Ｒesearch Progress in The Clinical Efficacy Of Traditional Chinese Medicine On The Treatment Of Postmenopausal Osteoporosis Ⅱ

2 Research progress on the molecular mechanisms of deep pathways involved in the prevention and treatment of PMOP by traditional Chinese medicine

 

2.1 Regulation of osteoblast-osteoclast balance by traditional Chinese medicine in the PMOP process

 

2.1.1 ＲUNX2, BMP-2 and GSK-3β: BMSCs have multi-directional differentiation potential［39］.

 

OB differentiation, repair and mineralization functions are mainly affected by signaling pathways such as Runt-related transcription factor 2 (ＲUNX2), bone morphogenetic protein (BMP) and Wnt/β-catenin. ＲUNX2 is one of the osteoblast-specific transcription factors. It regulates protein expression by binding to different core sites of promoters and enhancers, inducing BMSCs to differentiate into OBs. BMP-2 is a transforming growth factor, which also has the ability to induce BMSCs to differentiate into OBs［40］. Therefore, the inhibition of BMP-2 and ＲUNX2 can reduce the osteogenic activity of BMSCs, reduce OB, reduce bone formation, and fail to maintain the balance between OB and OC, which aggravates bone loss. Studies have found that costunolide mainly acts on BMSCs, which enhances the osteogenic activity of BMSCs and promotes OB differentiation to prevent and treat PMOP by upregulating the mＲNA expression of BMP-2 and ＲUNX2［41］.

 

Herbal Cistanche Supplements For Bone Health

 

Glycogen synthase kinase-3β (GSK-3β) is a serine/threonine protein kinase that mainly participates in bone metabolism through phosphorylation［42］. OB proliferation and differentiation are related to Lrp6 and β-catenin regulation, and GSK-3β mＲNA expression is related to GSK-3β phosphorylation. Wnt/β-catenin is an important signaling pathway for regulating bone remodeling and bone balance, which can regulate BMSCs differentiation into OB［43］. Wnt ligands bind to frizzled and lipoprotein receptor-related protein 5/6 (Lrp5/6) receptors, resulting in inhibition of GSK-3β activity, β-catenin translocation to the nucleus, and binding to the N-terminus of T cell factor/lymphocyte proliferation factor (Tcf/Lef) family DNA binding protein, regulating the transcription of OB-related target genes and promoting OB formation. Rehmannia catalpol is the main active ingredient of Rehmannia root for the treatment of PMOP, which may activate the Wnt/β-catenin signaling pathway, enhance BMSCs-specific gene expression, and promote osteogenic differentiation [44].
At the same time, the Wnt/β-catenin signaling pathway may promote OB differentiation by upregulating BMP-2 and BMP-4 expression, activating p38MAPK to inhibit GSK-3β, reducing OC activity, and regulating Wnt/βcatenin signaling to maintain bone metabolism balance. The active ingredients of Yougui Pills can increase β-catenin mRNA expression, reduce GSK-3β mRNA expression, promote GSK-3β phosphorylation, promote the transfer of β-catenin in the cytoplasm to the nucleus and activate the Wnt/β-catenin signaling pathway, promote OB differentiation, and improve PMOP［45］.

2.1.2 ＲANKL/ＲANK/OPG regulatory axis: NF-κB receptor (ＲANK) - NF-κB receptor activator ligand (ＲANKL) -OPG axis has been elucidated as a key regulatory axis for bone remodeling. ＲANKL is a member of the tumor necrosis factor receptor (TNFＲ) superfamily and is a cytokine necessary for the growth and development of OC. It is expressed by OB and is a membrane-associated factor.

