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Effects of electroacupuncture on bone mineral density, oestradiol level and osteoprotegerin ligand expression in ovariectomised rabbits
  1. Jing He,
  2. Lin Yang,
  3. Yuxi Qing,
  4. Chengqi He
  1. Department of Rehabilitation Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan, China
  1. Correspondence to Dr Chengqi He, Key Laboratory of Rehabilitation Medicine, Sichuan Province; Department of Rehabilitation Medicine, West China Hospital, No 37, GuoXue Xiang, Chengdu, Sichuan 610041, PR China; yeshj{at}sohu.com

Abstract

Objectives To investigate the effects of electroacupuncture (EA) on the oestradiol level, bone mineral density and osteoprotegerin ligand (OPGL) expression, and to explore whether EA might be a complementary method to prevent and treat osteoporosis.

Methods A total of 21 New Zealand rabbits were randomly divided into three groups: a normal control (NC) group undergoing no surgery or EA; an ovariectomised (OVX) group, in which rabbits were ovariectomised but did not receive EA; an EA group, in which rabbits were ovariectomised and treated with EA. Acupuncture was applied at ST35, BL20 and BL23 points bilaterally. EA (10 Hz, 2 mA) was applied bilaterally at BL20 and BL23 for 30 min a day for 14 days. After 14 days, all animals were killed. OPGL expression level was determined by immunohistochemistry. Blood serum levels of oestradiol were measured by ELISA and bone mineral density was detected by dual-energy x-ray absorptiometry.

Results After ovariectomy, the bone mineral density and oestradiol level decreased significantly in the OVX group compared with the NC group (p=0.001), whereas the OPGL expression level increased. After EA, the bone mineral density and oestradiol level increased compared with the OVX group (p=0.049 and p=0.012, respectively). The OPGL level OPGL level in the EA group was lower than that in the OVX group (p=0.022).

Conclusions EA restored bone mineral density towards normal and was associated with increased plasma oestradiol level and reduced OPGL expression in an ovariectomised rabbit model of osteoporosis.

  • Acupuncture
  • Osteoporosis Arthritis
  • Therapeutics

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Introduction

Osteoporosis is a systemic disease characterised by low bone mass and micro-architectural deterioration of bone tissue, resulting in reduced bone strength and increased susceptibility to fractures. WHO defines osteoporosis as a bone mineral density that lies ≥2.5 SDs below the mean peak bone mass (average for healthy adults) as measured by dual-energy x-ray absorptiometry.1 Osteoporosis is a common disease, with a high incidence among elderly postmenopausal women.2 The International Osteoporosis Foundation estimates that there are more than 200 million women around the world with osteoporosis, one-third of whom are aged 60–70 and the rest ≥80 years.3 People with osteoporosis have increased risk of fractures in their hips, spine and wrists, with relatively low levels of trauma. Some researchers have reported that between ages 45 and 64, hip and spine fractures are associated with decreased bone mineral density.4 Osteoporosis and other related complications result in increased morbidity and mortality and seriously affect the quality of life—socially, emotionally and financially—of the patients and their families.2 Given the health implications of osteoporotic fractures and the heavy burden on society, effective treatments that prevent or slow down bone loss, maintain bone strength and minimise the factors that may cause fractures would significantly improve the outcomes for subjects with postmenopausal osteoporosis.

A higher bone density, which is regulated by bone remodelling, is associated with higher bone quality. An imbalance between the activities of osteoblasts and osteoclasts causes alterations in bone remodelling. Bone mineral density decreases when the activity of osteoclasts predominates, which may cause osteoporosis. Several factors influence bone remodelling, including general (endocrine) and local (paracrine) factors. Each factor has an important role in the primary regulation and final pathway of bone remodelling—namely, the RANK/RANKL/OPG (receptor nuclear activator factor κ B/RANK κ B ligand/osteoprotegerin) system. NF-κB ligand (RANKL) is related to tumour necrosis factor that is identical to the receptor activator of OPG ligand (OPGL), which is in turn essential for the differentiation of osteoclast precursors into mature cells.5 ,6 OPGL plays a pivotal role in osteoclast formation, it increases osteoclast activity and prolongs the lifespan of osteoclasts by inhibiting apoptosis.7

