Objective To characterise the effects of early and late electroacupuncture (EA) treatment on serum 17β-oestradiol (E2), C-terminal cross-linking telopeptide of type I collagen (CTX-I), bone mineral density (BMD), biomechanical bone strength and mRNA expression of cathepsin K in ovariectomised (OVX) rats.
Methods Sixty Sprague-Dawley rats underwent ovariectomy (n=40) or sham surgery (n=20) and were randomly divided into two batches. Batch 1 (n=30) consisted of 10 sham-operated rats (Sham-0 group) and 20 OVX rats: half commenced EA immediately (early EA group, n=10) and half were left untreated (OVX-0 group, n=10). Batch 2 (n=30) consisted of 10 sham-operated rats (Sham-12 group) and 20 OVX rats: half commenced EA treatment 12 weeks after ovariectomy (late EA group, n=10) and half were left untreated (OVX-12 group, n=10). Rats in batches 1 and 2 were killed after 12 and 24 weeks, respectively. Serum E2, CTX-I, BMD, bone strength and cathepsin K expression were determined by ELISA, dual energy X-ray absorptiometry, biomechanical assessment and qRT-PCR, respectively.
Results Both early and late EA treatment increased serum E2 levels, reduced serum CTX- I levels and increased BMD and bone strength of the L5 vertebral body in OVX rats. Although early EA treatment similarly increased BMD and bone strength of the femur, late EA treatment did not. However, both early and late EA treatment reduced mRNA expression of cathepsin K in OVX rats.
Conclusions Early EA completely prevented and late EA partially prevented bone loss and deterioration of bone strength in OVX rats. The timing of initiation of EA treatment may be an important consideration for optimisation of effects. The influence of EA on bone strength appears to be at least partially mediated through regulation of the expression of cathepsin K.
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Osteoporosis is a metabolic disorder that is characterised by low bone mass, skeletal fragility and increased risk of fractures.1 Although some pharmacological interventions have been shown to increase bone mineral density (BMD) and thereby reduce the risk of fracture, such therapies also have undesirable side effects.2 Thus, there is a need to develop safe, effective therapies with novel mechanisms of action.
Acupuncture, one of the main modes of treatment in Traditional Chinese Medicine, is simple and safe and associated with minimal side effects. It has been widely used in China to treat disease for thousands of years and has been shown to improve BMD, regulate bone metabolism, relieve pain and improve quality of life in patients with osteoporosis.3 ,4 Animal studies have shown that acupuncture can increase serum 17β-oestradiol (E2) levels, BMD and bone strength in ovariectomised (OVX) rats.5 ,6 Electroacupuncture (EA), which combines acupuncture with electrical stimulation, has also been demonstrated to have positive effects on osteoporosis through inhibition of bone loss in OVX rats and rabbits.7–9 In a previous study we showed that EA prevents ovariectomy-induced bone loss and deterioration of bone architecture and strength in OVX rats through activation of the Wnt/β-catenin signalling pathway.10 Although there is a growing body of evidence in support of the beneficial effects of EA on osteoporosis, the underlying mechanisms remain elusive.
Cathepsin K, a member of the papain cysteine protease superfamily, is selectively and highly expressed by osteoclasts.11 During bone resorption, type I collagen, a major organic component of the bone, is degraded by cathepsin K in an acidic microenvironment. Mutations in the cathepsin K gene resulting in a loss of function are associated with a number of complications in humans characterised by higher BMD,12 and mice lacking cathepsin K exhibit increased bone mass.13 Conversely, transgenic mice overexpressing cathepsin K show accelerated turnover of metaphyseal trabecular bone and decreased bone mass.14 Expression of cathepsin K has been found to be increased in OVX mice and rats.15 ,16 Previous studies have shown that osteoclast-mediated bone resorption can be decreased through inhibition of cathepsin K activity.17 Consequently, it is clear that cathepsin K plays a key role in bone resorption. Accordingly, inhibition of cathepsin K represents a potentially attractive therapeutic approach for treating osteoporosis. To our knowledge, no study to date has assessed the effects of EA on cathepsin K expression in vivo. Furthermore, although beneficial effects of EA on osteoporotic bone have been reported, it is unknown whether the timing of the treatment is of importance in OVX rats. The aim of the present study was to characterise the effects of both early and late EA treatment on bone mass and the expression of cathepsin K in OVX rats.
