The pill contains botanical drugs including Drynariae Rhizoma, Cistanches Herba and Epimedii Folium, which are used to tonify kidney-yang and strengthen bones. According to the research, Yi Shen Juan Bi pill could affect the activation and differentiation of OCs caused by RA by regulating the phenotypic balance of T cells, upregulating the percentage of Tregs, such as IL-10 and TGF-β1 levels, and downregulating the percentage of Th1 and Th17 cells, such as IFN-γ and IL-17A levels. Wang, (2017); Zhao et al. (2018b) Both in vivo and in vitro experiments showed that Yi Shen Juan Bi pill directly inhibited the bone resorption function of OCs by inhibiting the JAK2/STAT3 pathway and upregulating the ephrin B2 pathway to downregulate the expression of OC transcription factors, such as c-Fos, c-Jun, NFATc1, RANK, HMGB1 and RAGE. Guo et al. (2018); Cao, (2021); Xia et al. (2021) Further coculture of Tregs and OCs showed that it could also inhibit the JAK2/STAT3 pathway in Tregs to enhance the immunosuppressive effect of Tregs and indirectly inhibit bone resorption function. Xia et al. (2021) Early studies have shown that it can target the inflammatory and immune regulatory responses of Mφs to reduce the production of TNF-α, IL-1 and NO derived from Mφs in the abdominal cavity. Perera et al. (2011).

Drynariae Rhizoma and Epimedii Folium are the most abundant in the formula. The Bu Shen Tong Luo formula could regulate CD3, CD4, and CD8 T cells to improve the joint score of CIA-rats (Su et al. 2010), and it could inhibit the differentiation and maturation of OCs and bone absorption, promote bone reconstruction, alleviate systemic inflammatory response and joint swelling to treat RA and PMOP through the Chemerin, NF-κB, OPG/RANK/RANKL pathways. Han, (2021); Liu et al. (2021a) Its osteoprotective effects may be related to the stimulation of HIF-1α/VEGF angiogenesis. Yuan et al. (2018).

The combination of RANKL and M-CSF was used to induce RAW264.7 cells to differentiate into OCs. Cervi Cornu and Epimedii Folium are the botanical drugs to tonify kidney-yang mainly in the three prescriptions. Yang et al. also found that Jia Wei Yang He decoction inhibited OC activity and reduced TRAP activity secreted by OCs through the NF-κB pathway. Yang et al. (2022) suggested that Deerhorn Glue pills could improve bone structure, increase bone strength, inhibit bone absorption and promote OC apoptosis through PI3K/AKT. Yu et al. (2018); Yu et al. (2020) It was found that Ai Ke Qing granules could inhibit OC to treat PMOP, which inhibited the binding of RANK and TRAF6 and affected the activation of downstream NF-κB, p38 and JNK pathways to inhibit functional genes, OC surface receptor genes, OC precursor cell fusion genes, bone resorption functional proteins and transcription factors. Liu (2021) There are many tonifying kidney yang formulae, but they focus on the role of OC differentiation and absorption, and research on immune cells and related cytokines is lacking.

In vivo experiments showed that the Chinese herbal prescription for tonifying kidney yang could regulate the inflammatory response, oxidative stress response and downstream pathway through the OPG/RANK/RANKL pathway and play a role in inhibiting OC differentiation. Psoraleae Fructus and Drynariae Rhizoma are the botanical drugs to tonify kidney-yang mainly in the two prescriptions. Yi Shen Bu Gu liquid upregulated IL-33 and downregulated IL-1, IL-7, and TNF-α levels to reduce the inflammatory reaction and increased serum superoxide dismutase (SOD) and catalase (CAT) levels to reduce the oxidation reaction to inhibit OC formation. Li (2021) Bu Shen Qu Han Zhi Wang decoction suppressed OC activity through OPG/RANK/RANKL, reducing the expression of MMP-13, TNF-α, IL-1, IL-2, IL-17, and RANKL to cure RA Li et al. (2017); Ma, (2021); Wu et al.,(2018); Yan, (2012); (Figure 2); (Table 1). These traditional Chinese medicine formulae of tonifying kidney-yang herbs for osteoclastogenesis are preliminarily studied from the cytokines, but the potential immunoregulation mechanisms need to be further explored using immune cells.

