基于METTL14-m^(6)A-FOXO3A信号通路介导的细胞自噬探讨补肾强督方抑制炎症治疗强直性脊柱炎的机制研究  

Bushen Qiangdu formula in suppressing inflammation in ankylosing spondylitis via METTL14⁃m^(6)A⁃FOXO3A signaling pathway mediated autophagy

作　　者：吴琳 李松倍[1] 苏晓庆 韩天然 李泽光[2] WU Lin;LI Songbei;SU Xiaoqing;HAN Tianran;Li Zeguang(Heilongjiang University of Chinese Medicine,Harbin 150040,China;The First Affiliated Hospital of Heilongjiang University of Chinese Medicine,Harbin 150040,China)

机构地区：[1]黑龙江中医药大学,黑龙江哈尔滨150040 [2]黑龙江中医药大学附属第一医院,黑龙江哈尔滨150040

出　　处：《海南医科大学学报》2025年第2期94-101,共8页Journal of Hainan Medical University

基　　金：白求恩医学科学研究基金(TY144EN)。

摘　　要：目的:观察补肾强督方对T淋巴细胞METTL14-m^(6)A-FOXO3A信号通路介导的细胞自噬的影响,从而明确其抑制炎症、治疗强制性脊柱炎的分子机制。方法:使用Jurkat细胞,采用抗CD3联合抗CD28抗体诱导24 h构建细胞模型,并给予补肾强督方含药血清和METTL14重组蛋白进行干预。采用ELISA法检测炎症因子(IL-23和IL-17A)和氧化应激指标的水平,流式细胞术检测ROS水平,Western blot法检测自噬相关蛋白、METTL14和FOXO3A的表达,RT-qPCR检测METTL14和FOXO3A的表达,斑点杂交实验检测FOXO3A的m^(6)A甲基化水平。结果:与模型组比较,补肾强督方显著降低Jurkat细胞上清中IL-23、IL-17A和MDA的含量与ROS的水平,增加SOD和GSH-Px的活性,差异均具有统计学意义(P<0.01)。此外,补肾强督方能显著上调Jurkat细胞中Beclin-1、FOXO3A和METTL14蛋白和基因的表达和增加LC3Ⅱ/LC3Ⅰ的比值,下调p62蛋白的表达,与模型组比较差异均具有统计学意义(P<0.01)。在此基础之上,与模型组比较,补肾强督方能显著上调Jurkat细胞FOXO3A的m^(6)A甲基化水平,差异均具有统计学意义(P<0.01)。结论:补肾强督方通过METTL14介导的FOXO3A的m^(6)A甲基化修饰促进T细胞自噬,从而达到治疗强直性脊柱炎的作用。Objective:To investigate the effect of Bushen Qiangdu formula on METTL14-m^(6)A-FOXO3A signaling pathwaymediated autophagy in T lymphocytes,and to elucidate its molecular mechanism in suppressing inflammation for the treatment of ankylosing spondylitis(AS).Methods:Jurkat cells were stimulated with anti-CD3 and anti-CD28 antibodies for 24 h to establish the cell model,followed by intervention with Bushen Qiangdu medicated serum and METTL14 recombinant protein.ELISA was used to measure the levels of inflammatory factors(IL-23 and IL-17A)and oxidative stress markers.ROS levels were assessed by flow cytometry.Western blot methodology was utilized to ascertain the levels of expression of autophagy-related proteins,namely METTL14 and FOXO3A.Real-time quantitative polymerase chain reaction was used to determine the mRNA and protein expression levels of METTL14 and FOXO3A,and dot blot hybridization was performed to assess the m^(6)A methylation level of FOXO3A.Results:Compared to the model group,Bushen Qiangdu medicated serum significantly reduced the levels of IL-23,IL-17A,and MDA,as well as ROS levels of Jurkat cells,all of which were statistically significant(P<0.01),while the activity of SOD and GSH-Px were increased.The results demonstrated statistically significant discrepancies between the experimental and control groups(P<0.01).Additionally,Bushen Qiangdu medicated serum notably upregulated the expression of Beclin-1,FOXO3A,and METTL14 proteins,and increased the LC3II/LC3I ratio while downregulating p62 protein expression(P<0.01).Moreover,Bushen Qiangdu medicated serum significantly enhanced the m^(6)A methylation level of FOXO3A in Jurkat cells compared to the model group(P<0.01).Conclusion:Bushen Qiangdu formula promotes T cell autophagy through METTL14-mediated m^(6)A methylation of FOXO3A,achieving therapeutic effects in AS.

关 键 词：补肾强督方 T淋巴细胞 METTL14 FOXO3A m^(6)A甲基化修饰 

分 类 号：R285[医药卫生—中药学]