Bushen Yijing Fang Reduces Fall Risk in Late Postmenopausal Women with Osteopenia: A Randomized Double-blind and Placebo-controlled Trial

Falls in late postmenopausal women with osteopenia usually cause fractures with severe consequences. This 36-month randomized, double-blind and placebo-controlled trial with a 10-year observational follow-up study aimed to investigate the long-term effect of herbal formula Bushen Yijing Fang (BSYJF) on fall risk in the late postmenopausal women with osteopenia. 140 late postmenopausal women (Femoral neck T-score, −2.5~−2 SD) were recruited and randomized to orally receive calcium carbonate 300 mg daily with either BSYJF or placebo for 36 months. The effect was further investigated for another 10-year follow-up. During the 36-month administration, there were 12 falls in BSYJF group and 28 falls in placebo group, respectively, indicating 64% lower risk of falls (RR 0.36 [95% CI, 0.18 to 0.71]; P = 0.004) in BSYJF group. During the 10-year follow-up, 36% lower fall risk (RR 0.64 [95% CI, 0.46 to 0.89]; P = 0.009) was observed in BSYJF group. No significant difference was found in safety profile between two groups. Thirty-six-month administration of BSYJF reduced fall risk with an increase in bone mass, and its latent effect on fall risk was continually observed in the 10-year follow-up in late postmenopausal women with osteopenia. This clinical trial was registered at Chinese clinical trial registry (ChiCTR-IOR-16008942).


Methods
Study design: All protocols and experimental procedures were approved by the Animal Experimentation Ethics Committee of the Chinese University of Hong Kong. All experiments were performed in accordance with the relevant guidelines and regulations outlined in the Animal Experimentation Ethics Committee Guide for the Care and Use of Laboratory Animals, The Chinese University of Hong Kong. Twenty-four 6-month old female Sprague Dawley (SD) rats were recruited. Ovariectomy was performed to mimic postmenopause. Eight rats were sacrificed as baseline group (BL) at 6 months post-surgery. The rest rats were assigned into 2 groups as follows, PBS group (n=8): daily oral administration of 2 ml PBS for three months started from 6 months post-surgery; BSYJF group (n=8): daily oral administration of BSYJF powder in PBS (100mg/kg) for three months started from 6 months post-surgery. All the rats were sacrificed after 3-month intervention. The left extensor digitorum longus (EDL) was collected for in vitro muscle testing and the right EDL was collected for hematoxylin and eosin (H&E) staining to determine the cross-sectional area (CSA) of the muscle fiber.

Evaluation protocols:
In vitro muscle functional testing: Dissected muscle with intact tendons was mounted in a specific chamber bathed with 95%O2/5%CO2-bubbled Krebs' solution (In Vitro Muscle Test System 1205, Aurora Scientific Inc., Aurora, ON, Canada). One end of the tendon was attached to a hook connected to the lever arm of a position feedback motor, and the other end was attached to a force transducer. The muscle was stimulated with 0.5 ms pulses of supramaximal intensity through two platinum plates that were parallel to the muscle. Peak twitch force at 1Hz and peak tetanic force at 100Hz for 400 ms were recorded with the ASI Dynamic Muscle Control Software (DMC v5.1 beta, Aurora Scientific Inc.).

Cryosectioning and H&E staining:
The dissected soleus muscles were snap frozen in liquid nitrogen-cooled isopentane and then embedded in OCT medium. Serial cross-sections (6 μm thickness) were cut from the mid-belly of the muscles on a cryostat at -20°C for histological and immunohistochemical staining. H&E staining was performed on sections to examine the general morphology. The software ImageJ was used to calculate the muscle fiber CSA.

Three-point bending test:
The three-point bending test was performed using the materials testing system (MTS, Eden Prairie, MN USA) following the established procedure. The femur was placed on its lateral surface on two rounded supporting bars that were 20 mm from each other. A preload of 1 N was applied by lowering the upper bar. A constant displacement rate of 6 mm/min was applied until bone break. Energy at ultimate force, determined by the area under the curve until ultimate force, was calculated.

Results:
The muscle fiber CSA of EDL from rats in PBS group was significantly decreased after the ovariectomy. BSYJF treatment attenuated the decline of muscle fiber CSA in aged OVX rats. In vitro muscle testing showed the twitch force of EDL in PBS group was dramatically decreased, while the twitch force of EDL in BSYJF group was significantly higher than that in PBS group. The time to peak (TTP) of EDL was unaltered (Appendix Figure 1).  ‡Values were presented as median (interquartile range). *The interaction of treatment group and times were analyzed by Linear generalized estimating equations (GEEs) **Values were analyzed by Wilcoxon rank-sum test unless otherwise indicated. §Values were calculated by analysis of covariance model (ANCOVA) at three time points. Sensitivity analysis using the multiple imputation method estimated the effect of missing values. The baseline age, time since menopause, body mass index (BMI), and history of falls were used as covariates in Linear GEEs and ANCOVA.

Methods
The chemical structures, molecular formula, and molecular weight of the compounds in BSYJF were collected and summarized from Traditional Chinese Medicine (TCM) Database (http://tcm.cmu.edu.tw/) 1  ). Briefly, all information of compounds in BSYJF was input into the UPLC-Q-TOF MS for finding compounds through their molecular features. The water extraction from BSYJF was run in the UPLC-Q-TOF MS and detected in the full scan mass spectra in both positive ion mode and negative ion mode. The scan results were analyzed by the Find by Molecular Feature program to identify the database-matching compounds with targeted molecular formula, molecular weight, as well as the corresponding isotopic pattern and fragmentation profile on the basis of the chemical structures 3 .
The herbs in BSYJF were classified into two categories based on the number of target genes in fall-related network, herbs target ≥ 3 genes (HT3G) or herbs target ≤ 2 genes (HT2G). HT3G were proposed as active component. HT3G and HT2G were composed with the original weight ratio in BSYJF. Further animal study was conducted to investigate the fictional roles of these two categories on musculoskeletal system. All protocols and experimental procedures were approved by the Animal Experimentation Ethics Committee of the Chinese University of Hong Kong. All experiments were performed in accordance with the relevant guidelines and regulations outlined in the Animal Experimentation Ethics Committee Guide for the Care and Use of Laboratory Animals, The Chinese University of Hong Kong.
To investigate the role of HT3G within BSYJF in regulating muscle mass and strength, OVX rat model was used. Briefly, thirty-two 6-month old female SD rats were recruited. Ovariectomy was performed to mimic postmenopause. Eight rats were sacrificed as BL group at 6 months post-surgery. The rest rats were assigned into 3 groups as follows, PBS group (n=8): daily oral administration of 2 ml PBS for three months started from 6 months post-surgery; BSYJF group (n=8): daily oral administration of BSYJF powder in PBS (100mg/kg) for three months started from 6 months post-surgery. BSYJF w/o HT3G group (n=8): daily oral administration of HT2G powder in PBS (36mg/kg) for three months started from 6 months post-surgery. All the rats were sacrificed after 3-month intervention. The left EDL was collected for in vitro muscle testing and the right EDL was collected for H&E staining to determine the CSA of the muscle fiber.
To investigate the role of HT2G within BSYJF in regulating bone mechanical property, OVX rat model was used. Briefly, thirty-two 6-month old female SD rats were recruited. Ovariectomy was performed to mimic postmenopause. Eight rats were sacrificed as BL at 6 months post-surgery.