Drynariae Rhizoma, the dried rhizome of Drynaria fortunei J. Sm., is a type of medicine for tonifying kidney yang, and its active substance is mainly naringin, which is widely used in the treatment of OP and fracture. Zhang et al. (2017b) A previous study showed that Drynariae Rhizoma possessed immunoregulatory activity, which stimulated the proliferation of the human osteoprecursor cells, promoted alkaline phosphatase activity and protein secretion, and stimulated cellular and humoral immunity. Jeong et al. (2005) Current studies found that it inhibited the differentiation of Mφs into OC by downregulating the expression of RANKL, NFATc1, Fra-1, Fra-2, c-fos, and CTSK and upregulating the expression of OPG, Wnt10b, and β-catenin. Xu et al. (2020); Cheng et al. (2019); Qiu et al. (2016b); Zhang et al. (2019b) Rhizoma Drynariae Flavonoids positively regulated the immune function of immunocompromised mice by increasing the number of leukocytes in peripheral blood, spleen index, thymus index, the expression of inflammatory factors and the ratio of CD4⁺ T/CD8+ T cell (Zhao et al. 2016), while it regulated the OC function of the OVX rats by regulating the OPG/RANKL/RANK pathway. Song et al. (2016a) Its active substance naringin could inhibit NF-κB activation, IκB-α degradation and ERK phosphorylation and downregulate the expression of related genes induced by RANKL to inhibit the differentiation of Mφs into OCs. Ang et al. (2011); Li et al. (2015) Other studies have found that naringin promotes OCs apoptosis by regulating the mitochondrial apoptosis pathway, downregulating BCL-2, and upregulating Bax, caspase-3 and cytochrome C. Li et al. (2014).

Icariin is the main active substances in Epimedii Folium, which inhibits the expression of various genes involved in OC formation and bone resorption through the immune response. Icariin reduced Th17 cells by inhibiting STAT3 activation and suppressed IL-17 production, which contributed to the inhibition of cartilage and bone degradation in RA. Chi et al. (2014) In vitro experiments showed that icariin inhibited reactive oxygen species and the NF-κB, MAPK and AKT pathways in OCs and promoted negative regulators to inhibit osteoclastogenesis genes and inflammatory factors. Ji et al. (2022); Xu et al. (2019); Hsieh et al. (2011); Li et al. (2022) The results of clinical trials showed that icariin plays an anti-inflammatory role by promoting the apoptosis of CD4⁺ lymphocytes in patients with ankylosing spondylitis. Wang et al. (2017) In addition, icariin decreased inflammatory factor expression to inhibit OC formation in titanium particle-charged calvariae and can be used to treat aseptic loosening after arthroplasty. Shao et al. (2015) Other studies have shown that icariin promoted osteogenic differentiation and inhibited OC differentiation by improving the osteogenic activity of MC3T3-E1 cells, inhibiting the OC activity of RAW264.7 cells, reducing the adipogenic differentiation level of bone marrow-derived mesenchymal stem cells (BMSCs), and blocking the osteoclastogenesis induced by MCF7 and MDA-MB-231 breast cancer cells. Kim et al. (2018); Zhang et al. (2017a).

