中国骨质疏松杂志

填精益气中药对BMSCs成骨分化及甲基化转移酶METTL3的影响

Effect of Chinese herbal medicine replenish essence and boost Qi on differentiation of BMSCs and methylation transferase METTL3

DOI：10.3969/j.issn.1006-7108.2023.07.001

中文关键词: 骨髓间充质干细胞填精益气 METTL3 甲基化骨质疏松症

英文关键词:bone marrow mesenchymal stem cells replenish essence and boost Qi METTL3 methylation osteoporosis

基金项目:国家重点研发计划（2018YFC1704301）；辽宁省自然科学基金面上项目（2020-MS-222）；辽宁省教育厅重点攻关项目（LNKZZ20220103）；沈阳市中青年科技创新人才支持计划（RC210129）；辽宁中医药大学自然科学重点项目（2021LZY035）；辽宁中医药大学中医脏象理论及应用教育部重点实验室开放基金（zyzx2201）

作者	单位
邓洋洋* 刘明欣王重一高巳东林浩楠刘德育	辽宁中医药大学，辽宁沈阳 110847

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中文摘要:

目的探究基于“精气互化”理论的填精益气中药对骨髓间充质干细胞（bone marrow mesenchymal stem cells，BMSCs）向成骨分化的影响以及其分化过程中对甲基化转移酶METTL3、WTAP、PPARγ影响的实验研究。方法将BMSCs随机分为对照组、诱导组、填精组、益气组以及填精益气组。填精组药物为鹿茸提取液，益气组药物为黄芪甲苷，填精益气组药物为鹿茸提取液联合黄芪甲苷。使用Elisa法检测6、12、18 d各组细胞碱性磷酸酶（alkaline phosphatase，ALP）含量变化；qRT-PCR法检测各组细胞METTL3、WTAP、PPARγ的基因转录；Western blot法检测各组METTL3、WTAP、PPARγ的蛋白表达。结果 ALP实验结果显示，填精益气组ALP活性优于填精组和益气组，且在第12天填精益气组的ALP活性达到了最高值；QRT-PCR及Western blot的实验结果表明，与对照组相比，各用药组甲基化转移酶METTL3和WTAP mRNA及蛋白表达水平均显著上调（P＜0.01），PPARγ mRNA及蛋白表达水平显著下调（P＜0.01）。结论填精法、益气法、填精益气法对BMSCs向成骨分化均具有促进作用，可能是通过提高甲基化转移酶METTL3和WTAP表达，降低PPARγ的表达，进而促进BMSCs向成骨分化增加，向成脂分化减少而实现的。三组中填精益气组明显优于单独用药组，说明填精益气组更有效提高BMSCs增殖向成骨分化，延缓成骨细胞凋亡进程，对绝经后骨质疏松症及其并发症的防治具有指导意义。

英文摘要:

Objective To investigate the effect of Chinese herbal medicine for replenish essence and boost Qi on the proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs) based on the theory of the essence and qi complement each other and the effect of methyltransferase METTL3, WTAP, and PPARγ on osteogenic differentiation. Methods BMSCs were randomly divided into control group, induction group, essence filling group, Qi supplementing group, and replenish essence and boost Qi group. The drugs in the essence filling group were Antler extract, the drugs in the Yiqi group were astragaloside IV, and the drugs in the Qi filling group were Antler extract combined with astragaloside IV. ELISA method was used to detect the changes of ALP content in each group at 6, 12 and 18 days. Gene transcription of METTL3, WTAP, and PPARγ in each group was detected with qRT-PCR. The protein expressions of METTL3, WTAP, and PPARγ in each group were detected using Western blotting. Results The ALP test results showed that the ALP activity in the replenish essence and boost Qi group was better than that in the essence filling group and the boost Qi group, and the ALP activity of the replenish essence and boost Qi reached the highest value on the 12th day. The results of qRT-PCR and Western blotting showed that compared to those in the control group, the mRNA and protein expression levels of methyltransferase METTL3 and WTAP in all drug groups were significantly up-regulated (P<0.01), while the mRNA and protein expression levels of PPARγ were significantly down-regulated (P<0.01). Conclusion All the essence filling method, boost Qi method, and replenish essence and boost Qi method promote the osteogenic differentiation of BMSCs, which may be achieved by increasing the expressions of METTL3 and WTAP and decreasing the expression of PPARγ, thus promoting the osteogenic differentiation of BMSCs and decreasing the lipogenic differentiation. Among the three groups, the replenish essence and boost Qi group is significantly better than the medication alone group, indicating that the replenish essence and boost Qi effectively improve the proliferation of BMSCs to osteogenic differentiation and delay the process of osteoblast apoptosis, which has guiding significance for the prevention and treatment of PMOP and its complications.

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