Jingui Shenqi Pills Prevents and Treats Postmenopausal Osteoporosis via Regulating the BMP/Smad Signaling Pathway

Background: Jingui Shenqi （ JGSQ for short ） pills is a traditional Chinese medicine formula, which has the functions of warming and tonifying kidney-yang, generating essence and filling marrow, warming tendons and veins and bones, and improving bone mineral density. The aim of this study was to investigate the effect and mechanism of JGSQ pills in preventing and treating postmenopausal osteoporosis. Methods: Twelve-week-old SPF female SD rats (n=48) were used in this study. Following the ovariectomy operation (n=40), the rats were randomly divided into the model group, (high, medium, and low dose groups) treated with Jingui Shenqi pills and estradiol group. Recorded the weight gain of rats and calculated the uterine coefficient; To detect the expression of serum calcium, phosphorus, ALP and OPG; HE staining was used to detect the pathological changes of femur; In all the groups, Micro-CT was used for detection of bone mineral density and bone microstructure; and gene expression of BMP-2, Smad1, and Runx2 in rat bone tissue was determined by RT-PCR and Western Blot methods. Results: Compared with the sham operation group, rats in the model group had the highest increase in body weight and the lowest uterine coefficient. Each of the treatment groups had a modest increase in weight gain. Micro-CT and HE staining showed a decrease in bone mineral density in the model group with shorter, thinner, broken, and osteoporotic trabeculae of the femur. The bone mineral density in Jingui Shenqi pills treatment groups had a significant increase with an improved bone microstructure and intact bone trabecular as compared to the model group. Serum ALP in the model group was significantly higher than in the sham operation group but the serum calcium, phosphorus and OPG was significantly lower. The Jingui Shenqi pills treatment groups and the estradiol treated group had lower serum ALP and increased serum calcium, phosphorus and OPG.There was decreased gene expression of BMP-2, Smad1, and Runx2 in the bone tissue of the model group compared to the sham operation group. BMP-2, Smad1, and Runx2 gene expression in Conclusion: Jingui Shenqi pills improve the microstructure of bone tissue and increase bone mineral density thus resolving osteoporosis. This is achieved by regulating BMP/Smad signaling pathway and increasing the expression of osteogenic factors BMP-2, Smad1, and Runx2. Graphical abstract: This study revealed that JGSQ promote bone formation, improves bone tissue microstructure, and increases bone mineral density by regulating the expression of the BMP/Smad signaling pathway. JGSQ offer clinical value in resolving osteoporosis. As a traditional Chinese medicine compound preparation with high safety and efficacy, JGSQ can be used as a preventive and therapeutic drug for osteoporosis for in-depth mining and development. :


