Objective To explore the role of p38 MAPK on the apoptosis of mouse embryo fibroblasts induced by myeloid-related protein 8 (MRP8)/myeloid-related protein 14 (MRP14).Methods MTT assay was performed to detect the cell viability of p38+/+ and p38-/-cells in the control group and groups in which cells were treated with 50 μg/mL MRP8, MRP14, and MRP8/14 for 24 h and 48 h;flow cytometry was applied to detect the apoptosis rate of p38+/+ and p38-/-cells in the control group and groups in which cells were treated with 50 μg/mL MRP8/14 for 24 h and 48 h;MTT assay was performed to detect the cell viability of p38+/+ cells in the control group, MRP8/14 group, MRP8/14+TAK242(TLR4 inhibitor) group and MRP8/14+RAGE neutralized antibody group for 24 h;MTT and flow cytometry were used to analyze the cell viability and the apoptosis rate of p38+/+ cells in the control group, MRP8/14 group, MRP8/14+SB203580 (p38 MAPK inhibitor) group for 24 h;Western blotting was used to detect the phosphorylation changes of p38 MAPK in p38+/+ cells treated with MRP8/14 for 0, 1, 2, 4, 6, and 8 h, respectively.Results The cell viability of p38+/+ and p38-/-cells in the groups treated with MRP8, MRP14, and MRP8/14 for 24 h and 48 h was lower than that of the control group (P<0.05).At the same time, the cell viability of p38-/-cells was significantly higher than that of p38+/+ cells in the MRP8/14 group at 24 and 48 h (P<0.05).The apoptosis rate of p38+/+ cells in the MRP8/14 group at 24 and 48 h was higher than that of the control group, especially at 48 h (all P<0.05).However, the apoptosis rate of p38-/-cells did not change after MRP8/14 treatment for 24 and 48 h.The cell viability of p38+/+ cells in the MRP8/14 group and MRP8/14+RAGE neutralized antibody group was lower than that in the control group and MRP8/14 +TAK242 group (P<0.05).The cell viability of p38+/+ cells in the MRP8/14+SB203580 group was higher than that of the MRP8/14 alone group, and the apoptosis rate of p38+/+ cells in the MRP8/14+SB203580 group was lower than that of the MRP8/14 alone group (P<0.05).The phosphorylation level of p38 MAPK in p38+/+ cells was significantly higher at 2, 4, and 6 h after MRP8/14 treatment than that at 0, 1, and 8 h (P<0.05).Conclusion MRP8/14 can induce the apoptosis of mouse embryo fibroblasts via the activation of TLR4-p38 MAPK pathway.%目的 探讨p38丝裂原活化蛋白激酶(p38 MAPK)对髓样相关蛋白8(MRP8)/髓样相关蛋白14(MRP14)诱导小鼠胚胎成纤维细胞凋亡的影响.方法 制备MRP8、MRP14、MRP8/14备用.分别取p38+/+和p38-/-细胞,随机分为空白对照组、MRP8组、MRP14组、MRP8/14组.MRP8组、MRP14组、MRP8/14组分别加入50 μg/mL MRP8、MRP14和MRP8/14.空白对照组加入等体积的DMEM培养液.各组干预24、48 h,采用MTT法检测p38+/+和p38-/-细胞活力.采用流式细胞术检测空白对照组及MRP8/14干预24、48 h组p38+/+和p38-/-细胞凋亡率.采用MTT法检测空白对照组、MRP8/14组以及TLR4受体抑制剂TAK242或RAGE中和抗体处理的MRP8/14(分别设为MRP8/14+TAK242组、MRP8/14+RAGE组)干预24 h p38+/+细胞活力.采用MTT法和流式细胞术检测空白对照组、MRP8/14组以及p38激酶抑制剂SB203580处理的MRP8/14(设为MRP8/14+SB203580组)干预24 h p38+/+细胞活力和凋亡率.采用Western blotting法检测MRP8/14干预0、1、2、4、6、8 h p38+/+细胞p38 MAPK磷酸化.结果 MRP8组、MRP14组、MRP8/14组干预24、48 h p38+/+、p38-/-细胞活力均低于空白对照组(P均<0.05),但无论MRP8/14组干预24 h还是48 h,p38-/-细胞活力明显高于p38+/+细胞(P均<0.05).MRP8/14干预24、48 h组p38+/+细胞凋亡率均高于空白对照组,且MRP8/14干预48 h组细胞凋亡率更高(P均<0.05);而MRP8/14干预24、48 h组p38-/-细胞凋亡率均未见明显变化(P均>0.05).MRP8/14组、MRP8/14+RAGE组干预24 h p38+/+细胞活力低于空白对照组、MRP8/14+TAK242组干预24 h(P均<0.05).MRP8/14+SB203580组干预24 h p38+/+细胞活力较MRP8/14组干预24 h明显升高,细胞凋亡率较MRP8/14组干预24 h明显降低(P均<0.05).MRP8/14干预2、4、6 h p38+/+细胞p38 MAPK磷酸化水平明显高于干预0、1、8 h(P均<0.05).结论 p38 MAPK能促进MRP8/14诱导的小鼠成纤维细胞凋亡,其机制可能与TLR4-p38 MAPK信号通路激活有关.
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