(Pro)renin Receptor Mediated Inflammatory Factors Alteration Via P38MAPKs During Hypoxia And Reoxygenation

Background and objective: Myocardial ischemia-reperfusion injury (IRI) was firstdiscovered by Jennings in canines in1960. It was reported that ischemia-reperfusionwould aggravate myocardial infarction, and is the major pathological basis of cardiacdysfunction and arrhythmia in clinical early reperfusion of acute myocardial infarction(AMI), angina pectoris, percutaneous transluminal coronary angioplasty, cardiactransplantation. Ischemia-reperfusion injury could occur in various important organs,including heart, liver, lung, kidney, gastrointestinal tract and so on. It was reportedfactors that affect IRI contains free radical injury, calcium overload, complementactivation, endothelin, angiotensin II, etc. Researches in the rat model of myocardialischemia reperfusion injury have shown that ischemia reperfusion injury could increaseapoptosis of cardiomyocytes and infarction area obviously. Nowadays, three millionpeople would die from ischemic cardiovascular and cerebrovascular diseases in chinaevery year, nearly40%of all deaths. So, IRI is a medical problem needing to be settledurgently.Renin-angiotensin system (RAS) plays an important role in IRI. It was reportedmyocardial ischemia could activate RAS, and so to protect systolic function ofcardiomyocytes.(Pro)renin and its receptor are new members of RAS. Ichiharadiscovered and cloned (pro)renin receptor [(P)RR] of mouse in2004. Researches hasproven (P)RR of human is highly homologous with mouse.(P)RR is widely expressedin human body, including kidney, brain cell, heart, placenta, and is expressed in liverand pancreas in a low level. Moreover, mRNA and protein of (P)RR was detected inmacrophages,T cell, granulocytes. It was proved activated (P)RR participates inERK1/2and P38MAPK pathway, and plays an important role in diabetic nephropathy,myocardial fibrosis and heart failure. Hirose et al discovered that mRNA level of (P)RR is increased in kidney and heart of heart failure rats,(P)RR is commonly foundin cardiomyocytes smooth muscle endothelial cells of cardiac vessel, as well as renaltubular cells. It indicates that activation of (P)RR correlates with cardiac and renaldiseases. However, there is no reports about whether (P)RR takes part in IRI.Mitogen-activated protein kinase system is a major intracellular signal transductionsystem, which could bring extracellular signals into cells and take parts in variousintracellular reactions. Many extracllular signals could activate MAPK, includesischemia reperfusion injury, cytokines, ultraviolet irradiation, lipopolysaccharides, heatshock, active oxygen, hyperosmotic state, ect. P38is a significant component of MAPKfamily, at the same time, it is a major stress activated protein kinase of MAPK system,which secreted by lipopolysaccharides stimulated white cells by Han et al in1993. Maet al firstly proved activation of p38MAPK plays a vital role in IRI. Although there aresome disputes, most researchers consider phosphorylation of p38would lead to cellularapoptosis and inflammation in ischemia reperfusion injury. Treatments with p38inhibitor could attenuate IRI, and so to decrease area of infarction. Some other reportspointed out that activation of p38MAPK would lead to increation of TNF-α, IL-1, IL-4,IL-6, IL-8, IL-12from monocyte and macrophages, and meanwhile to decrease of IL-10.It was reported inflammatory factors were closely correlated with ischemia reperfusioninjury and TNF-α, IL-1from neutrophile granulocyte played important roles ininflammatory reaction. In this study, we will investigate actions of (P)RR duringhypoxia and reoxygenation in H9C2cells, and its relationship with p38MAPK as wellas inflammatory factors.Aims:1. To prove the expression of (P)RR in H9C2cardiomyocytes of rats.2. Todetect alteration of (P)RR expression level during hypoxia and reoxygenation, and confirm the time point of maximum expression during reoxygenation.3. To investigate(P)RR participated mechanism of p38MAPK dependent signal transduction ininflammation induced by hypoxia and reoxygenation in H9C2cardiomyocytes,withthe combination of siRNA and p38MAPK inhibitor.4. To detect expression levels ofinflammatory factors in H9C2cardiomyocytes after2hours of hypoxia and6hours ofreoxygenation.Methods:1. Culture of rat cardiomyocytes (H9C2cells), develop cellular model ofhypoxia and reoxygenation to mimic state of ischemia-reperfusion.2. Immunocytochemistry and immunofluorescence technology were used to detect (P)RRexpression in H9C2cells.3. Western blot was used to detect the expression level of(P)RR at different time points during hypoxia and reoxygenation.4. Western blot wasused to detect phosphorylation levels of signal proteins of MAPK family.5. The levelsof TNF-α、TGF-β、IL-6in supernatant of cardiomyocytes were detected by ELISA.Results:1.(P)RR was expressed by H9C2rat cardiomyocytes. Immunocytochemistryshowed that H9C2rat cardiomyocytes had an elongated shape. Immunofluorescenceshowed that (P)RR had green fluorescence, and was distributed on cell membrane,cytoplasm and nuclear membrane in H9C2rat cardiomyocytes.2. Alteration of (P)RRprotein expression level during hypoxia and reoxygenation: compare with control group,hypoxia2h group (P)RR protein increased obviously;2h-6h group further increasedobviously,there were no significant differences between2h-3h group and2h group. Itindicated that (P)RR protein expression reached its maximum level in2h-6h group.Reoxygenation time was determined to6hours in the next experiments.3. Western blotresults showed that (P)RR siRNA or p38MAPK inhibitor SB203580coulddown-regulate p-p38protein. Hypoxia and reoxygenation could lead to increase ofphosphorylation of p38MAPK.(P)RR siRNA or p38MAPK inhibitor SB203580couldpartly block increase of p-p38MAPK protein induced by hypoxia and reoxygenation,indicating that activation of p38MAPK is at least partly depend on (P)RR.4. ELISAresults showed that compared to control group, IL-6, TNF-a, TGF-βconcentrationincreased obviously in2h group, and their levels further increased in2h-6h group.Using of (P)RR siRNA alone or combination with blocking of p-p38MAPK coulddecrease the secretion of IL-6, TNF-a, TGF-β.Conclusion:1.(P)RR is expressed in H9C2rat cardiomyocytes.2. Hypoxia and reoxygenation could increase the expression of (P)RR protein, and(P)RR protein expression reached its maximum level in2h-6h group.3.(P)RR partly mediates inflammatory reactions in H9C2cells via p38MAPK pathway.4. Hypoxia for2h, secretion of IL-6, TNF-a, TGF-β from H9C2increased, and thenreoxygenation for6h led to further increase.