Preparation Of Carbamylated Erythropoietin And Its Protective Effects To The SH-SY5Y Cells Damaged By Hydrogen Peroxide

Background Erythropoietin(EPO) is a 34.4 kDa glycoprotein with a 166 amino acid peptide and high-sialic acid content. EPO production and secretion occur foremost in the kidney. EPO can regulate bone marrow cell proliferation, differentiation, and survival through its binding to the EPO receptor (EPOR) on the erythroid progenitor cell surface. The EPOR also is found expressed in numerous non-erythroid cell lines that include neurons, microglias, astrocytes, and in cerebral endothelial cells as well as on myelin sheaths of radicular nerves in human peripheral nerves, suggesting that the protective role of EPO playing is not only in the central nervous system, but also in disease that involved in the peripheral nervous system. But in terms of cerebrovascular disease patients, EPO plays the role of promoting the proliferation of red blood cells which may increase blood viscosity, and even lead to thrombosis, which limite the further clinical application of EPO. However, EPO derivative, carbamylated EPO (CEPO), has been proved to have no erythropoetic effects, making it possible for the CEPO further clinical applications.Hydrogen peroxide is one of the body metabolized product, which is also a reactive oxygen species with the property of through the cell membrane easily. It is easy for hydrogen peroxide to bind with and react with the intracellular irons and causing cell damage. It has been used as an important instrument for the investigation of the cellular injury brought on by the oxygen free radical.SH-SY5Y cell line is come from human neuroblastoma cells, one kind of the tumor cells with low differentiation degree, the cellular morphology, physiological characteristics and biochemical functions are similar with human neurons. The cell line has been used for the experimental study of neurology in recent years.Objective The study was to be designed to investigate the protective effects of CEPO to the SH-SY5Y cells damaged by hydrogen peroxide, compare the degree of protective effects of CEPO with that of EPO, and draws the conclusion whether there is difference between their protective effects. And also some efforts were putted on the possible mechanisms of CEPO protection on the SH-SY5Y cells, such as apoptosis.Methods1 CEPO preparation Firstly, the mixture of EPO powder 50000IU (0.5mg) and the amount of sodium borate and potassium cyanate were mixed and incubated for 23 hours, then following dialysis. The protein concentration of CEPO was determined by the method of Coomassie brilliant blue G250. Then 50IU of CEPO and EPO standard protein preparation were mixed with the dithioerythritol and ethanamide, added into the Hi Trap G25 column, the first 1/3 to 1/2 column fluid was collected. Also the protein concentration of the collected protein was determined by the method of Coomassie brilliant blue G250.100IU EPO and CEPO were added to 0.4μg endoproteinase Lys-C, on 37℃incubated for 20 hours.CEPO identification By SDS-polyacrylamide gel electrophoresis (SDS-PAGE), samples were added as following:①Protein marker;②EPO not digested with endoproteinase Lys-C;③EPO digested with endoproteinase Lys-C;④EPO carbamylated and digested with endoproteinase Lys-C;⑤EPO carbamylated but not digested. After electrophoresis, the gel was removed and stained by silver.2 The research of the protective effects and mechanisms of CEPO to the SH-SY5Y cells damaged by hydrogen peroxide.(1) The estabalishment of hydrogen peroxide damaged SH-SY5Y cell model the SH-SY5Y cell was cultured in the 6-well plates for 12 hours, then added different concentration of H2O2 (0,50,200,300,400μmol/L), after 12 hours, the culture was termed and the cells were collected, the cell viability of SH-SY5Y cells were determined by the method of thiazole Portland(methylthiazoletetrazolium, MTT). Then according to the MTT value, the amount of the H2O2 concentration required for the estabalishment of of the cell model was come out.(2) The groups of cell culture According to the different hydrogen peroxide concentration, the cells were divided into two major groups, the first major group was cultured with the H2O2 concentration of 300μmol/L, and was subdivided into the following subgroups:①the control group, that only with hydrogen peroxide but not other agents groups;②EPO group;③CEPO group; each group was cultured with 300μmol/L H2O2 culture solution for 12 hours, replaced with the corresponding reagents culture medium for 24 hours then following the next step. The second major group was cultured with 400μmol/L H2O2, the others were the same with the first group.(3) The observation of cell morphology After the end of culturing the SH-SY5Y cells were observed by the microscope. The cell morphology can be described by the cell shape, nuclear shape and its axons.