Study design and EA treatment procedure
Sixty 3-month-old female Sprague-Dawley rats were purchased from the experimental animal centre of the University of South China and acclimatised to conditions for 1 week before the experiment. Animals were housed in an animal room maintained at a temperature of 24±2°C and a relative humidity of 55±5% under a 12 h light/dark cycle. Access to food and water were unrestricted. Body weight was checked once a week throughout the experimental period. After 1 week of acclimatisation, all rats underwent either bilateral ovariectomy or sham surgery in a 2:1 ratio, as previously described.18 Briefly, two longitudinal skin incisions of approximately 1.5 cm were made at the level of the kidneys 1 cm lateral to the dorsal midline. OVX rats had both the left and right ovaries removed whereas sham-operated rats underwent excision of just the fatty tissue surrounding each ovary and were left gonad-intact. Immediately following surgery the animals were randomly divided into two batches with three groups per batch, as per the experimental design illustrated in figure 1. The first batch (n=30) consisted of 10 sham-operated rats (Sham-0 group) and 20 OVX rats, half of which commenced EA treatment immediately (early EA group, n=10) and half of which were left untreated (OVX-0 group, n=10). All rats in batch 1 were killed 12 weeks after surgery. The second batch (n=30) comprised 10 sham-operated rats (Sham-12 group) and 20 OVX rats, half of which commenced EA treatment 12 weeks after ovariectomy (late EA group, n=10) and half of which were left untreated (OVX-12 group, n=10). All rats in batch 2 were killed 24 weeks following randomisation.
Rats in the early and late EA groups received EA treatment bilaterally at points SP6 (Sanyinjiao) and ST36 (Zusanli). As in our previous study,10 disposable sterile acupuncture needles (Huatuo, Suzhou Medical Instruments Factory, China) with a diameter of 0.25 mm and length of 25 mm were used. The depth of needle insertion varied with the thickness of the soft tissues at the needling sites but averaged 2–3 mm at SP6 and 3–5 mm at ST36. Electrical impulses with an intensity of 1 mA and frequency of 3 Hz were generated by a SDZ-V therapeutic stimulator (Huatuo, Suzhou Medical Instruments Factory, China). Both early and late EA treatments were administered for 30 min/day, 5 days/week for a total of 12 weeks. During EA treatment the rats remained conscious without anaesthesia. The EA procedure is illustrated in figure 2. No therapeutic intervention was given to rats in the OVX and sham groups. Rats in the early EA, sham-0 and OVX-0 groups were killed after 12 weeks and rats in the late EA, sham-12 and OVX-12 were killed after 24 weeks.
As previously described,18 blood was collected and serum was extracted after centrifugation at 2000 g and stored at −80°C pending analysis. Serum E2 and C-terminal cross-linking telopeptide of type I collagen (CTX-I) levels were measured using a commercial ELISA system according to the manufacturer's protocol (Shanghai QaYee Biological Technology Co, China).
As previously described,19 BMD of the L5 vertebral body and left femur was measured in all rats using dual energy X-ray absorptiometry (Lunar-DPX-IQ; Lunar, USA) with dedicated software for small animal research. After BMD analysis, bone specimens were stored at −20°C pending biomechanical examination.
The compression test and three-point bending examination were performed on the L5 vertebral body and the left femur, respectively, using an AG-IS biomechanical testing system (Shimadzu, Japan), as previously described.10 The load–displacement curve was generated by the instrument's software and extrinsic strength parameters including maximum load and energy absorption were calculated.
Real-time reverse transcription PCR (qRT-PCR)
Total RNA was extracted from the right femur using Trizol reagent (Invitrogen, USA) according to the manufacturer's protocol. First-strand cDNAs were synthesised from 5 μg total RNA in 20 μL reactions using the RevertAid First Strand cDNA Synthesis kit (Fermentas, USA) and oligo dT random primers. RT-PCR was performed using a FTC-2000 real-time PCR machine (Funglyn, California, USA) as previously described.19 Gene expression levels were normalised to the housekeeping gene β-actin. The data were analysed using the method of 2−ΔΔCT. Primer sequences and the expected RT-PCR products were as follows: cathepsin K (forward 5′-GAGACATGACCAGCGAAGAA-3′, reverse 5′- CACATATTGGAAGGCAGTGG-3′, product length 332 bp); β-actin (forward 5′-GCCAACACAGTGCTGTCT-3′, reverse 5′-AGGAGCAATGATCTTGATCTT-3′, product length 114 bp).
All data are presented as mean±SD. Groups were compared by one-way ANOVA followed by Tukey's post hoc test using SPSS V.18.0 (Chicago, USA). Differences were considered significant at p<0.05 for all analyses.