Introduction:
Osteoporosis is a metabolic disease that frequently occurs in elderly and postmenopausal women. It is mainly characterized by damage of the bone tissue microstructure, decrease of bone mineral density, and increased risk of fractures [1] [2]. Environmental and genetic factors accelerate the occurrence and development of osteoporosis, of which the imbalance of bone remodeling caused by reduced bone formation is a key factor in the occurrence of osteoporosis [3].
Statistics show that worldwide, 1,000 people are fractured due to OP every hour, and there are about 900 million fractures each year [4], of which the incidence of postmenopausal osteoporosis is about 30% in people over 50 years of age [5], and the resulting fractures are the main cause of middle-aged and elderly visits, which seriously affect the physical and mental health of middleaged and elderly people, bring a huge burden to families and society, and become an important public health problem currently faced .
Modern medicine believes that the dynamic balance of osteoblasts and osteoclasts is the key to maintain bone remodeling, thereby maintaining the balance of bone metabolism [6]. Bone remodeling is regulated by various factors, such as estrogen levels, intestinal bacteria, oxidative stress, and inflammatory responses [7]. At present, the commonly used anti-osteoporosis drugs in clinical practice include bisphosphonates, parathyroid hormone and hormone replacement therapy.
However, while exerting the role of targeted therapy, the above drugs ignore the integrity of the body. Long-term or high-dose administration will increase the risk of cardiovascular disease, cervical cancer, kidney stones and gastrointestinal diseases [8]. Therefore, it is important to continue exploring for safe and effective treatment.
Traditional Chinese medicine seeks the root cause of the disease, pays attention to the occurrence, development, and disease changes to bring a balance to yin and yang. Traditional Chinese medicine has the advantage of minimal side effects and high efficacy in the treatment of postmenopausal osteoporosis. The multiple compounds in a traditional Chinese medicine preparation act on multi-targets and multi-pathways to promote bone formation. Traditional Chinese medicine also relieves the bone pain associated with osteoporosis [9]. Traditional Chinese medicine theories, postulate that the kidney controls bone marrow generation by regulating bone metabolism. Jingui Shenqi pills(JGSQ for short) are a classic prescription for warming and act as a tonic for kidney yang which was first published in the synopsis of the Golden Chamber. It is composed of dried Rehmannia glutinosa, yam, Cornus officinalis, Poria, alisma, moutan, aconite, and cassia twig. Currently, it is clinically used in the treatment of kidney yang deficiency [10] [11] [12].
Additionally, long-term treatment with Jingui Shenqi pills in postmenopausal osteoporosis resulted in a similar increase in bone mineral density as alendronate [14]. Jingui Shenqi pills were also shown to increase estrogen in in vivo studies carried out in ovariectomized rats. Jingui Shenqi pills also improved osteoblast vitality, ALP activity, and Collagen I protein expression [15]. Jingui Shenqi pills were also shown to promote bone differentiation in an in vitro study, by promoting the expression of ALP, RUNX2, and OCN [16]. However, the mechanism of Jingui Shenqi pills in preventing and treating PMOP has not been fully elucidated. Current studies have shown that the mechanism of traditional Chinese medicine in preventing and treating postmenopausal osteoporosis is related to the BMP/Smad signaling pathway, which can regulate all processes of osteoblast proliferation, differentiation, and mineralization, and its abnormal function can cause decreased bone mass and reduced bone formation, leading to osteoporosis [17] [18]. The BMP/Smad signaling pathway can regulate downstream osteoblast transcription factors, promote bone formation, and increase bone mineral density [19] [20] [21]. Therefore, in this experiment, ovariectomy was used to duplicate the postmenopausal osteoporosis model to observe the effect of Jinkui Shenqi Pills on calcium, phosphorus and bone formation markers in the serum of OVX rats; HE staining was used to observe the pathological changes of the femur; micro-CT was used to detect the bone mineral density and bone microarchitecture of rats; qRT-PCR and Western blot were used to detect the gene expression of BMP-2, Smad1 and Runx2 in the femur of rats to further elucidate the mechanism by which Jinkui Shenqi Pills improved osteoporosis in OVX rats. It further enriches the theory of "kidney main bone" and provides theoretical basis and scientific basis for the prevention and treatment of osteoporosis by traditional Chinese medicine.

JGSQ pills preparation
All the traditional Chinese medicines of JGSQ were purchased from Shandong Xinzhonglu Hospital (Jinan, China). The preparation of JGSQ is composed of eight herbal medicines as follows: Rehmannia glutinosa 24g, Dioscorea zingiberensis 12g, Cornus officinalis 12g, Poria 9g, alisma alisma 9g, moutan bark 9g, aconite 3g, and cassia twig 3g. The herbal materials were decocted in eight volumes of distilled water for 1 hour. A filtrate was then obtained by double filtration. The filtrate was then concentrated to a high dose of 1.7g/ml (twice the clinical dose), a medium dose of 0.85g/ml (clinically used dose), and a low dose of 0.425g/ml (half the clinical dose). Medium dose is the original amount of classical prescription.  Table 1).

Animals model establishment and drug administration
12 week-old female Sprague Dawley rats (n=48) were obtained from the Laboratory Animal Center of Shandong University of Traditional Chinese Medicine (Shandong China). They were maintained in a 12 h light-dark cycle, at a controlled temperature (22-24℃) and humidity (50-60%). Standard laboratory rodent diet and water were provided ad libitum. The rats were subjected to adaptive feeding for three days before the experiment. Complete bilateral ovariectomy was then carried out in all rats (n=40) except the rats allocated to the sham group ( a sham operation involving removal of the same amount of fat near the ovary was carried out). Five days after the operation, the rats were allocated to six groups of 8 rats as follows: sham (shamoperated rats + saline), OVX (OVX rats + saline), high-dose JGSQ pills (OVX + JGSQ-H, 1.

Hematoxylin and eosin (H&E) staining
The left femur bones were collected, fixed in 10% neutral formaldehyde for 72 h, then removed and decalcified in 10% ethylenediaminetetraacetic acid solution (pH 7.4) at 4°C for 4 weeks. Paraffin was then used to embed the fixed samples. The samples were then cut into 4 μm slices which were stained with H&E stain and observed under a light microscope.

