| BackgroundCerebrovascular disease is one of three diseases which caused most huma n deaths of worldwide which stroke play a most important character. The inci dence of stroke is about140/10,000to200/10,000all over the world. As tim e goes by the developing country have a higher morbidity while the industry country have a better control of motality rate about stroke after great enocom y input. Now stroke is the second mainly cause of death in China while the patients are getting more younger that result in a huge impair to labor force with60%～70%patients may have a complicationg of physical disability whic h40%of them would completely lose man power. That is why the preventio n and treatment of stroke is the hot spot right now.Hypertension and atherosclerosis is the most important and common reaso n of cerebrovascular disease. Some data indicated that93%of patients with c erebral hemorrhage and86%patients with Cerebral thrombosis had hypertensi on history,70%patients with cerebrovascular disease had history of atheroscle rosis.The risk factors of atherosclerosis include hypertension, diabetes, hyperlip idemia, smoking, obesity and family genetic. Homocysteine (Hcy) is a new in dependent risk factor of atherosclerosis.The plasma homocysteine levels increas ed and serum folic acid, vitamin B6, B12levels of plasma decreased in patie nts with Coronary heart disease and cerebrovascular disease. Hcy caused ather osclerosis leading to the damage of the vascular endothelial cells. The possibl e mechanism of injury of Hcy on vascular endothelial cells include:1. Secret i nflammatory cytokines.2. Induce apoptosis.3. Cause Oxidation reaction, prod uce oxygen radicals.4. Reduce the production of endothelium-dependent vasodi lation factor (EDRF).5. Change the endothelial cell phenotype, interfere the pl asminogen activator binding sites.Erythropoietin (EPO) is widely used for treatment of anemia in patients with chronic kidney disease (CKD) and in cancer patients receiving chemothra py. The protective effect of erythropoietin was investigated in myocardial injur y, cerebral ischemic injury, acute brain injury, spinal cord injury by hypoxia,l ung injury and acute kidney injury. The possible mechanisms of protection fro m EPO in cardiovascular system may be reduces apoptosis,modifies inflammat ion,increases endothelial nitric oxide synthase, stimulates angiogenesis, promote s endothelial progenitor cells proliferation and differentiation, enhances antioxi dant enzyme expression and reduces the rate of free radical production. The p ossible mechanisms of how EPO protect nervous system maybe be normalizes cerebral blood flow, stimulates angiogenesis, affects neurotransmitter relese, m odifies inflammation and immune response, stimulates non-differentiated Schwa n cells to proliferate, reduces apoptosis.NF-κB was a kind of transcription factor which found in mature B lymp hocytes. NF-κB transcriptional regulation of important physiological processes and related with inflammation, immune response, stress response, cell prolifer ation,cell transformation and apoptosis which is the most important point topic in today’s study. NF-κB formed by two types of subunits homologous or het erologous dimer and the most common form of NF-κB subunit composition w as p65/p5or p65/p65. Inflammatory cytokines, growth factors or chemokines can activate NF-κB, and IκB-a phosphorylated at Ser32and Ser36, then degra ded by the ubiquitin-proteasome pathway. NF-κB and IκB-α was translated i n nucleus after there degradation. The experimental analysis pointed out that Hcy could stimulated the genetic expression of cell adhesion molecules, chem okines, inflammatory cytokines and then caused chronic inflammation of the v ascular wall and promoted the occurrence of AS. Researches show that NF-κ B signaling pathway exerts anti-apoptotic fuction via many other proteins, incl uding c-IAP,c-IAP2, IAXP, TRAF1, TRAF2, Bcl-2, A20, MnSOD and so on, but the exact mechanism remains unclear.ObjectiveTo investigate protective effect of erythropoietin on vascular endothelial c ells against damage induced by homocysteine, and investigate the role of NF-κB in this process, it is possible to provide a reference for the secondary pre vention of cerebrovascular disease.MethodsPart1:Human umbilical vein endothelial cells(HUVEC) were incubated i n vitro.Part2:Observe the damage of Hcy on Human umbilical vein endothelial cells(HUVEC). Human umbilical vein endothelial cells were treated with Hcy at different concentrations (1.25mmol/L、2.5mmol/L、5mmol/L、10mmol/L、2 Ommol/L) and cultured24hours. The cell viability was detected by MTT ass ay-Part3:Observe the protective effect of erythropoietin on vascular endoth elial cells against damage induced by homocysteine.The experiment was desig ned as different groups,coutrolgroup was treated with normal cell culture medi um, Hey treatment group was treated with10mmol/L Hey, EPO pretreatment group was treated with EPO at different concentrations (0.1U/ml、1U/ml、10U/ml、100U/ml) for1hour and then reated with10mmol/L Hcy.The cell viabil ity was detected by MTT assay, the expression of IL-6was assessed by enzy me linked immunosorbent assay,and the apoptosis rate of cells was measured by Hoechst35528staining.Part4:NF-κB mediated protective effect of erythropoietin on vascular en dothelial cells against homocysteine-induced apoptosis. Human umbilical vein endothelial cells(HUVEC) were incubated in vitro and treated with Hey. The experiment was designed as control group,Hcy treatment group, the group of pretreatment with EPO, and EPO treatment group. The cell viability was dete cted by MTT assay, the apoptosis rate of endothelial cells was measured by f low cytometry, the protein expression of NF-κB in nucleus was assessed by western blot.Results1. Human umbilical vein endothelial cells(HUVEC) were cultured in viro successfully.2. The damage induced by Hey was in a concentration-dependent manne r.3. Compared with control group,survival of cells of Hey treatment group decreased, while the cell apoptosis and the expression of IL-6of Hey treatm ent group incresed. Compared with Hcy treatment group, survival of cells of EPO pretreatment group increased, while the cell apoptosis and the expression of IL-6of EPO pretreatment group decresed.4. The western blot results showed that the NF-κB protein was hardly e xpressed in control group, and the protein expression of NF-κB in nucleus in Hcy treatment group was a little higher than control group. Thr protein expr ession of NF-κB in nucleus in the group of pretreatment with EPO and EPO treatment group was significantly higher than Hcy treatment group.Conclusion1.Hcy could cause the damage of HUVECs.2.EPO could protect vascular endothelial cells from Hcy toxicity, witch might related to Anti-inflammatory effects and Anti-apoptotic effects.3.EPO could protect vascular endothelial cells from Hcy toxicity, witch might related to signal transduction of NF-κB. |
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