Fangyu Hao,1,2,* Mengyu Guo,1,2,* Yuwei Zhao,1,2 Xingyu Zhu,1,2 Xiaofang Hu,1,2 Weihao Zhu,1,2 Chunmei Mei,1,2 Nong Zhou,3 Kunming Qin,4 Hui Zhu,1,2 Weidong Li1,2 

1School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, People’s Republic of China; 2Jiangsu Key Laboratory of Chinese Medicine Processing, Engineering Center of State Ministry of Education for Standardization of Chinese Medicine Processing, Nanjing University of Chinese Medicine, Nanjing, People’s Republic of China; 3College of Biology and Food Engineering, Chongqing Three Gorges University, Chongqing, People’s Republic of China; 4School of Pharmacy, Jiangsu Ocean University, Lianyungang, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Hui Zhu, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, People’s Republic of China, Email zhui_0826@njucm.edu.cn Weidong Li, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, People’s Republic of China, Email liweidong0801@163.com

Purpose: Bone metabolism disorders are strongly associated with T helper type 17/regulatory T (Th17/Treg) cell imbalance and inflammatory dysregulation. Qing’e pills (QEP) is a classical prescription for treating osteoporosis with both safety and clinical effectiveness. However, the mechanism of its immune action remains unclear.
Methods: QEP components were identified via HPLC. Anti-osteoporotic effects of QEP were assessed through biochemical, micro computed tomography, bone biomechanical and histopathological analyses. Th17/Treg balance and related inflammatory factors were analyzed using flow cytometric, biochemical, immunohistochemical, and quantitative real-time PCR assays. The effects of QEP on gut microbiota and endogenous metabolites were analyzed via 16S rRNA analysis, co-incubation experiments and untargeted metabolomics. Integrative correlations analysis was used to explore the relationships among gut-bone-Th17/Treg balance interactions.
Results: QEP improved bone mineral density and bone biomechanical properties and reduced bone conversion in ovariectomized rats. After treatment, QEP restored intestinal barrier integrity, and reduced serum LPS levels. QEP significantly decreased Th17-related inflammatory cytokines TNF-α, IL-17 levels, reduced the transcription of Th17-related genes RORγt and IL-17A and the percentage of CD4+IL-17A+ Th17 cells in the gut-bone axis, and concurrently restored the anti-inflammatory cytokines levels of TGF-β and IL-10, the expression of Foxp3 and the percentage of CD4+ CD25+ Foxp3+ Treg cells in the gut–bone axis. Notably, QEP improved the disorganization of gut microbiota composition and structure in ovariectomized rats. On genus level, QEP can significantly increase the relative abundance of Lactobacillus in vitro and in vivo. Furthermore, gut microbe-derived endogenous metabolites potentially mediating QEP’s regulation of Th17/Treg balance in gut-bone axis and anti-osteoporotic effects.
Conclusion: QEP ameliorates osteoporosis by improving the intestinal flora disorders and immune status, and restoring the balance of Th17/Treg in the gut-bone axis, highlighting its clinical potential in the treatment of postmenopausal osteoporosis.

Keywords: Qing’e pills, osteoporosis, Th17/Treg, gut microbes, Lactobacillus