OPG, a soluble decoy receptor, neutralises the effects of OPGL. The main mechanism by which oestrogen prevents bone loss is by inhibiting osteoclastogenesis—that is, it downregulates the activation of osteoclasts. Administration of exogenous oestrogen is the primary way of increasing oestrogen. A number of experiments have shown that oestrogen deficiency upregulates the formation of OPGL and promotes osteoclastogenesis by enhancing the expression of cytokines, including interleukin 1, interleukin 6 and tumour necrosis factor α.5 Because of the important role of oestrogens and other hormones in the prevention of bone loss, the administration of oestrogens, or hormone replacement therapy (HRT), has become a useful method for preventing bone loss, increasing bone density and preventing bone fractures in postmenopausal women.8

Despite observational evidence gathered over several decades, there is no defined optimal regimen—that is, time of onset, duration and dose—for oestrogen and HRT. Moreover, the Women's Health Initiative and the Million Women Study have suggested that HRT increases the risk of cardiovascular disease, stroke, breast cancer and venous thrombosis. Some selective oestrogen receptor modulators, such as raloxifene, may also induce vasomotor symptoms (such as hot flashes, sweating and leg cramps) and other adverse reactions, which affect patients’ quality of life.9 These adverse reactions greatly reduce patient compliance and the effectiveness of treatment.10 Therefore, this strategy requires further research.

Acupuncture is a technique based on the theory of traditional Chinese medicine that has existed in China for more than 2500 years and has been continuously improved and developed.11 Chinese medicine emphasises the treatment of overall physical and mental aspects. Some researchers have shown that acupuncture regulates neuropeptides in the central nervous system and influences physiological outcomes.12 ,13 In clinical studies, acupuncture has been used to decrease the pain of postmenopausal osteoporosis.11 ,14 The study by Sunay et al showed that acupuncture effectively reduces menopausal complaints and increases oestrogen levels in postmenopausal women.15 The results suggest that acupuncture may ameliorate the process of menopausal osteoporosis. A further study demonstrated that gonadotrophin releasing hormone is downregulated after electroacupuncture (EA) and that corticotrophin releasing hormone is upregulated only when EA is given together with oestrogen and not when given alone.16 The study by Bokmand and Flyger showed that acupuncture relieves menopausal discomfort (hot flashes and sleep disturbances) in patients with breast cancer, but the effect was not correlated with levels of plasma oestradiol.17

In this study, we used ovariectomised rabbits as a model of the menopausal processes in women. We investigated the effects of EA on the oestradiol level, bone mineral density and OPGL expression level, and examined whether EA might be a complementary method for preventing osteoporosis.

Materials and methods

Animal model

Female New Zealand White rabbits (21.5 months old) were used in this study. All rabbits weighed about 2 kg (2140.3±179.6 g) and were purchased from the Animal Experiments Center of the West China Hospital, Sichuan University, under licence number SYXK (Sichuan) 2009-045.

The rabbits were all housed separately in iron cages at a temperature of 20–26°C on a 12 h cycle, with food and water provided according to the Laboratory Animal Standards. The Animal Center and the People's Republic ethics commission approved the experiment.

Twenty-one New Zealand White rabbits were randomly divided into three groups: a normal control (NC) group, in which the rabbits were not ovariectomised and did not receive treatment; an ovariectomised (OVX) group, in which rabbits were ovariectomised but did not receive EA treatment; and the EA group, in which rabbits were ovariectomised and received EA. Before the surgery, the rabbits were fixed in a supine position with 5% chloral hydrate anaesthesia (3 mL/kg) and a 0.5 cm incision was made in the central abdomen to ovariectomise both ovaries (except for the NC group). The rabbits were sutured after surgery. Gentamicin (40 000 units) was intramuscularly injected for 3 days to prevent infection after surgery. All rabbits were given a normal diet and adequate water and were allowed to move freely in their cages. The study outline is given in figure 1.

Figure 1

Flow chart. BMD, bone mineral density; OPGL, osteoprotegerin ligand.

Treatment and techniques

Eight weeks after they were ovariectomised, the rabbits were fixed in the prone position in the holder for 30 min. The EA group received acupuncture treatment. Three points were chosen: Pishu (BL20), Shenshu (BL23) and Zusanli (ST36). The acupuncture sites were shaved and disinfected (75% alcohol); the operators’ hands were also disinfected. Disposable, sterile plastic handle needles (Jiajia Medical Instrument Co, Wuxi), 25 mm long with a diameter of 0.30 mm, were used. A dried, sterilised cotton ball was held around the needle tip with the thumb and index fingers of the left hand, fixing the needle tip directly over the selected point. The needle handle was held with the right hand, while the left hand inserted the needle tip swiftly into the skin via uniform twisting. When the acupuncturist felt the de qi sensation of the subject, two leads of an EA 6805 stimulator (Medical Equipment Factory Co, Shantou Production) were connected to the needles at BL20 and BL23 on the same side. A frequency of 10 Hz with a 2 mA current was used. The acupuncture needles were removed after 30 min and pressure was applied to the site with cotton. The subjects received treatment every day for 2 weeks.