The active substances of Psoraleae Fructus are neobavaisoflavone and psoralen. Psoraleae Fructus, as a Chinese botanical drug for treating RA, reduced the percentages of CD4⁺ IL-17A+, CD4⁺ TNF-α+, and CD4⁺ IFN-γ+ cells in the spleen and the expression of inflammatory factors by increasing myeloid-derived suppressor cells (MDSCs) (Pai et al. 2021), four of which can inhibit RANKL-induced osteoclastogenesis. Lee et al. (2021) Psoralen promoted the balanced development of CD4⁺ CD25⁺ Treg/Th17 to CD4⁺ CD25⁺ T cells by promoting Foxp3, TGF-β and IL-10 levels and inhibiting RORγt, IL-17 and TNF-α levels in CD4⁺ T cells to downregulate OC differentiation-related genes through AP-1 pathways (Li et al. 2018; Chai et al. 2018), and reduced the expression of IL-8 and PTHrP to inhibit the interaction between OCs, OBs and cancer cells and to reduce the burden of bone metastasis caused by breast cancer. Wu et al. (2013) Neobavaisoflavone, another potential bioactive compound, was proven to inhibit osteoclastogenesis and OC function in vitro and in vivo. Neobavaisoflavone was found to block NFATc1 nuclear translocation and inhibit the activation of the NF-κB, MAPK and AKT pathways and the expression of MMP9, CTSK, CTR and TRAP by disrupting the recruitment of TRAF6 and c-Src molecules by the RANK receptor at the cell membrane. Chenet al. (2020a).

Curculigoside is an important active substance of Curculiginis Rhizoma, which is used to treat OP, arthritis and osteolysis. In vivo studies have found that curculigoside reduced the spleen and thymus indices and the expression of inflammatory factors in the serum of CIA rats, such as TNF-α, IL-1β, IL-6, IL-10, IL-12 and IL-17A, while inhibiting the expression of inflammatory factors in Ti-induced osteolysis model mice and restoring the RANKL/OPG ratio. The results showed that curculigoside inhibited osteoclastogenesis and alleviated oxidative stress by activating Nrf2 and inhibiting the NF-κB pathway, alleviated the inflammatory response in MH7A cells through the JAK/STAT/NF-κB pathway, alleviated the expression of inflammatory factors and OC genes in BMSCs, and weakened mitochondrial damage in MC3T3-E1 cells. Liu et al. (2021a); Zhu et al. (2021); Tan et al. (2019) In addition, studies have shown that curculigoside can reverse the immunosuppression caused by cyclophosphamide and proinflammatory cytokines in serum and has anticancer potential against cancer cell lines, such as HepG2, HeLa and MCF-7 (Murali and Kuttan 2015; Hejazi et al. 2018), which may have certain therapeutic value for bone metastasis after the development of a tumour.

Morinda officinalis polysaccharide, bajijiasu and rubiadin-1-mrthyl ether are the active components of Morindae Officinalis Radix, which is commonly used to treat RA and OP and has obvious therapeutic effects on senile, postmenopausal, and glucocorticoid osteoporosis. Morinda officinalis could improve atrophy of the spleen and thymus by increasing the number of CD4⁺ cells and reducing the number of CD8⁺ cells in the peripheral blood of immunosuppressive mice induced by cyclophosphamide. Wei et al. (2019) Morinda officinalis polysaccharide could enhance the immune and anti-inflammatory function of psoriasis patients, enhance the growth capacity of Tregs, and weaken immune and proinflammatory functions. Yin, (2017) It protected bone loss and biomechanical dysfunction in OVX-rats and had antiosteoporosis activity through OPG/RANK/RANKL (Zhang et al. 2020; Peng and Zhong 2020), while inhibiting OC differentiation through the exosomes of mesenchymal stem cells, which was closely related to the expression of PTGS2 and miR-101-3p. Wu et al. (2022c) Bajijiasu and Rubiadin-1-mrthyl ether inhibited the expression of OC-specific genes induced by RANKL through the NF-κB pathway to inhibit OC generation and function and treat osteolytic bone diseases. Hong et al. (2017); He et al. (2018).

