RasGRP1启动子区甲基化对脂多糖诱导人T淋巴细胞炎症的作用及机制

Role of RasGRP1 promoter methylation in lipopolysaccharide-induced human T lymphocyte inflammation and its mechanism

目的探讨RAS鸟苷酸释放蛋白1(RasGRP1)启动子区甲基化对脂多糖(LPS)诱导人T淋巴细胞炎症的作用及机制。方法人T淋巴细胞白血病细胞JurkatE6-1培养至对数生长期,分为对照(完全培养基,C)组、1 mg/L(低浓度)LPS(L1)组、10 mg/L(高浓度)LPS(L2)组及10μmol/L甲基化转移酶抑制剂5-Aza-2′-脱氧胞苷+10 mg/L LPS(LA)组,干预5 d,置于荧光显微镜下观察细胞形态变化;离心收集细胞、留取上清液,采用酶联免疫吸附试验(ELISA)和比色法检测各组细胞中肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)浓度及乳酸脱氢酶(LDH)活性,采用实时荧光定量PCR(RT-qPCR)法检测各组细胞中RasGRP1、细胞外调节蛋白激酶(ERK)1/2、TNF-α、IL-6、DNA甲基转移酶(DNMT)1、DNMT3a mRNA的表达,采用Western blotting法检测各组细胞RasGRP1、ERK1/2及p-ERK1/2蛋白的表达,采用甲基化特异性PCR(MSP)法和琼脂糖凝胶电泳检测RasGRP1启动子区甲基化状态。结果光学显微镜结果显示,C组JurkatE6-1细胞生长状态良好,L1组、L2组细胞数量明显减少,细胞形态趋于碎片化,死亡细胞逐渐增加,随LPS浓度增加而增加;LA组较L2组细胞数量及形态明显好转;与C组比较,L1组JurkatE6-1细胞炎症因子表达升高(P<0.05),细胞RasGRP1、p-ERK1/2表达升高(P<0.05),DNMT1和DNMT3a mRNA表达无差异(P>0.05),RasGRP1启动子区未检测到甲基化表达;与L1组比较,L2组细胞炎症因子表达升高(P<0.05),细胞RasGRP1、p-ERK1/2表达降低(P<0.05),DNMT1和DNMT3a mRNA表达升高(P<0.05),RasGRP1启动子区呈现高甲基化表达;与L2组比较,LA组细胞炎症因子表达降低(P<0.05),细胞RasGRP1、p-ERK1/2表达升高(P<0.05),DNMT1和DNMT3a mRNA表达降低(P<0.05),RasGRP1启动子区未检测到甲基化表达;各组间ERK1/2总量比较无差异(P>0.05)。结论抑制RasGRP1启动子区高甲基化可减轻LPS诱导的人T淋巴细胞炎症损伤,其机制可能是与调控下游ERK信号通路有关。

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 10μmol/L methyltransferase inhibitor 5-Aza-2'-deoxycytidine+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 DNMT 1 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.