Analysis of serum E2 and CTX-I levels
As shown in figure 3A, B, ovariectomy significantly decreased serum E2 levels compared with the sham procedure (OVX-0 and OVX-12 vs Sham-0 and Sham-12). Serum E2 levels were significantly increased in the early EA group (p<0.05 vs OVX-0 group) and late EA group (p<0.05 vs OVX-12 group). Effects on CTX-I, an osteoclast marker, are illustrated in figure 3C, D. Serum CTX-I levels were higher (p<0.01) in OVX rats compared with sham-operated rats. After 12 weeks EA treatment, CTX-I concentrations were significantly lower in the early EA group (p<0.01 vs OVX-0 group) and late EA group (p<0.05 vs OVX-12 group).
Bone mineral density examination
As shown in figure 4A, B, BMD of the L5 vertebral body was significantly lower in the OVX-0 group (p<0.01 vs Sham-0 group) and OVX-12 group (p<0.01 vs Sham-12 group). Furthermore, L5 vertebral body BMD values were significantly elevated in both the early EA and late EA groups compared with the OVX-0 (p<0.01) and OVX-12 (p<0.05) groups. As shown in figure 4C, D, BMD of the femur was significantly decreased in the OVX-0 group (p<0.01 vs Sham-0 group) and OVX-12 group (p<0.05 vs Sham-12 group). Femur BMD values were significantly elevated in early EA versus OVX-0 groups (p<0.01) but did not differ significantly between late EA and OVX-12 groups (p>0.05).
As shown in figure 5A–D, biomechanical compression testing of the L5 vertebral body showed that ovariectomy significantly reduced maximum load and energy absorption (p<0.01 vs sham control), whereas both early and late EA-treated OVX rats had significantly greater maximum load and energy absorption compared with OVX rats (p<0.01). As shown in figure 5E–H, the three-point bending examination of the femur showed that OVX rats had significantly reduced maximum load and energy absorption compared with the sham control (p<0.01 or p<0.05). Early EA treatment of OVX rats resulted in significantly greater maximum load and energy absorption compared with OVX rats (p<0.05), while late EA treatment did not improve maximum load and energy absorption compared with OVX rats.
mRNA expression of cathepsin K
As shown in figure 6A, B, mRNA expression of cathepsin K was significantly higher in the OVX-0 group (p<0.01 vs Sham-0 control) and OVX-12 group (p<0.01 vs Sham-12 control). Moreover, mRNA expression of cathepsin K was significantly decreased in both the early EA group (p<0.01 vs OVX-0 group) and late EA group (p<0.01 vs OVX-12 group).
This study was designed to explore the potential mechanisms by which EA impacts osteoporosis in OVX rats. Our results showed that early EA treatment completely inhibited bone loss and deterioration of bone strength in the OVX rats, while late EA treatment partially slowed down the progression of bone loss. Although the late EA treatment starting at 12 weeks after ovariectomy inhibited further bone loss and deterioration of bone strength in the L5 vertebral body over time, it did not achieve a full reversal of such changes in the femur at the end of the experiment. Furthermore, we observed that both early EA and late EA treatment suppressed cathepsin K mRNA expression in the femur of OVX rats.
In this study, the serum E2 level in the OVX groups was much lower than the sham control, which is consistent with our previous studies.18 ,19 This significant reduction in oestrogen reflects the success of OVX surgery. We found that both early EA and late EA increased the serum E2 level in OVX rats. Previous studies have shown that EA can modulate reproductive hormone levels in patients with primary ovarian insufficiency20 and regulate activities of the hypothalamus-pituitary-ovarian axis and the signal transduction pathway of the hypothalamic neuroendocrine system in OVX rats.21 ,22 Although it appears that EA can increase the serum E2 level in OVX rats, the precise mechanism of action needs to be investigated further.
In collagen degradation, CTX-I generated by cathepsin K is used as a marker of bone resorption.23 In agreement with an earlier study,24 we found that serum CTX-I levels significantly increased in OVX rats compared with sham-operated rats. Both early EA and late EA treatment of 12 weeks duration significantly attenuated serum CTX-I levels, suggesting that EA ameliorated OVX-induced bone loss in rats through inhibition of osteoclast function and bone resorption.