Sample preparation

Blood samples from all rabbits were collected before ovariectomy, 8 weeks later (after ovariectomy and before treatment) and 10 weeks later (after treatment) to determine the oestrogen levels. The plasma was centrifuged for 1 h and stored at −20°C. At each time point, 2 mL serum was collected. Ten weeks after ovariectomy, all rabbits were killed by air embolism under general anaesthesia. The left femur was soaked in 10% buffered formalin for decalcification, cut into 5 µm thick paraffin-embedded vertical slices and mounted on glass slides for histological and immunohistochemical analysis.

Oestradiol test

Oestradiol was detected with an ELISA kit (R&D, USA).

For each serum sample 25 µL aliquots were added to 200 µL of enzyme reaction hinge material. The reaction was allowed to proceed for 2 h at a temperature of 36°C. Substrate material (100 µL) was added to each sample, the samples were incubated for 15 min at room temperature and finally, the reaction was stopped by addition of 50 µL Stop Solution. The optical density was read with a microplate reader at 450 nm and measured twice for each sample.

Immunohistochemical detection of OPGL

After the intervention, immunohistochemistry was used to detect OPGL and the grey value (optical density value) was determined with an electron microscope.

The test method was as follows: each glass slide containing the sample was treated with xylene and graded alcohol, was dewaxed, rehydrated and then soaked in 3% peroxide methanol at room temperature for 10 min to inhibit the endogenous peroxidase activity. The slides were washed with phosphate-buffered saline, and bovine serum albumin was added to each sample, followed by 30 min incubation at 37°C to reduce non-specific antibody binding. A drop of an anti-OPGL antibody (Boster Biological Technology Co, Wuhan, Hubei, China, 50 µL, 1:40 dilution) was added and then, the samples were incubated at 4°C overnight. The slides were washed fully next day, incubated with the full secondary antibody (anti-rabbit IgG biomarkers) at 37°C for 40 min and were then ready for analysis. After anti-dilution of the cytoplasm with 3,3’-diaminobenzidine the slides appeared brown and stained purple after treatment with haematoxylin nuclear stain; the slides were then dried and sealed with a coverslip. Separate specimens that had not been incubated with the primary antibody were used as a negative control group.

Bone mineral density test

The right femurs of the rabbits were chosen for the bone mineral density (BMD) tests using dual-energy x-ray absorptiometry (GE Lunar iDXA Densitometer). Each sample was completely and independently immersed in a plastic box filled with deionised water. The femur neck and head were oriented perpendicular to the dual energy X harness.18 ,19

Statistical analysis

SPSS 17.0 statistical analysis software was used for the statistical analysis. The data are expressed as the mean±SD. The oestradiol data were analysed using two t tests. Based on the Bonferroni adjustment, p<0.025 was considered significant. The OPGL and BMD values were compared among the groups with one-way analysis of variance followed by least significant difference post hoc tests. Results were considered significant for p<0.05.

Results

The rabbits in each group did not differ significantly in their body weight before and after ovariectomy. All experimental rabbits were calm and tolerated acupuncture treatment.

After 10 weeks, the BMD of rabbits in the NC group was higher than that of rabbits in the OVX group and this difference was statistically significant (p=0.001). The bone mineral density of rabbits in the EA group was higher than that of rabbits in the OVX group and this difference was statistically significant (p = 0.049). The difference between the EA and NC groups was not statistically significant (p = 0.067) (see figure 2).

Figure 2

Comparison of bone mineral density (BMD) between three groups after 10 weeks. EA, electroacupuncture, NC, normal control; OVX, ovariectomised.

* NC compared with OVX, p=0.001. # EA compared with OVX p=0.049.

There were no significant differences in the baseline levels of serum oestradiol among the different groups. In the NC group, there were no statistically significant changes over time (baseline, 8 weeks and 10 weeks) (p>0.05). In the OVX and EA groups, the levels at 8 weeks were lower than in the NC group and the differences were statistically significant (p=0.000 and p=0.001, respectively). After 10 weeks, the level in the OVX group was still lower than that in the NC group (p=0.017). However, the level in the EA group was higher than the level in the OVX group and this difference was statistically significant (p=0.012) (see figure 3).

