Study design
This is a randomized, sham-controlled, patient and assessor-blinded trial. A target sample of 104 participants will be recruited from the acupuncture clinic at the first affliated hospital of Dali university and Yunnan Provincial hospital of Traditional Chinese Medicine . The present protocol followed the SPIRIT guidelines and fulfilled the SPIRIT checklist(Additional file 1). The flow chart is shown in Fig.1. The protocol is in line with the principles of the Declaration of Helsinki and has been approved by Institution review board (IRB) of Yunnan Provincial hospital and Traditional Chinese Medicine (approval no.2018-003-01). This trial was registered at the Chinese Clinical Trial Registry (ChiCTR1800017581). Any changes which need to be made in the trial protocol will be communicated to all researchers, the ethics committees, and the trial registry. Written informed consent will be signed by each participant.
Sample size, randomization, and blinding
According to the preliminary research [15], we hypothesize that the effective rate after OA is 77.8% and the effective rate after SA is 50%. The calculation equation we used was α = 0.05,β = 0.20 and we determined that a sample size of 42 patients in each group would be sufficient to detect the statistical difference between the two groups, allowing for a 20% withdrawal rate. We will plan to enroll a total of 104 participants with 52 participants in each group. According to the random number sequence generated by computer, the patients will be randomly divided into either the OA group or the SA group in a 1:1 ratio. In order to minimize potential sources of bias ,participants, doctor, outcome assessors, and data analysts will be blinded to the treatment allocation. Due to the special nature of acupuncture, acupuncturists can not be blinded to the treatment.
Inclusion criteria
Participants who meet all the following requirements will be allowed for enrollment:
1. Women aged 45-70 years.
2. Patients who meet the diagnostic criteria for PMOP:
①Pain occurs mostly with the hips, lower back and ribs, and slowly onset;
②Brittle fracture occurs under the action of slight external force;
③There is obvious tenderness point on the lower back or there is a large area of tenderness;
④Scoliosis, kyphosis and other deformities;
⑤DXA(dualenergy X-ray absorptionmetry) is used to detect femoral neck bone mineral density lower than -2.5. Severe osteoporosis is accompanied by two or more fractures;
⑥Imaging examination showed obvious osteoporosis or fragility fracture;
⑦non-secondary osteoporosis
3. Meet the " atrophic debility of bones" in Chinese medicine diagnostic criteria;
4. Natural menopause for more than 1 year;
5. Other medications or other related interventions for postmenopausal osteoporosis were not received within 4-6 months;
6. Patients voluntarily, informed consent, and signed informed consent, with good compliance.
Exclusion criteria
Participants meeting any of the following criteria will be excluded:
1. Except for women with abnormal lumbar anatomy (such as severe scoliosis);
2. Exclude related endocrine disorders (such as diabetes, hyperthyroidism, hypothyroidism, thyroid cysts, etc.) that can cause secondary osteoporosis;
3. After uterine or ovarian ablation surgery;
4. Concomitant with diseases such as rickets, rheumatoid arthritis or other diseases that affect the dynamic balance of bone metabolism;
5. In the past 3 months, he has taken drugs that can interfere with bone metabolism (such as glucocorticoids, calcitonin, estrogen, etc.);
6. Female patients with severe underlying diseases and mental illness who are unable to cooperate with treatment research programmes;
7. Drug or alcohol abuse;
8. Those who had received acupuncture treatment;
9. Participating in other clinical trials.
Interventions
Both OA and SA groups will receive acupuncture sessions for a total of three months. All the pa- tients will receive herbal medicine twice a day for three months. The same doctor (the professor of acupuncture in Yunnan Provincial hospital and Traditional Chinese Medicine who has 20 years of work experience) will slightly adjust the formula each week depending on the changes of the symptoms, pulse, and tongue coating of the patients (main herbal formula components are shown in Table 1). During the trial, patients will be based on pattern differentiation:(1) spleen-kidney Yang deficiency will be given Yougui Pill;(2) liver-kidney Yin deficiency will be given Zuogui Pill;(3)kidney deficiency and blood stasis will be given Bushen Huoxue decoction . Participants will get all herbs from the same hospital and the doctor will instruct them how to decoct.
According to the overall adjustment of acupuncture and moxibustion [15], and taking opinions from Chinese acupuncture experts, the essential acupuncture points selected are as follows: BL11(Dazhu); BL23 (Shenshu); ST36 (Zusanli). The additional individualized acupuncture points will be chosen by the practitioners according to the patterns of identification: (1) kidney Yang deficiency will plus DU04(Mingmen),GB34(Guanyuan); (2)spleen-kidney Yang deficiency will plus DU04(Mingmen), GB34(Guanyuan); (2)liver-kidney Yin deficiency will plus SP06(Sanyinjiao),LU11(Taixi);(3)kidney deficiency and blood stasis will plus SP06(Sanyinjiao),BL17(Geshu)(Table 2). A placebo device [16] will be applied in both groups for better implementation of blindness. The schematic diagram of acupuncture treatment is shown in Fig.2.The acupuncture treatment in both groups will take 30 min per session, twice per week. Three months is a course of treatment, a total of one course .
