Objective To investigate the therapeutic effects of Qing'e formula on postmenopausal osteoporosis, analyze its influence on bone metabolism, gut microbiota, and inflammation.

Methods An ovariectomized (OVX) rat model of osteoporosis was established. Animals were divided into a normal group, an OVX model group, and low-, medium-, and high-dose Qing'e formula treatment groups. Bone mineral density (BMD) and micro-architectural parameters were assessed by micro-computed tomography. Hematoxylin-eosin staining was used to observe histopathological changes in bone tissue. Serum levels of procollagen type I N-terminal propeptide (PINP), osteocalcin (BGP), bone-specific alkaline phosphatase (BALP), collagen type I cross-linking C-telopeptide (CTX), tartrate-resistant acid phosphatase (TRAP), calcium, phosphorus, and the inflammatory cytokines IL-1β, IL-6, and TNF-α were measured by ELISA. Changes in the gut microbiota were analyzed by bacterial culture. Expression of p-P65 was determined by Western blot.

Results Compared with the normal group, OVX rats exhibited sparse, disordered trabeculae and significantly lower BMD, bone volume/tissue volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and serum PINP. Trabecular separation (Tb.Sp), serum BGP, BALP, CTX, TRAP, IL-1β, IL-6, TNF-α, and p-P65 expression were markedly increased. Compared with the model group, the high-dose Qing'e formula group exhibited regularly arranged trabecular bone morphology, with significant increases in BMD, BV/TV, Tb.Th, Tb.N, and PINP, as well as significant decreases in Tb.Sp, BGP, BALP, CTX, TRAP, IL-1β, IL-6, TNF-α, and p-P65. The abundances of the probiotic bacteria Lactobacillus and Bifidobacterium were significantly increased, whereas those of the pathogenic bacteria Proteus and Bacteroides were significantly decreased.

Conclusion Qing'e formula exerts anti-osteoporotic potential by modulating gut microbiota composition and suppressing inflammatory cytokine release, thereby attenuating inflammation and enhancing bone formation.

1. 中华医学会骨质疏松和骨矿盐疾病分会. 原发性骨质疏松症诊疗指南(2022)[J]. 中国全科医学, 2023, 26(14): 1671-1691.Chinese Society of Osteoporosis and Bone Mineral Research. Guidelines for the diagnosis and treatment of primary osteoporosis (2022)[J]. Chinese General Practice, 2023, 26(14): 1671-1691. DOI: 10.12114/j.issn.1007-9572.2023.0121.

2. 中国康复医学会骨质疏松预防与康复专业委员会. 骨质疏松性骨折二级预防中国专家共识[J]. 中华医学杂志, 2022, 102(45): 3581-3591.Professional Committee on Osteoporosis Prevention and Rehabilitation, Chinese Association of Rehabilitation Medicine. Chinese expert consensus on secondary prevention of osteoporotic fractures[J]. National Medical Journal of China, 2022, 102(45): 3581-3591. DOI: 10.3760/cma.j.cn112137-20220923-02011.

3. MannER, LamYK, UhligHH. Short-chain fatty acids: linking diet, the microbiome and immunity[J]. Nat Rev Immunol, 2024, 24: 577-595. DOI: 10.1038/s41577-024-01014-8.

4. ZhangYW, WuY, LiuXF, ChenX, SuJC. Targeting the gut microbiota-related metabolites for osteoporosis: the inextricable connection of gut-bone axis[J]. Ageing Res Rev, 2024, 94: 102196. DOI: 10.1016/j.arr.2024.102196.

5. 姚阳阳, 李天友, 王延宙. 双膦酸盐治疗成骨不全的疗效与争议[J]. 中华小儿外科杂志, 2021, 42(10): 956-960.YaoYY, LiTY, WangYZ. Efficacy and controversy of bisphosphonates in the treatment of osteogenesis imperfecta[J]. Chinese Journal of Pediatric Surgery, 2021, 42(10): 956-960. DOI: 10.3760/cma.j.cn421158-20200430-00304.

6. GuanZ, XuanqiZ, ZhuJ, et al. Estrogen deficiency induces bone loss through the gut microbiota[J]. Pharmacol Res, 2023, 196: 106930. DOI: 10.1016/j.phrs.2023.106930.

7. 郭世明, 吴志君, 何亚标, 等. 青娥方对去卵巢大鼠骨组织Src/p130Cas/CRK信号通路的影响[J]. 中华中医药杂志, 2020, 35(9): 4347-4351.GuoSM, WuZJ, HeYB, et al. Effects of Qing'e Formula on the Src/p130Cas/CRK signaling pathway in bone tissue of ovariectomized rats[J]. China Journal of Traditional Chinese Medicine and Pharmacy, 2020, 35(9): 4347-4351. DOI: 10.13288/j.11-2166/r.2020.09.012.

8. 唐崇曦, 戴燚, 唐光平, 等. 青娥方对膝骨关节炎伴骨质疏松模型大鼠早期软骨下骨整合素及抑癌基因表达的影响[J]. 中国中医骨伤科杂志, 2022, 30(5): 1-5.TangCX, DaiY, TangGP, et al. Influence of Qing'e Formula on integrin and tumor-suppressor gene expression in early subchondral bone of rats with knee osteoarthritis and osteoporosis[J]. Chinese Journal of Traditional Medical Traumatology & Orthopedics, 2022, 30(5): 1-5. DOI: 10.12209/j.issn.1005-0205.202205001.