(4) The MTT assay After the termination of the cell culturing, the cell viability was determined by MTT as following operations:MTT powder was dissolved into the phosphate buffer saline, then was added to each cell well and was incubated for 4 hours. Then the medium was removed and dimethylsulfoxide was added each well of the plate, after the dissolving of the dimethylsulfoxide and blue formazan produced. Absorbance of the product was measured at a reference wavelength of 570nm.(5) The value of lactate dehydrogenase(LDH) release The LDH release was measured according to the operations described by the reagent description. The SH-SY5Y cells were cultured in 24-well plates. After the end of the culture, the culturing medium was collected and the LDH release was measured as described.(6) AnnexinⅤ/PI immunofluorescent staining To test the apoptosis rate of the SH-SY5Y cells, after cultured, the cells were treated by the apoptotic reagent then the apoptosis rate was tested by the flow cytometer according the kit discription. AnnexinⅤcan show the early phase of cell apoptosis, while the PI staining is usually used to measure the cell necrosis, the two are used simultaneouly in order to distinct the apoptosis cells from the necrosis cells.(7) Analysis of Bcl-2 and cell cysteine protease(Caspase)-8 mRNA expression by the method of fluorescence quantitative RT-PCR detection After cultured, the total RAN was extracted from the SH-SY5Y cells. The manipulation of RT-PCR was according the drscription of the reagent. The Bcl-2 shows the cell ability of anti-apoptosis on the gene level and the Caspase-8 protein shows one of the cells apoptosis phase.(8) Statistical analysisAll experiments were performed in triplicate. Data are presented as mean±SD. The Duncan test and one-way ANOVA were used for multiple comparisons using SPSS 11.0 software (SPSS, Chicago, USA).Results1 The result of the identification of CEPO by SDS-PAGE electrophoresis Results indicated that EPO could be digested by the Lys-C endoproteinase and showed allover degradation, the band of relative molecular mass of about 3.4×104 was completely disappeared, replaced by the band of relative molecular mass of about 1.4×104 of the protein spectrum; but CEPO can not be digested by the Lys-C endoproteinase, so the bands of CEPO wether being digested or not digested by the Lys-C endoproteinase showed only one band on the site of relative molecular mass of about 3.4×104. Especially for the digested CEPO, there were no bands on the site of relative molecular mass of about 1.4×104, which indicated that the EPO carbamoyl reaction was completely accomplished, all of the EPO were converted into CEPO.2 The research of the protective effect and mechanisms of CEPO to the SH-SY5Y cells damaged by hydrogen peroxide.(1) The estabalishment of H2O2 damaged SH-SY5Y cell model The results showed that with the increase concentration of H2O2, MTT values showed a downward trend. According to the results, we selected a concentration of 300μmol/L and 400μmol/L of H2O2 to estabalish the cell model for the further experiments.(2) The effects of CEPO to the cell morphology The SH-SY5Y cells cultured without any treatment was polygonal, and multi-angled. When treated with H2O2, the cells were mostly round with cell nuclear shrinkage. When treated with different concentrations of CEPO and EPO, the cell morphology became better than treated by H2O2, the number of axons was increased, and with the increaing concentration of CEPO and CEPO, the cell morphology became better, but still could not be as good as the untreated. Also, we can find that the influence of CEPO to the cell morphology was as similar as that of EPO.(3) The effects of CEPO to the MTT value The results showed, compared with the other groups, the cell viability of controlled groups(H2O2 groups) was significantly lower than that of the CEPO and EPO groups. Also, with CEPO and EPO concentration increasing, the cell viability was accordingly rising, but when the CEPO and EPO concentration reached 100IU/mL from 80IU/mL, the cell viability did not rise obviously; during the same concentration of the CEPO and EPO groups, the cell viabili ty did not differ significantly, which proved that the influence of CEPO to the MTT value of cells be similar with that of EPO.(4) The effects of CEPO to the measurement of the release of LDH When the cells are injuried, the cell necrosis results in a disruption of the cytoplasmic membrane and the necrotic cells release cytoplasmic LDH and other cytotoxic substances into the medium. We examined the existence of LDH in the SH-SY5Y cells culturing medium. The LDH of the CEPO and EPO groups were significantly more reduced than that of the controlled groups. Also, with CEPO and EPO concentration increasing, the LDH release were accordingly decreasing, but when the CEPO and EPO concentration reached 100IU/mL from 80IU/mL, the LDH release did not show obviously decreasing; during the same concentration of the CEPO and EPO groups, the LDH release did not differ significantly, which proved that the influence of CEPO to the LDH release be similar with that of EPO.