ＲANK is the signal receptor of ＲANKL. It is composed of type I transmembrane proteins and is a typical TNFＲ superfamily. ＲANKL and ＲANK are contacted by cells or bound to soluble ＲANK, resulting in ＲANK aggregation, and finally activating NF-κB, c-Jun N-terminal kinase (JNK), p38 and extracellular signal-related kinase (ERK) through TRAF recruitment, which ultimately leads to the activation of T cell nuclear factor c1 (NFATc1), thereby promoting the differentiation and activation of OC［46-47］.
OPG is a member of the TNFＲ superfamily and is mainly produced by OB. Estrogen can increase OPG levels［48-49］. OPG interacts with membrane and soluble ＲANKL as a decoy receptor, blocking the binding of ＲANKL to its cell receptor ＲANK, inhibiting the activation of ＲANK signaling, and affecting OC differentiation. The mechanism of action of traditional Chinese medicine is closely related to the RANKL/RANK/OPG regulatory axis. The bone-drying herb in the Yishen Jiangu recipe can regulate the ratio of OPG to RANKL in BMSCs, inhibit OC maturation and promote OB differentiation [50]. Jiawei Yanghe Decoction can increase the levels of ALP, OPG, etc., so that the OPG/RANKL ratio is increased, which plays a bone protective role [51]. Cucumber seed polypeptide (CSP) can effectively inhibit the expression of cathepsin-K (CTS-K), matrix metalloproteinase 9 (MMP9) and TRAP. At the same time, the activation of the NF-κB pathway downstream of RANK is also inhibited. Different concentrations of CSP can effectively reduce the levels of RANKL and macrophage colony stimulating factor (M-CSF) in the serum and BMSCs of ovariectomized rats, increase the expression of OPG, inhibit the formation of OC, and thus improve the symptoms of PMOP [52].
Icariin can increase the proliferation of endoplasmic reticulum-dependent cells, ALP activity, and OPG gene expression, resulting in an increase in OPG protein levels and a decrease in RANKL protein levels, thereby increasing the OPG/RANKL ratio and affecting the progression of PMOP [53]. Garcinia cambogia capsule can increase the immune regulatory molecule IL-33 in PMOP model rats to inhibit the upregulation of NFATc1 and inhibit the differentiation of OC [54].

2.1.3 MAPK and NF-κB pathways: MAPK and NF-κB are important downstream signaling pathways of the ＲANKL/ＲANK/OPG axis.

When OC precursor cells are exposed to M-CSF and its ligand ＲANKL, ＲANK on the cell membrane is activated, causing a cascade reaction of the pathway. These pathways jointly promote the production of reactive oxygen species (ROS). These activated signal factors stimulate the activation of OC marker genes, such as activated NFATc1 and c-Fos, and ultimately promote the formation and activation of OC. Regarding the relevant mechanism of action of this pathway, Tanshinone I can inhibit the activation of the NF-κB signaling pathway by reducing the level of ROS, preventing the translocation of p65 to the nucleus, and inhibiting the phosphorylation of the MAPK signaling pathway induced by ＲANKL, thereby inhibiting the formation and function of OC［55］.

Puerarin inhibits the activation of TRAF6/ROS-dependent MAPK and NFκB signaling pathways in RANKL-induced bone marrow macrophages (BMMs), thereby regulating the expression of NFATc1, MMP9, CTS-K, and c-Fos to inhibit OC differentiation and alleviate PMOP symptoms [56]. Regarding the mechanism of action on RANKL-induced BMMs, astragalus membranaceus (CAG) can effectively inhibit the formation of OCs in RANKL-stimulated BMMs, and this mechanism is related to CAG's inhibition of NF-κB, calcium and activation of NFATc1 pathways [57].

 

Fig． 1 The mechanism of traditional Chinese medicine in regulating PMOP

 

 

In general, RANKL is mainly expressed by stromal cells and OBs. Under certain conditions, T cells are an important source of RANKL. CD4+T cells can bidirectionally regulate OC production by secreting factors such as RANKL and OPG. Animal experimental results showed that CD4+T cells isolated from OVX mice and treated with serum derived from Bushen Ningxin Decoction showed an increase in OPG mＲNA and a decrease in ＲANKL mＲNA levels, increasing the OPG/ＲANKL ratio［58］.
In short, TCM treatment of PMOP is a process of balancing the relationship between OC and OB, as shown in Figure 1. Inhibiting OC growth and promoting OB differentiation are the key ideas of TCM treatment of PMOP.

2.2 TCM affects PMOP through intestinal flora

The human intestinal flora (GM) is diverse and large in number, and plays an important role in human metabolism［59-60］. When estrogen declines, the abundance of bacteria with the ability to degrade mucin increases, and mucin decreases.
When the number of bacteria in the intestinal epithelium increases, it triggers its pattern recognition receptors, leading to the activation of the NF-κB pathway, the increase of inflammatory cytokines such as TNFα, IL-1, and γ interferon, the increase of intestinal permeability, and the entry of various substances into the submucosal layer, causing abnormal inflammatory reactions [61-62]. These inflammatory factors can activate ＲANKL and M-CSF and reduce OPG, thereby promoting OC proliferation [63]. At the same time, GM can affect CD4+ T cell subsets, such as segmented filamentous bacilli (SFB), which can induce the differentiation of helper T cells (Th17) [64]. Th17 cells differentiate from CD4+ T cells and secrete IL-17, which can not only induce RANKL expression on the surface of OBs and activate RANK signaling on OCs, but also promote IL-6 and IL-8 secretion through the NF-κB and P13K/AKT pathways, thereby accelerating bone resorption［65-66］. See Figure 2.