There are other tonifying kidney-yang botanical drugs with the functions of tonifying the kidney, strengthening the yang and strengthening the muscles and bones, which are often used to treat immune-mediated bone diseases, but the mechanism of action on immunity needs to be further studied. Eucommiae Cortex can be used to treat OP and RA, and its active components include polysaccharides, iridoid glycosides, and flavonoids. It was found that ethanol extracts from Eucommiae Cortex alleviated the inflammatory response through the OPG/RANK/RANKL and NF-κB pathways to delay the destruction of intraarticular cartilage and bone. Zhang et al. (2021); Wang et al. (2022a); Zhang et al. (2014) The ethanol extracts and polysaccharides from Eucommiae Cortex increased the coefficient of the thymus and spleen and improved the clearance ability and phagocytosis ability of peritoneal Mφs in mice. Qiu et al. (2008); Ye et al. (2015) Osthole may restore BMSC-mediated T-cell migration and apoptosis by stimulating the expression of Fas/FasL, enhancing the immune activity of BMSCs in healthy or inflammatory microenvironments to treat OP (Yu et al. 2021), and downregulating c-Scr, β3-integrin and OC genes to inhibit OC formation and bone resorption. Ma et al. (2019); Zhao et al. (2018a); (Figure 2); (Table 2).

Rehmanniae Radix Praeparata is the main nourish kidney-yin component of the decoction. The Bu Shen Ning Xin decoction can treat PMOP by regulating osteoclastogenesis. A previous study suggested that Bu Shen Ning Xin decoction abrogated RANKL-mediated NFATc1 and NF-κB pathways through selective oestrogen receptor (ER)α. Wang et al. (2015) Later, CD4⁺ T cells were cocultured with BMMs to investigate the immune modulatory effects of Bu Shen Ning Xin decoction on CD4⁺ T cells and osteoclastogenesis, and the findings suggest that Bu Shen Ning Xin decoction has an immune-regulating effect on the bone phenotype of OVX mice by changing the immune environment, increasing CTLA-4+ Tregs and the apoptosis rate of CD4⁺ T cells and regulating the RANKL/OPG imbalance in CD4⁺ T cells. Qiu et al. (2016a); Zhang et al. (2018).

Rehmanniae Radix Praeparata is the main nourish kidney-yin component of the Zuo Gui Wan, which prevented bone loss by downregulating IL-6 and RORγt, upregulated Foxp3 in OVX mice and skewed the Th17/Treg paradigm to Treg (Lai et al. 2015), which may be related to the OPG/RANKL pathway mediated by β2-adrenergic receptor (β2AR). Liu et al. (2018) Further experimental results show that Zuo Gui Wan inhibited bone resorption by inhibiting OC proliferation and differentiation and promoting OC apoptosis. Zhang, (2019).

Rehmanniae Radix Praeparata is the main nourish kidney-yin component of the Yukmijihwang-Tang, which inhibited RANKL-induced OC differentiation, resorption and fusion and OC differentiation in the coculture system of bone marrow cells (BMCs) and OBs and BMMs and downregulated OC-related genes, which may be related to the inhibition of the p38, JNK, ERK, and NF-κB signalling pathways. Han and Kim, (2021); Shim et al. (2011) Moreover, the study found that it upregulated the expression of the immune-related gene IRF1 in the JAK/STAT pathway in PMOP patients with kidney yin deficiency. Chen et al. (2017) However, its mechanism has not been explored with regard to immune cells.

Ligustri Lucidi Fructus and Ecliptae Herba make up Erzhi formula, which is used to treat kidney yin deficiency. Erzhi formula improved PMOP by controlling the OPG/RANK/RANKL signalling axis, and suppressing the expression of OC differentiation genes, such as CTSK, TRAP and NFATc1, which may be related to inhibiting cell autophagy, controlling oestradiol, and nourishing the uterus. Qin et al. (2021); Tian et al. (2020) Meanwhile, Erzhi formula had a protective effect on immunocompromised mice and could improve the activity level of T lymphocyte factor. Yao et al. (2014) At present, there is only network pharmacological analysis of the Erzhi formula in RA, and basic research to prove the relevant mechanism is lacking. (Figure 2); (Table 3).

