Osteoporosis is a metabolic bone disorder that is characterized by low bone mass and microarchitectural deterioration of bone tissue with a consequent increase in bone fragility and susceptibility to fracture (1). A recent study has reported that the prevalence of osteoporosis among those aged 40 years or older is 5.0% among men and 20.6% among women in mainland China (2). Osteoporosis-related fractures are associated with increased disability, increased mortality, and low quality of life (3). Due to population aging, osteoporosis has become a major public health issue globally (4, 5).

Postmenopausal osteoporosis (PMOP), resulting from estrogen deficiency, is the most common type of osteoporosis (6). Estrogen deficiency increases bone turnover with an imbalance between bone formation and resorption, and it is the main cause of osteoporosis in women aged 50 years or older (7, 8). An active role of the immune system as well as increased production and release of a variety of cytokines in the bone marrow environment, such as granulocyte-macrophage colony stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-alpha), interleukin-1, interleukin-6, and receptor activator nuclear kappa-b ligand (RANKL), are implicated in sex hormone deficiency-induced bone loss, which stimulates bone resorption by increasing the number and/or the activity of bone-resorbing osteoclasts (9, 10).

Several drugs are licensed to reduce fracture risk by slowing down bone resorption (such as bisphosphonates and denosumab) or by stimulating bone formation (such as teriparatide, abaloparatide, or romosozumab) (11). Moreover, menopausal hormone therapy (MHT) has also been demonstrated to be effective for the maintenance of skeletal health and prevention of future fractures, and the application of MHT is confined to women who have recently (<10 years) become menopausal, who have menopausal symptoms, and who are less than 60 years old with a low baseline risk for adverse events (12, 13). Given the adverse effects of prolonged estrogen therapy, including an increased risk of cardiovascular events, thromboembolic disease, and breast cancer, as well as the rare but potentially serious side effects of common osteoporosis medications (bisphosphonates, raloxifene, denosumab, and teriparatide) (14, 15), there is an urgent need for alternative treatments to increase bone mineral density (BMD) and prevent fractures with a good safety profile.

Acupuncture has a long history in traditional Chinese medicine (TCM) and has gained increasing acceptance and popularity worldwide in recent decades. With increasing evidence of its clinical efficacy, acupuncture is now a widely practiced convenient and safe treatment modality within complementary and integrative medicine worldwide (16). Application of acupuncture in the treatment of osteoporosis was described in Huangdi Neijing, the ancient classical text of acupuncture. In recent years, many studies have investigated the efficacy of acupuncture for the treatment of primary osteoporosis in clinical practice, and the findings have shown that acupuncture is effective in improving osteoporosis (17–20). Moreover, a previous study on secondary osteoporosis in patients with spinal cord injuries has reported an increase in BMD after 3 months of adjunctive acupuncture treatment, but the difference is not statistically significant (21).

Overall adjustment acupuncture (OA) was originally proposed by our research group based on our clinical exploration and experience for more than 20 years (22). OA combines the etiological and pathophysiological aspects of osteoporosis with kidney and spleen deficiency according to TCM theory, and it stimulates the meridian system, including the skin, collaterals, and meridians. OA has been shown to improve the clinical symptoms and quality of life in patients with osteoporosis (23). A systematic and comprehensive clinical study is also being conducted by our team to study the efficacy, safety, and mechanism of OA in patients with PMOP to provide more rigorous clinical evidence to promote its application (24). In the present study, we investigated the anti-osteoporotic effect of OA and explored its mechanism of action through endocrine modulation in a rat model of PMOP induced by bilateral ovariectomy.

In the present study, we demonstrated that OA improved osteoporosis in terms of BMD, BMC, bone tissue biomechanical properties, and bone tissue histomorphology. We also demonstrated that OA increased systemic estrogen levels and that OA modulated the HPA axis function in ovariectomized rats. Beneficial effects of acupuncture on osteoporosis in ovariectomized animals have been previously reported, demonstrating improvements in BMD, bone strength, bone structure, and bone turnover (28, 29, 32, 33). Consistent with these results, the present study demonstrated that OA improved osteoporosis in the OVX rat model despite differences in types of acupuncture and specificity of acupoint selection.

The present study demonstrated that OA increased the uterus index and serum estrogen levels in the OVX rat model, which agreed with previous studies on acupuncture treatment of ovariectomized animals (34, 35). Moreover, the efficacy of acupuncture has been reported to reverse osteoporosis in the SAMP6 mouse model of senile osteoporosis by enhancing the secretion of testosterone and decreasing bone turnover (36). These studies suggest a regulatory effect of acupuncture on hormonal profiles, and the improvement of PMOP by acupuncture may be closely related to this modulatory effect of sex hormone levels. However, these results were obtained from studies on non-human subjects, and evidence in human subjects is lacking. Our ongoing clinical trial was designed to collect data, including estradiol levels, in patients with PMOP upon OA treatment to cover this gap (24).