(5) The influence of CEPO to the results of SH-SY5Y cell AnnexinⅤ/PI AnnexinⅤ/PI immunofluorescent staining The results showed that the apoptosis cells in the CEPO and EPO groups were significantly more reduced than that of the controlled groups. Also, with the CEPO and EPO concentration increasing, the apoptosis rate of cells was accordingly decreasing, but when the CEPO and EPO concentration reached 100IU/mL from 80IU/mL, the apoptosis cells did not show obviously decreasing; during the same concentration of the EPO and CEPO groups, the apoptosis rate of cells did not differ significantly, which proved that the influence of CEPO to the apoptosis rate of cells be similar with that of EPO.(6) The influence of CEPO to the results of SH-SY5Y cell analysis of Bcl-2 and Caspase-8 expression by the method of fluorescence quantitative RT-PCR detection The expression of Bcl-2 results showed, the expression of Bcl-2 mRNA of the CEPO and EPO groups was more increased than that of the controlled groups. Also, with CEPO and EPO concentration increasing, the expression of Bcl-2 was accordingly increasing, but when the CEPO and EPO concentration reached 100IU/mL from 80IU/mL, the expression of Bcl-2 did not show obviously increasing; during the same concentration of the EPO and CEPO groups, there was no significant difference, which proved that the influence of CEPO to the Bcl-2 expression be similar with that of EPO.As to the Caspase-8, the expression of Caspase-8 of the CEPO and EPO groups was significantly more decreased than that of the controlled groups. Also, with CEPO and EPO concentration increasing, the expression of Caspase-8 was accordingly decreasing, but when the CEPO and EPO concentration reached 100IU/mL from 80IU/mL, the expression of Caspase-8 mRNA did not show obviously increasing; during the same concentration of the EPO and CEPO groups, there was no significant defference, which proved that the influence of CEPO to the Caspase-8 expression be similar with that of EPO.Conclusion1 CEPO could be prepared by the reaction with sodium borate and identified by the Lys-C endoproteinase digestion and SDS-PAGE electrophoresis. EPO can be digested by Lys-C endoproteinase and then degradated into two low relative molecular mass peptides, while CEPO can not be digested by Lys-C endoproteinase. The result of SDS-PAGE electrophoresis analysis showed that all of the EPO were digested by the Lys-C endoproteinase, so there was one corresponding band; but CEPO can not be digested by the Lys-C endoproteinase, there was another corresponding band at the same site with EPO, which proved that CEPO was prepared successfully and had high purity.2 By the rusults of MTT and the LDH, it was proved that both CEPO and EPO could evidently protect SH-SY5Y cells from oxygen-derived free radicals damage, there was no difference between the two groups. With the increasing concentration of CEPO, the protective effects were accordingly increasing, which showed that the protective effects were increased with the increase of the concentration. The dose-dependent protective effects of CEPO could increase with the concentration and reach the maximal at the concentration of 100IU/ml,and there were no significant difference between that of the CEPO and EPO.3 By the results of Annexin V/PI, we can find that the inhibition of apoptosis was one of the protective mechnisms. With the increasing concentrations of CEPO, the apoptosis inhibition was accordingly increasing, which proved that the apoptosis-inhibiting effect was increased with the increase of the CEPO concentration. The dose-dependent protective effects of CEPO was increased with the concentration and reach the maximal at the concentration of 100IU/ml, and the inhibiting of apoptosis degree of CEPO was similar to that of EPO.4 By the measurement of Bcl-2 and caspase-8 mRNA, we can prove that CEPO and EPO could inhibit the cell apoptosis by the way of Caspase-8 and bcl-2 expression, and there were no significant difference between the two groups. With the increasing concentration of CEPO, the influence on the Bcl-2 and Caspase-8 mRNA was accordingly increasing, which showed that the apoptosis-inhibiting effect was increased with the increase of the CEPO concentration. The dose-dependent protective effects of CEPO were increased with the concentration and reach the maximal at the concentration of 100IU/mL, and the apoptosis-inhibiting degree of CEPO is similar to that of EPO.Significance We first conducted the study of the whether CEPO had protective effects to SH-SY5Y cells and the possible mechanisms. From the study, we can conclude that CEPO can evidently protect SH-SY5Y cells from oxygen-derived free radicals damage, and there be no difference between CEPO and EPO groups. The dose-dependent protection effects of CEPO reach the maximal at the concentration of 100IU/mL. One of the protective effect mechanisms has relation with the inhibition of apoptosis, such as the expression of Bcl-2 and Caspase-8.