Ligustri Lucidi Fructus and Ecliptae Herba are two single botanical drugs of Erzhi formula. Wedelolactone is the active ingredient of Ecliptae Herba. Wedelolactone inhibited the production of Sema3A, Sema4D and Sema7A and the number of Sema4d-plexinB1 and plexinA1-DAP12 complexes in BMSCs, and inhibited RANKL-induced F-actin ring formation and bone resorption pits to inhibit osteoclastogenesis and bone resorption. Deng et al. (2018); Liu et al. (2016a); Liu et al. (2016b) Wedelolactone inhibited breast cancer-mediated osteoclastogenesis and OC activity in CD14⁺ monocytes through the AKT-mTOR pathway to treat bone metastasis after breast cancer. Hsieh et al. (2015).

Studies have shown that Ligustri Lucidi Fructus could upregulate the gene expression of the T-cell receptor signalling pathway. Zheng et al. (2018) The extract of Fructose Ligustri Lucidi upregulated the levels of IL-2 and IFN-γ produced by Th1 lymphocytes, and downregulated the level of IL-10 produced by Th2 lymphocytes (Wang et al. 2012), while it inhibited OC differentiation and bone resorption by inhibiting RANKL-induced OC formation and TRAP activity and reduced OC differentiation genes in RAW264.7 cells. Xu et al. (2016a).

Polygonatum sibiricum polysaccharide are some of the main components of Polygonati Rhizome. Polygonatum polysaccharide promoted the development of immune organs, lymphocyte proliferation, natural killer cell (NK) activity and phagocytosis of Mφs, increased the ratio of CD4+/CD8+, and mediated the release of cytokines at the cellular and transcriptional levels to reverse immunosuppression (Chen et al. 2020b; Zhang et al. 2019a), and it inhibited OC differentiation in BMMs through the Hippo signalling pathway based on miR-1224 and maintained bone mass in LPS-induced skull osteolysis-mice. Du et al. (2016); Li et al. (2019).

Two polysaccharides in Rehmanniae Radix Praeparata promoted phagocytosis of RAW264.7 cells and increased lysozyme activity in a dose-dependent manner. Zhou et al. (2021) At the same time, they have immune activity by promoting the activation of human dendritic cells and T cells and inducing the expression of costimulatory molecules and the production of proinflammatory cytokines. Wang et al. (2018a) Catalpol extracted from Rehmanniae Radix Praeparata upregulated the activity of phosphatase and tensin homologue (PTEN) by reducing ubiquitination and degradation, subsequently suppressing the NF-κB and AKT pathways induced by RANKL but had no effect on the MAPK pathway, leading to an inhibition of OC activity to avoid bone loss. Meng et al. (2020) Its immune mechanism may be related to the fact that catapol could inhibit the traditional differentiation and inflammatory factors of Th17 and inhibit the transdifferentiation of Treg-to-Th17 cells associated with STAT3 by upregulating let-7g-5p. Di et al. (2022) Acteoside is the active substance of Rehmanniae Radix Praeparata, which downregulated NFATc1, c-Fos, TNF-α, IL-6 and ROS in Mφs through the RANKL downstream pathway to inhibit osteoclastogenesis. Lee et al. (2013) Studies have shown that acteoside suppresses the differentiation of macrophage-like cells induced by IL-32 and the inflammatory response through the JAK/STAT pathway. Nam et al. (2015); Qiao et al. (2019) The immune regulation mechanism may selectively enhance the production of IFN-γ by T cells and promote the production of IL-10 derived from B cells by regulating the TLR4/PI3K pathway. Wu et al. (2022b); Song et al. (2016b); (Figure 2); (Table 4).