Objective To investigate role of RasGRP1 promoter methylation in lipopolysaccharide(LPS)-induced human T lymphocyte inflammation and its mechanism.Methods Human T lymphocytic leukemia Jurkat E6-1 cells were cultured to the logarithmic growth phase and divided into a control group(complete medium,C group),1 mg/L LPS(low concentration,L1 group),10 mg/L LPS(high concentration,L2 group),and 10 µmol/L methyltransferase inhibitor(5-Aza-2'-deoxy cytidine+10 mg/L LPS(LA group).After 5 days of intervention,the morphological changes of cells were observed under a fluorescence microscope.Cells were gathered by centrifugation.The supernatants were collected for enzyme-linked immunosorbent assay(ELISA)and colorimetry to detect the concentrations of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)as well as lactate dehydrogenase(LDH)activity.Real time fluorescent quantitative PCR(RT-qPCR)was used to detect mRNA expressions of RasGRP1,extracellular-regulated kinase(ERK)1/2,TNF-α,IL-6,DNA methyltransferase 1(DNMT1),and DNMT3a in each group.Western blot was applied to detect protein expressions of RasGRP1,ERK1/2,and p-ERK1/2 in each group.Methylation specific PCR(MSP)and agarose gel electrophoresis were used to detect the methylation status of RasGRP1 promoter region.Results Light microscope results showed that Jurkat(E6-1)cells grew well in Group C.The cell numbers in L1 and L2 groups were significantly decreased,and cell morphology tended to be fragmented,and the number of dead cells were increased with the increase of LPS concentrations.The number and morphology of cells in LA group were significantly improved when compared to L2 group.When compared with C group,L1 group had increased expressions of inflammatory factors in Jurkat E6-1 cells(P＜0.05),increased the expressions of RasGRP1 and p-ERK1/2(P＜0.05),non-distinct differences in mRNA expressions of DNMT1 and DNMT3a(P＞0.05).No methylation was detected in the RasGRP1 promoter region.When compared with L1 group,L2 group had increased expressions of inflammatory factors(P＜0.05),decreased expressions of RasGRP1 and p-ERK1/2(P＜0.05),increased mRNA expressions of DNMT 1 and DNMT3a(P＜0.05)and a highly methylated promoter of RasGRP1.When compared with the L2 group,LA group had decreased expressions of inflammatory factors(P＜0.05),increased expressions of RasGRP1 and p-ERK1/2(P＜0.05),decreased mRNA expressions of DNMT1 and DNMT3a(P＜0.05).No methylation was detected in the RasGRP1 promoter region.There were no differences in total ERK1/2 expression levels between groups(P＞0.05).Conclusion Inhibiting high methylation of the RasGRP1 promoter region can alleviate LPS-induced inflammatory damage in human T lymphocytes,and its mechanism may be related to regulating the downstream ERK signaling pathway.
关键词
脓毒症/脂多糖类/DNA甲基化/炎症/RAS鸟苷酸释放蛋白1/JurkatE6-1细胞
Key words
sepsis/lipopolysaccharide/DNA methylation/inflammation/Ras guanyl-releasing protein 1/JurkatE6-1 cells
维普
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