In postmenopausal women, the hypothalamic-adrenal-ovarian (HPO) axis tends to function at a low level and is accompanied by a significant decrease in ovarian-derived estrogen levels. Estrogen is then synthesized in extraglandular sites (a process named extragonadal aromatization), such as adipose tissue, liver, muscle, and several parts of the brain, thus providing a pathway for target tissues to synthesize and metabolize estrogen according to local needs (37–39). This process requires the involvement of a microsomal member of the cytochrome P450 superfamily, the aromatase cytochrome P450 (P450arom) (40). Aromatase catalyzes the demethylation of carbon 19 of androgen to produce phenolic 18-carbon estrogens (41). It has been reported that acupuncture promotes extragonadal aromatization and increases blood estrogen concentrations in OVX rats due to the contribution of abdominal subcutaneous fat and liver tissue (42). However, as the expression levels of aromatase vary among tissue types, the biosynthesis and function of estrogen are regulated locally in a paracrine or intracrine manner within individual tissue. The regulation of this process is unique in each target tissue, and circulating synthetic estrogens may also affect local metabolism, leading to a complex physiological situation that makes the interpretation of circulating estrogen levels more difficult (40, 43). To date, few studies have reported the aromatization in bone tissue upon acupuncture, and our future study aims will focus on this aspect.

C19-steroids, synthetic estrogen precursors, are produced mainly by adrenal glands (44, 45). Dehydroepiandrosterone (DHEA) and its sulfate (DHEA-S), which are secreted by the adrenal glands, are converted to androstenedione (ASD) and then to androgens and estrogens in peripheral tissues, thus providing autonomous production and metabolism of sex steroids according to local needs (46). In postmenopausal women, estrogen is almost exclusively synthesized locally in peripheral target tissues from inactive adrenal C19-steroid precursors secreted by the adrenal cortex, particularly DHEA (47). In addition, the circulating levels of adrenal C19-steroids may directly reflect the parameters of adrenal sex precursor steroid secretion levels (48). In a previous trial reporting a 12-month evaluation of the efficacy of DHEA replacement therapy in 14 women aged 60–70 years who used 10% DHEA cream daily, hip BMD increased in the absence of stimulatory effects on the endometrium, which correlated with a 20% reduction in plasma bone alkaline phosphatase and a 28% reduction in the urinary hydroxyproline/creatinine ratio (49).

Because the HPA axis is responsible for stimulating adrenal corticosteroids in response to changes and stresses in the body, we investigated whether OA also exerts a modulatory effect on HPA axis hormone secretion in the OVX rat model by measuring the serum levels of CRH, ACTH, and CORT. Similar to the results of our study, Zhao et al. reported that electroacupuncture increases the release of CRH from the paraventricular nucleus and increases blood estrogen levels in OVX rats. By comparing the effects of electroacupuncture and natural substitution on OVX rats, a previous study has reported that electroacupuncture may accelerate the process of endogenous substitution of estrogen synthesis (50, 51). Chen et al. reported a stimulatory effect of electroacupuncture on nucleolar organizer regions (NORs) of the adrenal cortex in ovariectomized rats, and they reported that the vaginal smears showed a response similar to estrogen-induced response with the appearance of exfoliative cells and that this response was not observed in normal rats upon electroacupuncture treatment, suggesting that electroacupuncture may promote the synthesis and secretion of adrenal steroid hormones. The produced androgen will then be transformed into estrogen in other tissues, thus compensating the deficiency of estrogen induced by ovariectomy (52).

In general, stress is considered to be an effective stimulus for HPA axis activity and CORT secretion. In addition, CORT is often referred to as the stress hormone. However, HPA axis research suggests that it may not be feasible to simply equate elevated CORT with stress. For example, some conditions that are not usually considered stressors, such as exercise and anti-anxiety medications, also cause CORT secretion; whereas some conditions that are usually considered stressful, such as chronic neuropathic pain, are not necessarily associated with acute CORT secretion (53, 54). More specifically, in the presence of a hypofunctional HPO axis, the HPA axis is an important bridge for the coordination of the central nervous system and neuroendocrine systems with each other due to its activation of endogenous adrenal compensatory effects and potential role in maintaining dynamic homeostasis (38, 40). Importantly, acupuncture, as a benign regulator, may accelerate this ability to regain dynamic balance (51).

Acupuncture improves osteoporosis while increasing circulating estrogen levels, which may be closely related to postmenopausal extragonadal aromatization. It has been shown that chondrocytes and osteoblasts of bone also have the ability to express aromatase and thus can locally synthesize estrogen (55). In particular, the risk of osteoporosis and the incidence of fractures are increased for patients treated with aromatase inhibitors for breast cancer (56, 57). Therefore, the pathway of acupuncture stimulation to activate extragonadal aromatization still needs to be further explored.

In conclusion, the present study demonstrated that OA improves osteoporosis and exerts a modulatory effect on systemic estrogen levels and HPA axis activity in OVX rats. Further studies are needed to determine whether OA acts through increasing the aromatization in bone tissue locally or increasing estrogen produced by aromatization in other tissues as well as to determine how OA regulates bone metabolism.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.

The animal study was reviewed and approved by The Ethics Committee of Yunnan University of Chinese Medicine (ethical code no. R-062021070).

All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.

This study was supported by the National Natural Science Foundation of China (81560799, 82060895, 31960178, 82160923); Applied Basic Research Programs of Science and Technology Commission Foundation of Yunnan Province (2019FA007); Key Laboratory of Traditional Chinese Medicine for Prevention and Treatment of Neuropsychiatric Diseases, Yunnan Provincial Department of Education; Scientific Research Projects for High-level Talents of Yunnan University of Chinese Medicine (2019YZG01); Young Top-Notch Talent in 10,000 Talent Program of Yunnan Province (YNWR-QNBJ-2019-235); National Science and Technology Innovation 2030 Major Program (2021ZD0200900); Yunnan Key Research and Development Program (202103AC100005); Yunnan Province Fabao Gao Expert Workstation Construction Project (202105AF150037).

We would like to express our sincere gratitude to all those who participated in this study or helped with this study.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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