BMD measurement is accepted as a standard tool in the diagnosis and evaluation of osteoporosis.25 Biomechanical strength reflects bone fragility and fracture risk,26 so the impact of a treatment on bone strength is an important determinant of its therapeutic utility in the treatment of osteoporosis. In the present study, BMD and bone strength were significantly decreased in the femur and L5 vertebral body of OVX rats, confirming the development of osteoporosis. We demonstrated that early EA treatment significantly increased BMD and bone strength of both the L5 vertebral body and the femur in OVX rats. Although late EA treatment inhibited further bone loss and deterioration of bone strength at L5, it did not improve BMD or bone strength in the femur. It is known that BMD is an important component of bone strength,27 hence improvement in BMD may, at least in part, account for the increase in bone biomechanical strength. The relatively higher amount of cortical bone compared with trabecular bone in the femur versus the L5 vertebral body may account for the site-specific discrepancy in BMD and bone strength. The different outcomes from early and late EA treatment in OVX rats suggest that future clinical studies in women should explore the importance of timing of treatment initiation to optimise the management of postmenopausal osteoporosis. The present study suggests that early EA treatment may be desirable to optimise treatment outcomes.
To explore the mechanism of action underlying the effects of EA in OVX rats at a molecular level, we quantified mRNA expression of cathepsin K, which is intimately involved in the process of bone absorption. Our results showed that mRNA expression of cathepsin K was significantly increased in the femur of OVX rats, and was attenuated by both early and late EA treatment. A previous study demonstrated that synthesis of cathepsin K was negatively regulated by serum E2 at the mRNA level and deficiency of serum E2 resulted in augmentation of cathepsin K in OVX mice.15 Since the association between E2 and cathepsin K is apparent, we can infer that cathepsin K expression increased due to the low serum E2 level in OVX rats, and that the sharp rise in serum E2 levels following EA treatment may be responsible for the reversal of changes in cathepsin K expression thereafter.
In this study we examined the expression of cathepsin K in bone of OVX rats after application of EA and revealed for the first time that EA suppressed cathepsin K expression in OVX rats. However, cathepsin K may not be the only factor regulated by EA and it is feasible that other signalling pathways may be affected. It is known that many signalling pathways are involved in the pathological process of osteoporosis.28 Our previous studies have found that the receptor activator of nuclear factor κB (RANK)/RANK ligand (RANKL)/osteoprotegerin and Wnt/β-catenin signalling pathways play important roles in determining bone mass and strength in OVX rats.18 ,19 For example, we previously found that EA can prevent OVX-induced bone loss and deterioration of bone architecture and strength through stimulation of the Wnt/β-catenin signalling pathway.10 Further investigation is needed to explore the influence of EA on other signalling pathways.
In conclusion, these data demonstrate that EA can partially prevent bone loss and deterioration of bone strength in OVX rats. Although the beneficial effects of EA on osteoporotic bone have previously been reported in both clinical and animal studies, the present study suggests that timing of treatment initiation may be important to optimise the beneficial effects of EA. Unfortunately, no strong conclusions can be made in this respect due to limitations in the study design. As OVX rats in the early and late EA groups were killed 12 and 24 weeks following ovariectomy, respectively, these groups also differed with respect to duration of exposure to OVX and the age at necropsy in addition to timing of EA treatment, which lasted 12 weeks in both groups. Consequently, the potentially confounding influence of such additional factors and their possible interactions remain unknown. We also found that EA suppressed cathepsin K expression in the bone of OVX rats. Given the important role of cathepsin K in the regulation of bone resorption, we suspect that EA may modulate the process of osteoclast activation and subsequently bone resorption, at least partially, through regulation of the expression of cathepsin K. This in vivo study lays the groundwork for future clinical studies of EA therapy in postmenopausal osteoporosis.
Electroacupuncture (EA) improves oestrogen levels, bone mineral density and bone strength in a rat model of osteoporosis induced by ovariectomy.
The timing of EA treatment initiation appears to be an important factor, which needs further investigation.
The positive impact of EA on ovariectomised rats is at least partially mediated by effects on mRNA expression of cathepsin K.
We thank Shiju Chen for assisting in language editing of this manuscript.
Contributors JZ and XL performed most of the experimental work. JZ drafted the manuscript. YL, WF and XG were actively involved in the study including the animal protocols, laboratory work and data analysis.
Funding The study was supported by Health Department of Hunan Province (B2014-052) and State Administration of Traditional Chinese Medicine of Hunan Province (2013112), China.
Ethical approval All surgical and therapeutic procedures were approved by the ethics committee at the University of South China.
Competing interests None.
Provenance and peer review Not commissioned; externally peer reviewed.
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