Figure 3

Comparison of levels of serum oestradiol between three groups. EA, electroacupuncture, NC, normal control; OVX, ovariectomised.

* OVX and EA compared with NC at 8 weeks (p=0.000 and p=0.001, respectively). # EA compared with OVX at 10 weeks (p=0.012).

Comparison of the OPGL levels among the three groups after 10 weeks showed that the OPGL level in the NC group was lower than that in the OVX group (p=0.001). The OPGL level in the EA group was lower than that in the OVX group (p=0.022). The difference between the EA and NC groups was not statistically significant (p=0.056) (see figure 4).

Figure 4

Comparison of osteoprotegerin ligand (OPGL) between three groups after 10 weeks. EA, electroacupuncture, NC, normal control; OVX, ovariectomised.

* NC compared with OVX, p=0.001. # EA compared with OVX, p=0.022.

Discussion

Our study showed that the level of OPGL significantly increased in the OVX group, whereas BMD and the oestrogen level decreased. However, expression of OPGL decreased significantly after EA treatment, whereas BMD and the level of oestrogen increased in the ovariectomised rabbit model of osteoporosis. These results suggest that EA might suppress the process of osteoclastogenesis by upregulating the level of oestrogen and/or directly modulating the expression of OPGL in osteoclastic cells.

BL20, BL23 and ST36 are the points most commonly chosen for the prevention and treatment of osteoporosis in postmenopausal women.20 EA has an advantage in that the stimulus parameters can be controlled. The increases in serum oestrogen levels and BMD and the decreased expression of OPGL in rabbits over 14 days, may be pertinent to the EA treatment of postmenopausal women.15 ,21

Acupuncture is known to promote natural functional homeostasis and is reported to modulate neurotransmitter function in the central nervous system.10 ,15 ,17 Clinical data have suggested that acupuncture is an effective treatment for menopausal and perimenopausal syndromes as well as osteoporosis.13 ,14 ,17 In addition, laboratory evidence shows that acupuncture can affect endocrine secretion and hormones.22 ,23

In clinical practice, acupuncture is commonly used to relieve the pain, hot flashes and sleep disturbances in postmenopausal women in China and some Western countries.13 ,14 ,2,4 However, the clinical application of acupuncture for the prevention and treatment of osteoporosis is still being developed, with most studies focusing only on improving the symptoms. A few studies have explored the mechanism of action of EA in the treatment of osteoporosis but the results for its effect on oestrogen levels are inconsistent.20 ,2,4 Animal experiments have indicated that acupuncture can enhance oestradiol levels in ovariectomised animals.

In this study, we investigated the effects and mechanism of action of EA in the treatment of osteoporosis. Reductions in BMD and oestradiol levels and an increase in OPGL expression were seen in ovariectomised rabbits but EA was associated with increased oestradiol levels, suppressed OPGL and increased BMD in ovariectomised rabbits. EA might be a complementary treatment for the prevention of osteoporosis, if these same changes occur in postmenopausal women. The mechanism and effects of EA treatment in osteoporosis in human subjects should be further explored. Additionally, the optimal parameters for EA need to be validated. It is worth noting that EA includes two components—namely, needle location (traditional acupuncture) and electrical current stimulation—and it is not known whether the effects of EA are induced by the former, the latter, or by both.

Summary points

  • After ovariectomy, bone mineral density (BMD) is reduced owing to osteoclastic activity induced by the osteoprotegerin ligand (OPGL).

  • After electroacupuncture in a rabbit model, BMD increased and OPGL decreased over 14 days.

Acknowledgments

We thank all those who helped us to complete this study: Tingwu Qin, Professor, Regenerative Medicine Research Center, West China Hospital, Sichuan University, for assistance with the design of the experiments; Hua Guo, PhD; Lu Xia, master graduate student; and Jun Zhou, doctoral student, Department of Rehabilitation Medicine, West China Hospital and Sichuan University, for assistance with the animal model in this experiment; Odessa, an English teacher from 51 talk, for assistance with our English grammar.

References

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Footnotes

  • JH and LY contributed equally to this work and should be considered joint first authors.

  • Contributors JH prepared and carried out the experiments, wrote and revised the manuscript; LY carried out the experiments, collected and anlysed the data; YQ prepared the model for testing; CH provided methodology support and managed the whole process.

  • Funding The work was supported by the National Natural Science Foundation of China, grant No 81177865.

  • Competing interests None.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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