中国骨质疏松杂志

续苓健骨方调节骨组织m6A甲基化机制研究

Study on the mechanism of Xuling strong-bong formula in regulation of m6A methylation in the bone tissue

DOI：10.3969/j.issn.1006-7108.2025.01.001

中文关键词: 绝经后骨质疏松症 m6A甲基化修饰续苓健骨方甲基化RNA免疫共沉淀

英文关键词:postmenopausal osteoporosis m6A methylation modification Xuling strong-bone formula MeRIP

基金项目:国家自然科学基金项目（82374483）；福建省科技厅公益类科研院所基本科研专项（2022R1003002，2023R1003004）；国家中医药管理局高水平中医药重点学科建设项目(中医骨伤科学)和福建中医药大学中医骨伤科学学科开放课题资助(zyyzdxk-2023106)

作者	单位
屈丽1 谢丽华2，3 叶云金2，3 葛继荣2，3*	1.福建中医药大学，福建福州 350003 2.福建省中医药科学院基础研究所，福建福州 350003 3.福建省中西医结合防治骨质疏松重点实验室(福建省中医药科学院，福建中医药大学附属康复医院)，福建福州 350003

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中文摘要:

目的利用表观转录组学微阵列芯片技术检测续苓健骨方对绝经后骨质疏松症模型大鼠中骨组织mRNA的m6A甲基化修饰和基因表达的变化。方法雌性SPF大鼠随机分为模型组、续苓健骨方组，采用去卵巢法构建绝经后骨质疏松症模型。治疗组以续苓健骨方药液进行灌胃给药，模型组给予等量生理盐水，运用MeRIP-qPCR和表观转录组学微阵列芯片技术，检测2组大鼠骨组织中mRNA的m6A甲基化和表达水平变化，并对这些mRNA进行GO和KEGG分析，基于检测结果选取６个差异甲基化基因进行验证。结果与对照组相比，续苓组中差异表达的mRNA共有4479个，其中2298个上调，1497个下调；1215个基因m6A甲基化修饰水平显著变化，其中592个高甲基化，623个低甲基化；联合分析发现，825个基因同时发生mRNA表达和m6A修饰变化，433个m6A高甲基化且mRNA表达上调，392个m6A低甲基化且mRNA表达下调；GO和KEGG分析显示，m6A高甲基化且表达上调的基因参与骨髓细胞分化、线粒体中释放细胞色素C、红细胞分化等过程；m6A低甲基化且表达下调的基因参与甘油三酯分解代谢过程的调节、PPAR、磷脂酰肌醇、甲状腺激素、胆固醇代谢等信号通路；MeRIP-qPCR验证显示续苓组中Ggt1、Hps4、H1fx、Selp、Hras的相对m6A甲基化水平高于对照组。结论续苓健骨方对绝经后骨质疏松模型大鼠骨组织中m6A甲基化修饰影响显著，其中Ggt1、Hps4、H1fx、Selp、Hras等基因的m6A甲基化修饰变化可能是其治疗绝经后骨质疏松症的机制之一。

英文摘要:

Objective To detect the m6A methylation modification and gene expression changes of the bone tissue mRNA in postmenopausal rats using epitranscriptomics microarray technology. Methods Female SPF rats were randomly divided into model group and Xuling strong-bong formula group. The postmenopausal osteoporosis model was constructed using ovarian removal method. In this study, rats in the treatment group received the same amount of normal saline. MeRIP-qPCR and epitranscriptomics microarray technology were used to detect the m6A methylation and expression level changes of mRNA in the bone tissues of rats in the two groups. mRNAs were analyzed with GO and KEGG. Six differential methylated genes were selected for verification based on the detection results. Results Compared with those in the control group, a total of 4479 differentially expressed mRNAs were in the Xuling group, of which 2298 were up-regulated and 1497 were down-regulated. The m6A methylation modification levels of 1215 genes were significantly changed, including 592 hypermethylation and 623 hypomethylation. The joint analysis showed that 825 genes had mRNA expression and m6A modification changes at the same time. Among those, 433 m6A were hypermethylated and mRNA expression was up-regulated, and 392 m6A were hypomethylated and mRNA expression was down-regulated. GO and KEGG analysis showed that m6A hypermethylated and up-regulated genes were involved in bone marrow cell differentiation, cytochrome C release in mitochondria, and red blood cell differentiation. m6A hypomethylated and down-regulated genes were involved in the regulation of triglyceride catabolism, PPAR, phosphatidylinositol, thyroid hormone, cholesterol metabolism, and other signaling pathways. MeRIP-qPCR showed that the relative m6A methylation levels of Ggt1, Hps4, H1fx, Selp, and Hras were higher than those in the control group. Conclusion Xuling strong-bone formula has a significant effect on the methylation of m6A in the bone tissue of postmenopausal osteoporosis rats. The m6A methylation modification of Ggt1, Hps4, H1fx, selp, Hras and other genes may be one of the mechanisms for the treatment of PMOP.

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