Study On The Protective Effect Of CEPO In Human Umbilical Vein Endothelial Cells Damaged By Serum From Patients After PCI And The Mechanism

Background and purpose:Acute myocardial infarction (acute myocardial infarction, AMI) has become theworld’s highest incidence of fatal heart disease, the pathological changes are oftensecondary to acute coronary thrombosis on the basis of the occurrence of atherosclerosis,resulting in coronary blood supply drastically reduced or even interruption of blood flow,resulting in a corresponding long-lasting and severe myocardial blood supply to acuteischemia, causing a large number of cardiac cell death. Currently, percutaneous coronaryintervention conducted extensive clinical (percutaneous coronary intervention, PCI) toquickly restore blood perfusion in acute myocardial ischemia, maximize save dyingmyocardial cells is to limit infarct size and improve the prognosis of patients The key, ithas become the best therapeutic strategy for acute myocardial infarction. However, in therescue and treatment of acute myocardial infarction, and again after the culprit artery torestore blood supply, excess free radicals, which attack to regain part of the tissue cellswithin the blood supply will cause further injury, clinical manifestations: myocardialstunning, reperfusion arrhythmias, microcirculation and lethal reperfusion injury. Theseinjuries can lead to decreased left ventricular systolic function, left ventricular pressure anddecreased inhibition of myocardial function.At present, the mechanism of ischemia-reperfusion injury is not fully clear. Therewere many scholars have done a lot of animal experiments and clinical studies, manyresearchers have reduced the reperfusion injury in different ways, encouraging conclusionsdrawn. In addition, research in the field of molecular mechanisms continues, this will alsohelp people discover new ischemia reperfusion media.Erythropoietin (erythropoietin, EPO) is a hypoxia-inducible factor-induced familyof multifunctional cytokine superfamily. Carbamylated erythropoietin (carbamylatederythropoietin, CEPO) is erythropoietin (erythropoietin, EPO) of a derivative. EPO andCEPO by regulating inflammation, anti-apoptosis, angiogenesis, protect nerve cells and stimulate the proliferation and differentiation of cardiomyocytes in heart, brain plays animportant protective role in ischemia and hypoxia. In addition to the classic hematopoieticfunction, EPO and CEPO maintain homeostasis of the whole organism also plays animportant role. EPO as an angiogenic factor, directly involved in wound healing, themenstrual cycle, inflammation, cancer, retinopathy and other physiological andpathological angiogenesis, and has a considerable and vascular endothelial growth factoreffects. EPO directly or promote endothelial cell proliferation by regulating cell cycleproteins, protecting the integrity of the endothelial cells. EPO binding to its receptor canpromote angiogenesis. As EPO derivatives, CEPO effect on endothelial cells how to makethe main content of this research.Current research studies on CEPO is still relatively limited field of animal testing inpatients with acute myocardial infarction after PCI that myocardial ischemia-reperfusioninjury in patients Application rarely reported, the use of such attacks in patients with serumvascular endothelial cell damage caused research model is still rare. Therefore, the presentstudy human umbilical vein endothelial cells selected as the research object, in vitro fullysimulated clinical pathophysiological processes, using serum pharmacological method toestablish cell damage model to investigate the protective effect of CEPO, and its signaltransduction mechanisms were discussed.Methods:In this study, ECV340endothelial cells as research subjects, were given to surgeryafter PCI12h,24h,48h and72h, select the logarithmic growth phase cells were serumeffect24h and48h, the rate of apoptosis was observed by TUNEL, flow cytometry toobserve the cell cycle, NO content and NOS activity was measured by radioimmunoassay,ELISA was used to detect IL-1, IL-6and other inflammatory cytokines content screeningafter PCI vascular endothelial cell injury and cell peak time duration of action. Afteraddition of different concentrations of EPO (100IU/ml,50IU/ml,20IU/ml,10IU/ml,1IU/ml) and different concentrations of CEPO (100IU/ml,50IU/ml,20IU/ml,10IU/ml,1IU/ml). was observed by TUNEL apoptosis rate, flow cytometry cell cycle,radioimmunoassay NO content and NOS activity, ELISA was used to detect IL-1, IL-6and other inflammatory factor levels, screening for EPO and CEPO PCI Postoperativeserum-induced cell damage protective effects ECV304optimum concentration.EPO andCEPO on ECV304cell damage protective mechanisms places biochemical assay for EPOand CEPO LDH leakage rate; Western blotting analysis to detect JAK2and phosphorylation protein content; immunohistochemistry were used to detect endothelialcells pERK, pJNK, pP38content.Result:1.Compared with the negative control group, respectively,12h,24h,48h and72hafter serum treatment ECV304cells24h and48h, the apoptosis rate was a significantincrease (P <0.01) with after PCI; G0/G1phase, G2/percentage of M phase cellsdecreased significantly increased S phase G2/M phase (P <0.05or P <0.01).2.Compared with the negative control group were treated with PCI,12h,24h,48h,72h and24h after treatment serum48h, NO ECV304cells was significantly reduced, NOSactivity was significantly decreased (P <0.05or P <0.01), IL-1and IL-6levels were verysignificantly increased (P <0.01) in the serum of patients treated ECV304cells48h24h,the most significant effect.3.Compared with the control group, using1IU/ml-100IU/ml EPO administration,the absorbance values were very significantly increased (P <0.01), which50IU/ml EPOeffect is most significant.4.Using I0U/ml-100IU/ml EPO or CEPO administration, the apoptosis rate wassignificantly lower (P <0.05or P <0.01)); using20I0U/ml-100IU/ml EPO or CEPOafter administration, significantly reduced the proportion of S-phase cells (P <0.05or P<0.01), using50U/m lCEPO after administration of G2/M phase lower (P <0.01).5.Using I0U/ml-100IU/ml EPO or CEPO after administration, NO and NOSwere significantly increased (P <0.05or P <0.01)), IL-1and IL-6was significantly lower(P <0.05or P <0.01)), which CEPO20IU/ml and EPO50IU/ml most significant effect.6.Compared with the model group, the use of50IU/ml EPO or20IU/ml CEPOafter administration, LDH leakage rate is very significantly lower (P <0.01).7.Compared with the model group, JAK2and P-JAK2expression levels weresignificantly lower.8.Compared with the model group, EPO and CEPO treated pERK, pJNK, pP38positive expression levels were significantly lower (P <0.01).Conclusion:1, with PCI,12h,24h,48h and72h serum treated ECV304cell damage model canbe successfully established by the PCI patients serum induced human vascular endothelialcells;2,48h serum treated ECV304cells apoptosis after24h the most significant, the most obvious endothelial injury;3, EPO treatment ECV304cells after24h, can promote the proliferation of normalcells to EPO50IU/ml most significant effect; and CEPO does not promote normalendothelial cell proliferation;4, CEPO also can effectively alleviate the cellular damage caused after PCI inpatients with serum to CEPO20IU/ml most significant effect;5, CEPO reduces endothelial cell apoptosis index, a significant increase of NO andNOS levels, significantly reduced IL-1and IL-6;6, CEPO can reduce expression of JAK2p-JAK2, suggesting CEPO can play a rolein cardiovascular protection through the regulation of JAK2/STAT5signaling pathway;CEPO can reduce pERK, pJNK, pP38expression, suggesting that CEPO can play throughthe regulation of MAPK signaling pathway cardiovascular protection effect.