Overall adjustment acupuncture(OA) group
Acupuncture manipulation will be applied to the acupoints of the body until the patient achieves Qi, including soreness, soreness and distension, pain, numbness, tingling and even comfort. Huatuo brand needles (0.25 mm x 40 mm) were manufactured by Suzhou medical device company in Suzhou, Jiangsu province, China. After the patient achieves Qi in Shenshu and Zusanli, warm acupuncture will be used, fix the 2cm-long moxa-stick on the handle of the needle and light it at the root of the needle for 20 minutes.
The parameters of the skin needle are set as follows: Qixing needle: tapping along the first side line of the urinary bladder meridian in the back; moderate stimulation will be applied for the blood stasis and qi stagnation type, until redness of the skin and petechiae is observed in the tapping part; mild stimulation was used for the rest of the syndromes until redness in the skin is observed.
The parameters of the cupping are set as follows: moving cupping: smear the tapping spot evenly with Vaseline and move the cup along the first lateral line of urinary bladder meridian back and forth until red blood stasis appears on the skin, according to the patient’s tolerance (feeling comfortable, without an obvious pain). Retaining cupping: Leave the cup for 8 to 10 minutes in Dazhui, Shenshu, and a severely painful area.
Sham acupuncture (SA)group
The procedure and duration of treatment in the SA group will be identical in the OA group except the needles (0.25 × 25 mm, blunt tip, manufactured by Suzhou medical device company in Suzhou, Jiangsu province, China)、Qixing needles tip wrapped with cotton(show in figure3) are blunt tip and there will be no skin penetration or manual stimulation,at the same time cupping is applied to the skin with a small negative pressure comparing with the OA group(show in figure 4).
Practitioner background
The OA and SA treatments will be handled by acupuncturists who are registered by the Chinese medicine practitioner qualification certificate and who are specialized in acupuncture with more than three years of clinic experience. They will have studied acupuncture for more than 10 years and graduated from a TCM university. All operators will be trained in the overall adjustment of the needle operation in advance and qualified, and will undergo intensive and customized training for a full understanding of the SA procedure and a sham needle device. The techniques for the entire treatment procedure will be standardized between practitioners.
Outcome measures
Outcomes will be collected at baseline, the middle of treatment, after treatment, and follow-up six months after the completion of the treatment.The overview of the outcome measurement at the different time points is shown in Table 3.
Primary outcome
The primary outcome is the bone mineral density(BMD), BMD was measured by a dual energy X-ray absorptiometry (manufacturer: DMS, France; model: CHALLENGER C 313). BMD of the lumbar vertebrae (L1-L4) in the two groups will be measured before and after treatment.
Secondary outcome
Pain score The pain score will be assessed for the lower back. A Visual Analogue Scale (VAS) will be used to measure each group before and after treatment (see attached Table 4).
The TCM syndrome scores will be recorded by the same professor once a week to adjust the acupuncture and herbal treatment [17] and evaluate the improvement of the patients’ TCM syndromes (see attached Table 5).
Indicators for Quality of life Based on Quality of Life (QOL) and 36-Item Short Form Survey (SF-36), combined with the specificity of osteoporosis and clinical situations, a self-rating scale in line with Chinese ethnicity was developed (see attached Table 6).
Blood test
(1) Sex hormones The fasting blood samples will be drawing in every month before and after treatment by the nurse. The chemiluminescence immunoassay (CLIA) will be used to determine serum sex hormone levels include estradiol (E2), FSH (Model: Switzerland ELECSYS2011).
(2) Antioxidant index Before and after intervention, 5 cc of venous blood sample were obtained in non-fasting condition. Samples were frozen in -70° C. Advanced oxidation protein products (AOPP), and total antioxidant capacity (TAC) (with 2, 2-diphenyl-1-picrylhydrazyl [DPPH] oxidation) were measured with the following methods: Walwadkar [18], Kataaha et al. [19], Girbal et al. [20], Kitajima [21] and Janaszewska et al. [22].
(3) Bone metabolism index The fasting venous blood was collected before treatment, one course of treatment and at the end of treatment respectively. The enzyme-linked immunosorbent assay (ELISA) (biocell enzyme standard instrument) was used to determine BGP and BALP before and after treatment.
(4) Pathway signaling proteins Western blot was used to detect the expression of β-Catenin, FoxO3a and PPAR γ2 protein in bone tissue(Multiskan spectrum,American(Multiskan spectrum)).
Follow-up Six months after the completion of the treatment, follow-up will be performed by telephone to ask if the participants were fracture and the recurrence of pain.
Adverse events
Safety will be assessed by body temperature,pulse,breathe, blood pressure,renal function test, liver function test. These indicators are detected during the period of screening and after 12-weeks’ treatment. We will monitor adverse events for each treatment during the trial, including hematomas and acute pain. Any adverse events or reactions that are thought to be causally associated with the intervention will be recorded, managed, and reported to the study coordinators. Serious adverse reactions will be reported to the ethical committee.
Data analysis
All collected data results were compiled using SPSS21.0 statistical software package, and collated, checked and statistically analyzed. The comparison between the data groups was performed by χ2 test, and the measurement data was consistent with the normal distribution and the homogeneity of the variance. The mean ± standard deviation (±S) indicates that the rank sum test is used for comparison between grade data sets that do not satisfy the normal distribution and the homogeneity of the variance. P<0.05 was significant for the difference. The variance analysis will be performed for the difference between the two groups and within the group. We will perform a regression analysis to examine the causal relationship between the E2, TAC and β-Catenin, and the primary outcome.The statisticians who are independent of the research team will conduct data analysis.