9. 吴永贵, 胡霞, 李章青, 等. 补肾法对绝经后骨质疏松症肠道菌群的影响[J]. 时珍国医国药, 2022, 33(2): 504-507.WuYG, HuX, LiZQ, et al. Effect of kidney-tonifying therapy on intestinal flora in postmenopausal osteoporosis[J]. Lishizhen Medicine and Materia Medica Research, 2022, 33(2): 504-507. DOI: 10.3969/j.issn.1008-0805.2022.02.71.

10. 孙鑫, 杨铠宁, 王庆谚, 等. 基于"肾⁃血⁃骨"理论探究GH/IGF-1系统在青娥丸防治骨质疏松症中的作用机制[J]. 海南医学学报, 2022, 28(23): 1785-1791.SunX, YangKN, WangQY, et al. Mechanism of GH/IGF-1 system in Qing'e Pill against osteoporosis based on "kidney-blood-bone" theory[J]. Journal of Hainan Medical University, 2022, 28(23): 1785-1791. DOI: 10.13210/j.cnki.jhmu.20220825.003.

11. BhatnagarA, KekatpureAL. Postmenopausal osteoporosis: a literature review[J]. Cureus, 2022, 14(9): e29367. DOI: 10.7759/cureus.29367.

12. ChenM, GergesM, RaynorWY, et al. State of the art imaging of osteoporosis[J]. Semin Nucl Med, 2024, 54(3): 415-426. DOI: 10.1053/j.semnuclmed.2023.10.008.

13. 中国中西医结合学会骨伤科专业委员会. 骨质疏松症中西医结合诊疗指南[J]. 中华医学杂志, 2019, 99(45): 3524-3533.Professional Committee of Orthopedics and Traumatology, Chinese Association of Integrative Medicine. Guidelines for integrated traditional Chinese and Western medicine diagnosis and treatment of osteoporosis[J]. National Medical Journal of China, 2019, 99(45): 3524-3533. DOI: 10.3760/cma.j.issn.0376-2491.2019.45.002.

14. 陆思成, 向昱阳, 唐正. 青娥丸联合钙尔奇D3治疗绝经后骨质疏松症肾阳虚型临床观察[J]. 实用中医药杂志, 2019, 35(12): 1428-1430.LuSC, XiangYY, TangZ. Clinical observation of Qing'e Pill combined with Caltrate D3 in treating postmenopausal osteoporosis of kidney-yang deficiency type[J]. Journal of Practical Traditional Chinese Medicine, 2019, 35(12): 1428-1430. DOI: 10.3969/j.issn.1004-2814.2019.12.012.

15. XieH, HuaZ, GuoM, et al. Gut microbiota and metabonomics used to explore the mechanism of Qing'e Pills in alleviating osteoporosis[J]. Pharm Biol, 2022, 60(1): 785-800. DOI: 10.1080/13880209.2022.2056208.

16. LuJ, HuD, MaC, et al. Modified Qing' e Pills exerts anti-osteoporosis effects and prevents bone loss by enhancing type H blood vessel formation[J]. Front Endocrinol (Lausanne), 2022, 13: 998971. DOI: 10.3389/fendo.2022.998971.

17. 陈帆, 魏戌, 崔鑫, 等. 青娥丸治疗绝经后骨质疏松症的系统评价及Meta分析[J]. 中国骨质疏松杂志, 2021, 27(2): 179-189.ChenF, WeiX, CuiX, et al. Systematic review and Meta-analysis of Qing'e Pill in the treatment of postmenopausal osteoporosis[J]. Chinese Journal of Osteoporosis and Bone Mineral Research, 2021, 27(2): 179-189. DOI: 10.3969/j.issn.1006-7108.2021.02.006.

18. ZhuF, LiuH, CaoY, et al. The combination of Butyricicoccus pullicaecorum and 3-hydroxyanthranilic acid prevents postme-nopausal osteoporosis by modulating gut microbiota and Th17/Treg[J]. Eur J Nutr, 2024, 63(5): 1945-1959. DOI: 10.1007/s00394-024-03400-3.

19. FengR, WangQ, YuT, et al. Quercetin ameliorates bone loss in OVX rats by modulating the intestinal flora-SCFAs-inflammatory signaling axis[J]. Int Immunopharmacol, 2024, 136: 112341. DOI: 10.1016/j.intimp.2024.112341.

20. WangN, YangH, TongX, et al. Ginkgolide B modulates the gut-bone axis to ameliorate bone loss in ovariectomized mice[J]. J Orthop Surg Res, 2025, 20(1): 804. DOI: 10.1186/s13018-025-06215-y.

21. ChenM, WangJ, YangY, et al. The interplay of estrogen, gut microbiome, and bone immunity in osteoporosis[J]. Cell Commun Signal, 2025, 23(1): 516. DOI: 10.1186/s12964-025-02538-9.

22. YuM, PalS, PatersonCW, et al. Ovariectomy induces bone loss via microbial-dependent trafficking of intestinal TNF+ T cells and Th17 cells[J]. J Clin Invest, 2021, 131(4): e143137. DOI: 10.1172/JCI143137.

23. ZaissMM, JonesRM, SchettG, et al. The gut-bone axis: how bacterial metabolites bridge the distance[J]. J Clin Invest, 2019, 129(8): 3018-3028. DOI: 10.1172/JCI128521.

24. TyagiAM, YuM, DarbyTM, et al. The microbial metabolite butyrate stimulates bone formation via T regulatory cell-mediated regulation of WNT10B expression[J]. Immunity, 2018, 49(6): 1116-1131. DOI: 10.1016/j.immuni.2018.10.013.