Regulatory effect of Traditional Chinese Medicinal Formula Zuo-Gui-Wan on the Th17/Treg paradigm in mice with bone loss induced by estrogen deficiency

Bone loss is a common pathological condition induced by estrogen deficiency. The Th17/Treg paradigm, which can be skewed by estrogen, plays an important role in regulating bone metabolism The purpose of this study was to determine the role of the Th17/Treg shift in estrogen deficiency-induced bone loss in mouse models and to elucidate the immunopharmacologic mechanism of Zuo-Gui-Wan (ZGW) in preventing bone loss in this process by regulating Th17/Treg paradigm. Splenocytes of ovariectomized (Ovx) mice and naturally aged mice were isolated and Flow cytometry was used to detect the Th17/Treg subsets. In addition, serum estrodiol (E2) and serum C-terminal telopeptides of type Ι collagen (CTx) were detected by ELISA assay. Bone mineral density (BMD) of the left tibiae was measured by dual-energy X-ray absorptiometry. Moreover, Ovx mice were administrated with different doses of ZGW for 12 weeks, and BMD and Th17/Treg subsets were determined. Bone histomorphometry was observed by Hematoxylin and eosin (H&E) staining and serum protein levels of IL-6 were analyzed by ELISA assay. In addition, the mRNA and protein expression of RORγt and Foxp3 were detected by RT-PCR and Western blot respectively. The Th17/Treg paradigm shifted to Th17 in estrogen-deficient mice both in the Ovx mice and the naturally aged mice. BMD and E2 levels negatively correlated with the Th17/Treg ratio. After ZGW administration, the BMD was enhanced markedly in the Ovx mice as well as in the naturally aged mice. Both the mRNA and protein expressions of IL-6 and RORγt were decreased, whereas those of Foxp3 were increased significantly after ZGW administration. Th17/Treg shift involved in the bone loss induced by estrogen deficiency. ZGW prevented bone loss efficiently and skewed Th17/Treg paradigm towards Treg without enhancing E2.