| Objective :Experiential administration of methionine enkephalin(MENK) on mouse model improved disease manifestations to some extent. In order to gain more insight on the significance of MENK application, a capillary electrophoresis-mass spectrometry(CE-MS) technique was employed to profile intracellular metabolite fluctuation in 5 astrocytoma cell lines challenged by MENK.Methods: This study selected five different glial cells that is human glioblastoma cell line(U87-MG), human glioma cell line(U251), human glioma cell lines(H4), and rat astrocytoma cell line(C6), rat glioblastoma multiforme cell lines(RG2) challenged by MENK. Cells were divided into two groups, test and control groups. test group was treated by 10 u MM-enk, and the control group was added with the same volume of sterile water. After incubation 24 hours,a capillary electrophoresis-mass spectrometry(CE-MS) technique was employed to profile intracellular metabolite fluctuation in 5astrocytoma cell lines.The processed data were first evaluated through a bioinformatic process to ensure their compatibility with the study aims and then subjected to multivariate analysis. To further investigate the significant change in metabolite(Glycylglycine) whether cell growth and proliferation effect, will significantly change the metabolite configured for different concentrations of the gradient, was added sensitive cells using WST-1 method Cell Viability Assay, drawing growth curve, to determine the optimum concentration. Exemplified by U87 cells, Cells were divided into two groups, test and control groups. test group was treated by 10 u MM-enk, and the control group was added with the same volume of sterile water. After incubation24hours, in order to test Optimalconcentrations ofthe glycylglycine that affect gilia nitric oxide releasment using Nitric oxideassay kit.nitric oxide quantification as recommended by the manufacturer. Also set thetest group and the control group, in which the test group by adding a certain concentration of glycylglycine.Control group plus an equal volume of sterile water. After incubation in the incubator for 24 h, using RT-PCR to detect the change some myelin-related genes of the glial cells challenged by Glycylglycine. The concentration of certain Glycylglycine added to U87-MG, Cells cultured using the same media but replacing MENK with equalvolume of water were utilized as controls, incubated in the incubator for 12 h, 24 h, 48 h, 72 h, the cells were collected by Flow cytometry detecting glial cell cycle distribution at different time.Results: The results indicated that MENK administration increased intracellulartyrosine, phenylalanine, methionine and glycylglycine. Exemplified by U87 cells, glycylglycine inhibited cell proliferation as well as MENK but it also decreased cell nitric oxide excretion which could not be evoked by MENK. The neuron protective effects were also mirrored by the increased expression of some genes related to remyelination.Conclusion: In this assay, taking 5 astrocyte related glioma cell lines as examples, effects of MENK administration on astrocyte metabolism was studied pair-wised in vitro. Based on the thought that most of the intracellular metabolites are polar compounds and they are compatible with capillary electrophoresis – mass spectrometry(CE-MS) detection, a CE-MS platform was employed. Through sequential data quality appraisal and multivariate analysis, intracellular glycylglycine, phenylalanine, tyrosine and methionine were found to be increased after MENK treatment. Most importantly, exemplified by U87 cells, we found that extra glycylglycine could suppress cell growth, decrease the nitric oxide releasing and enhance the expression of some genes related to remyelination. This study exhibited the potential neuron protective benefits from MENK beyond the well studied ligand-receptor pathways and the promising roles of metabolomic study in therapeutic researches on multiple sclerosis. |
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