Fig． 2 The effect of gut microbiota on PMOP

 

2.3 Iron and PMOP

Iron, as an essential element, plays an important role in bone metabolism [72]. Iron overload is considered a risk factor for PMOP [73]. Iron overload can downregulate the expression levels of RUNX2, ALP, and OPN [74], inhibiting OB activity. Free iron can catalyze the Fenton reaction to produce ROS, acting as a mediator in the RANKL-induced signaling pathway. OCs are rich in mitochondria. OC precursors increase the production of ROS in mitochondria and induce the activation of cAMP response element binding protein through iron uptake by ferroportin 1 (FPN1).
cAMP response element binding protein is a key transcription factor downstream of RANKL-induced calcium signaling, which are important pathways for OC maturation. Iron can also induce the upregulation of peroxisome proliferator-activated receptor γ coactivator 1β (PGC-1β), promoting OC differentiation, as shown in Figure 3. E2 can enhance the activity of intracellular antioxidant enzymes [such as superoxide dismutase, glutathione peroxidase (Gpx4), etc.], reduce the production of ROS and protect bone cells from oxidative stress damage [75-76].

Icariin, as an active ingredient in epimedium, can reverse the downregulation of Gpx4, Runx2, ALP and OPN caused by iron overload, inhibit the overexpression of FPN1, reduce oxidative stress, and has the dual functions of downregulating iron overload and promoting osteogenic differentiation [77-78]. The active ingredients of the Chinese herbal medicine Ligustrum lucidum, salidroside and teruginosa, can inhibit NF-κB activation and the levels of c-Fos and NFATc1, thereby inhibiting the production of ROs downstream of NF-κB and the activation of cAMP response element binding protein, thereby alleviating the effect of iron overload on PMOP. At the same time, Ligustrum lucidum can inhibit iron overload in ovariectomized rats through the Nrf2/HO-1 signaling pathway, thereby alleviating PMOP [79-80]. The active ingredients of Ligustrum lucidum can also activate the BMP signaling pathway, upregulate osteogenic genes such as BMP-6, pSmad1/5, and Smad4, and promote osteogenic differentiation [80]. Therefore, Ligustrum lucidum also has the dual functions of downregulating iron overload and promoting osteogenic differentiation.

 

2.4 The relationship between kidney and bone in traditional Chinese medicine and its impact on PMOP

According to traditional Chinese medicine theory, kidney essence can promote human growth, development and repair, and BMSCs have a high degree of similarity in function [81].
The Yellow Emperor's Classic of Internal Medicine points out that "kidney essence produces marrow, and marrow is connected to the brain", "kidney governs bones", "kidney produces bone marrow", "bone is the home of marrow", "all marrow belongs to the brain", etc., and explains the impact of the "kidney-brain-bone" axis on bone metabolism from the relationship between kidney, brain, and bone. Modern medicine has found that the brain hippocampus, nervous system and bone metabolism are closely related [82-83]. At the same time, the Suwen also introduces the relationship between female kidney and Tiangui. Tiangui comes from kidney essence. When kidney qi is strong, Tiangui arrives, and Chongren is filled and poured into the uterus, triggering menstruation [84]. When Tiangui is exhausted, the uterus is malnourished, menstruation stops, and women enter menopause. Modern medicine calls this a decline in the function of the hypothalamus-pituitary-ovarian axis, ovarian decline, and a decline in estrogen, which becomes an important cause of PMOP [85].
Researchers have made many explorations on the specific pathogenesis of PMOP based on the above two axes. For example, Shang Qi et al. [86] used tortoise shell extract to intervene in female naturally aged mice, and extracted their hypothalamic hippocampal neurons for cell culture. They found that tortoise shell promoted hippocampal neuron proliferation and increased the expression of hippocampal neuron marker Nestin protein, while improving the BMD of mice. Similarly, Wu Zixian et al. [87] used tortoise shell extract to intervene in experimental mice and found that tortoise shell could significantly reduce the expression of aging proteins in the hippocampus and effectively improve the BMD of the intervention group. Li Shuzhen et al. [88] used 25-flavor Podophyllum Pills to intervene in PMOP model rats and found that 25-flavor Podophyllum Pills could reduce serum progesterone, pituitary secretion of follicle-stimulating hormone, and gonadotropin-releasing hormone levels, which played an important role in the endocrine function of the hypothalamus and pituitary. Zhang Yan et al. [89] used the Kidney-Nourishing and Phlegm-Removing Recipe to intervene in the PMOP mouse model and found that the Kidney-Nourishing and Phlegm-Removing Recipe could inhibit the expression of hypothalamic neuropeptides and neuropeptide Y receptors, thereby improving PMOP.

