability of myocardial cells after overexpression or knockdown of BNIP3 was used H9C2 myocardial cells in oxygen glucose deprivation reperfusion injury.Finally,by detecting the expression of related pathway proteins,we further explore the pathway of HIF-1α/BNIP3 signaling pathway in oxygen glucose deprivation reperfusion injury,and clarify the role of HIF-1α/BNIP3 signaling pathway in oxygen glucose deprivation reperfusion injury,so as to better understand the molecular mechanism of the occurrence and development of oxygen glucose deprivation reperfusion injury.Part One Expression of HIF-1α and BNIP3 in myocardial ischemia-reperfusion injury of SD ratsObjective: To verify the expression of HIF-1α and BNIP3 protein in myocardial ischemia-reperfusion of SD rats.Methods: 1.SD rats were divided into four groups randomized with 12 rats in each group: Sham group,ischemia-reperfusion group(I/R group),ischemia-reperfusion + DMOG group(I/R + DMOG group),ischemia-reperfusion + 3-MA group(I/R + 3-MA group).2.TTC and HE staining were used to detect the area of myocardial ischemia and the level of myocardial cell injury in SD rats.3.Western Blot method was used to detect the expression of HIF-1α、BNIP3、LC3-I、LC3-II and Beclin-1 in myocardial ischemia-reperfusion injury in SD rats.Results: 1.TTC results showed that DMOG pretreatment could significantly reduce the infarct area of I/R injury in SD rats,while 3-MA pretreatment could significantly increase the infarct area.2.The results of HE staining showed that DMOG pretreatment could significantly reduce the I/R injury of SD rats,while 3-MA pretreatment could aggravate the I / R injury of SD rats.3.DMOG pretreatment can increase the expression level of BNIP3 and autophagy regulatory proteins LC3-I、LC3-II and Beclin-1 by increasing the level of HIF-1α,and activate autophagy of cardiomyocytes to protect myocardium;3-MA pretreatment can reduce the expression of autophagy regulatory proteins LC3-I、LC3-II and Beclin-1 by inhibiting autophagy,which aggravates myocardial injuryConclusions: 1.The levels of HIF-1α and BNIP3 protein were significantly increased in the condition of myocardial I/R injury in SD rats.DMOG,the activator of HIF-1α can increase the levels of HIF-1α,and then increase the levels of BNIP3 protein and autophagy regulatory proteins LC3-I、LC3-II and Beclin-1.2.Increasing the level of HIF-1α and BNIP3 protein will increase the level of autophagy,further reduce the I/R injury of myocardial cells in SD rats,and inhibiting the level of autophagy will aggravate the I/R injury of myocardial cells in SD rats..Part two The effect of HIF-1 α on the regulation of BNIP3 in the glucose oxygen deprivation reperfusion injury of H9C2 cell line.Objective: To study the effect of increasing and knocking down BNIP3 on the cell viability and injury of H9C2 cardiomyocytes in the state of oxygen glucose deprivation reperfusion injury.Methods: 1.H9C2 cells were assigned randomized into 6 groups: control group,OGD/R(24h/2h)group,OGD/R(24h/2h)+DMOG group,OGD/R(24h/2h)+ YC-1 group,OGD/R(24h/2h)+Tre group,OGD/R(24h/2h)+BNIP3-si RNA group;2.The expression of HIF-1α and BNIP3 were detected by immunochemical staining and Western blot;3.MTS and LDH experiments were used to detect the effects of BNIP3 on the cell viability and damage of H9C2 cells in oxygen glucose deprivation reperfusion injury;Results: 1.Successfully constructed H9C2 cardiomyocyte deprivation and reperfusion damage model that raised and inhibited HIF-1α protein expression levels.2.Increasing BNIP3 protein expression level can reduce LDH release in the oxygen glucose deprivation reperfusion injury of H9C2 cardiomyocytes,and knocking down BNIP3 protein expression level can increase LDH release in the oxygen glucose deprivation reperfusion injury of H9C2 cardiomyocytes.3.Increasing BNIP3 protein expression level will increase the activity of H9C2 cells in oxygen glucose deprivation reperfusion injury,and knocking down BNIP3 protein expression level will reduce the activity of H9C2 cells in oxygen glucose deprivation reperfusion injury.Conclusions: 1.HIF-1α synchronously regulates BNIP3 expression in H9C2 cells during oxygen glucose deprivation reperfusion injury.2.Increasing BNIP3 protein level reduced the oxygen glucose deprivation reperfusion injury of H9C2 cells,and knocking down BNIP3 protein expression aggravated the oxidative glucose deprivation reperfusion injury of H9C2 cells.Part Three BNIP3 attenuates oxygen glucose deprivation reperfusion injury by activating mitochondrial autophagy.Objective: To further study the effect of BNIP3 on autophagy of H9C2 myocardial cells during oxygen glucose deprivation reperfusion injury.Methods: 1.H9C2 cells were randomly divided into 5 groups: control group,OGD/R(24h/2h)group,OGD/R(24h/2h)+Tre group,OGD/R(24h/2h)+ BNIP3-si RNA group,OGD/R(24h/2h)+ 3-MA group;2.Western Blot assay was used to detect BNIP3 knocking down,mitochondrial autophagy inhibition and BNIP3 expression by elevation and transfection techniques.3.The damage of H9C2 myocardial cells and the expression of autophagosomes were detected by Transmission electron microscope under different intervention conditions.Results: 1.Increasing BNIP3 protein can increase the autophagy level of H9C2 cells in OGD/R injury,and knocking down BNIP3 protein can reduce the autophagy level of H9C2 cells in OGD/R injury.2.Further application of Transmission electron microscope to verify that increasing BNIP3 protein will increase the autophagy of H9C2 cells in OGD/ R injury,knocking down BNIP3 protein will reduce the autophagy of H9C2 cells in OGD/R injury;inhibiting the autophagy of H9C2 cells will increase the OGD/R injury of H9C2 cells,and moderately increasing the autophagy of H9C2 cells will reduce the OGD/R injury.Conclusions: 1.Increasing the expression of BNIP3 protein can improve the autophagy level in the oxygen glucose deprivation reperfusion injury of H9C2 cells,so as to reduce the oxygen glucose deprivation reperfusion injury of H9C2 cardiomyocytes..2.Knocking down the expression level of BNIP3 protein can reduce the autophagy level of H9C2 cells,thus aggravating the oxygen glucose deprivation reperfusion injury of H9C2 cells.Part Four Expression of serum HIF-1α in patients with acute myocar-dial infarctionObjective: To verify the expression of HIF-1α in patients with ST-Elevation Myocardial Infarction-Executive Summary.Methods: 1.The expression of HIF-1α in serum of were examined by enzymelinked immunosorbent assay(ELISA)in 36 patients with infarction(STEMI),32 patients with these kind of infarction were examined by coronary angiography(CAG)patients and 35 controls without coronary heart disease(NC).Results: 1.There was no significant difference in clinical data among the three groups.2.Compared with NC group,the expression of HIF-1α in peripheral serum was up-regulated in STEMI + PCI group and STEMI + CAG group,and the level of HIF-1α in STEMI + PCI group was higher significantly.Conclusions: 1.In patients with ST-Elevation Myocardial Infarction-Executive Summary,the level of HIF-1 α in the peripheral blood will increase after the coronary blood flow is restored.