 

3 Summary

In recent years, with the deepening of clinical and basic research on the treatment of PMOP with traditional Chinese medicine, traditional Chinese medicine has better efficacy and safety in the treatment of PMOP, such as Qinge Pills, Epimedium and acupuncture. At the same time, traditional Chinese medicine has great potential in the treatment of PMOP, and has the mechanism of promoting bone formation, inhibiting bone destruction and resisting oxidative stress, which is worthy of further exploration and discussion.

 

References

[1] Ensrud KE． Bisphosphonates for postmenopausal osteoporosis［J］． JAMA． 2021,325( 1) : 96．
[2] He Jiaojiao,Chen Yifa,Chen Yulin,et al． Osteoimmunological mechanism of postmenopausal osteoporosis［J］． Chinese Journal of Osteoporosis,2023,29( 7) : 1032-1036．
[3] LeBoff MS,Greenspan SL,Insogna KL,et al． The clinician's guide to prevention and treatment of osteoporosis［J］． Osteoporos Int,2022,33( 10) : 2049-2102．
[4] Sun Jing,Yang Mingxia,Zhang Zhihai． Meta-analysis of zoledronic acid in the treatment of postmenopausal osteoporosis［J］． Chinese Journal of Osteoporosis, 2021, 27 (9): 1333-1338, 1365．
［5］ Jing Nan, Liu Lichang, Ye Bingzhao, et al. Effect of alendronate on bone strength in postmenopausal patients with osteoporosis and osteoarthritis［J］． Chinese Journal of Osteoporosis, 2021, 27(1): 119-122．
［6］ Zhang Yanjie, Gu Shanling, He Ziyi, et al. Effect of drugs for the treatment of osteoporosis on fracture healing［J］． Chinese Journal of Geriatrics, 2023, 43 (18): 4596-4599．
［7］ Guo Yiming, Hu Yunqiu, Li Miao, et al. Efficacy of alendronate followed by zoledronic acid in the treatment of postmenopausal osteoporosis: A 2-year retrospective study [J]. Chinese Journal of Osteoporosis and Bone Mineral Diseases, 2021, 14(4): 337-344.
[8] Wei Yuanxun, Chen Feng, Lin Zonghan, et al. Notch signaling pathway and osteoporosis and prevention and treatment with traditional Chinese medicine [J]. Chinese Journal of Tissue Engineering Research, 2024, 28(4): 587-593.
[9] Du Xin, Wang Huidan, Han Xu, et al. Acupoint selection rules for acupuncture treatment of ovarian dysfunction in women of childbearing age and its effect on the reproductive axis [J]. Shizhen Traditional Chinese Medicine, 2019, 30(11): 2784-2786.
[10] Wang Zhen, Wang Cong'an, Wang Yongquan, et al. A network meta-analysis of the effectiveness and safety of oral kidney-tonifying Chinese patent medicines in the treatment of postmenopausal osteoporosis［J］． Chinese Herbal Medicine,
2022,53( 13) : 4084-4099．
［11］ Chen Fan, Wei Xu, Cui Xin, et al. Systematic review and meta-analysis of Qing'e Pills in the treatment of postmenopausal osteoporosis［J］． Chinese Journal of Osteoporosis． 2021,27( 2) :179-189．
［12］ Xie Lihua, Chai Hao, Ye Yunjin, et al. Study on the efficacy and transcriptional mechanism of Xuling Jiangu Granules in the treatment of postmenopausal osteoporosis of kidney deficiency and blood stasis type［J］． Chinese Journal of Osteoporosis． 2022,28( 4) : 558-561,600．