BMD and Micro-CT examination
Local bone mineral density (g/cm 2 ) and trabecular microstructure (BV/TV, BS/BV, Tb.Th, Tb.N, SMI, Tb.Sp and BMD ) of the right distal femur were measured and analyzed by Bruker-Micro CT (NEMO, Pingsheng Medical Technology Co., Ltd.).Following the CT scan, the cortical and trabecular bones of the femur were analyzed by Skyscan NRecon software. The 3D and 2D sections of femoral cortical and trabecular bone were reconstructed.

qRT-PCR
Total RNA was extracted from the bone tissue with Vazyme R401-01RNA isolator per the manufacturer's instructions. Reverse transcription was then carried out using Vazyme R323-01kit.
Primer sequences were designed based on GenBank sequences with Primer Premier 5.0 software (Table 2) by Shanghai Shenggong Co., Ltd., China. ACTB was used as an internal control.
SYBRGreen PCR kit (Vazyme Q711-02/03) was used to prepare the reaction master mix, Fluorescencequantitative PCR (Bio-Rad, USA) was used for mRNA amplification and quantification. The reaction conditions were: denaturation at 95°C for 30 s, followed by 40 cycles of denaturation at 95°C for 5-10 s, annealing at 60°C for 30 s, and separation at 95°C for 15 s, 60°C for 60 s and 95°C for 15 s. Relative gene expression was determined using the 2 −ΔΔct method.

Western blot
The total protein was extracted from bone tissue using RIPA lysis buffer (P0013B, Beyotime) after pulverizing in liquid nitrogen. The concentration of the protein was determined using a BCA protein kit (G2026, Servicebio). The protein was then mixed with loading buffer and boiled at 100°C for 5 min to denature it. 30μg of total protein was separated by 10% SDS-PAGE and transferred into 0.45 µm polyvinylidene fluoride (A29280264, GE Healthcarelifescience) membranes. After blocking with 5% nonfat dried milk for one and a half hours, membranes were incubated with primary antibodies at room temperature: rabbit anti-BMP-2 (Ab14933, Abcom), rabbit anti-Smad1 (10429-1-AP, Proteintech), and rabbit anti-Runx2 (Bs-1134R, Bioss) respectively for 2 hours. The membranes were then incubated with goat anti-rabbit IgG (LY192274, Servicebio) as the secondary antibody for 1 h at room temperature. The blots were developed with ECL reagent (B500023, Proteintech). The total protein was compared with β-actin as an internal reference, and the gray values of internal reference and target bands were quantified by Image-ProPlus6.0 software.

Statistical analysis
SPSS 22.0 (SPSS Inc., Chicago, IL, USA) was used to perform the statistical analyses and the data were expressed as mean ± SEM. Differences between two groups were compared by t-test, while the comparison between three or more groups were performed through one-way analysis of variance (ANOVA) followed by Dennett's test in order to detect intergroup differences. P<0.05 was considered as statistically significant.

Identified compounds in JGSQ pills
The components of the JGSQ (Salsolinol, choline, uracil, gallic acid, caffeic acid, et al) filtrate was verified by Q-Orbitrap high-resolution liquid chromatography-mass spectrometry under optimized conditions. The main chemical components in JGSQ freeze-dried powder were detected by comparing with the retention time and UV spectrum of the reference standard. A total of 24 peak signals were obtained. The chemical components were also identified( Table 3 and Fig   1).

The general condition of rats
Rats in the Sham group, had soft, shiny hair, were sensitive to activity. Rats in the OVX group had a significant decrease in activity as compared to the other treatment groups. The body weight of the rats was recorded weekly during the 3 months. Compared with the Sham group, the OVX group had the highest weight gain and lowest uterine coefficient, while JGSQ-H, JGSQ-M, JGSQ-L, and E2 groups recorded a lower weight gain ( Fig.2A-2B), and an insignificant change in the uterine coefficient (Fig.2C).

Biochemical index
After 3 months of treatment, the serum levels of calcium and phosphorus in the OVX group were significantly lower than in the Sham group. However, the serum levels in the JGSQ-H group were significantly higher than in the OVX group. There was no significant difference between the treatment groups ( Fig.3A-3B).

Serum bone formation markers
Compared with the Sham group, the serum ALP levels in the OVX group were significantly higher, and the serum OPG was significantly lower. The JGSQ-H, JGSQ-M, JGSQ-L, and E2 treated groups had a decrease in the levels of ALP and an increase in serum OPG (Fig.3C-3D).

Figure.3 Effect of Jingui Shenqi Pills on Serum Parameters in Ovariectomized Rats. A,B.
Effect of Jingui Shenqi pills on calcium and phosphorus in serum of ovariectomized rats. C,D.

Histopathological analysis
The results of HE staining showed that the bone tissue structure in the Sham group was stable, the trabecular was dense and tidy, and the cortical and cancerous bones could be seen. In the OVX group, the bone tissue structure was seriously damaged, with a reduced trabecular volume and thickness, and a damaged medullary cavity space. Treatment with Jingui Shenqi pills showed an improvement in the bone microstructure, increased bone trabeculae, and an increase in the bone connection (Fig.4).

Figure.4 Effect of Jingui Shenqi Pills on Pathological Changes of Femur in Ovariectomized
Rats.Representative photographs of H&E stained femoral sections from each group (×100).Arrow (black): bone.

BMD and bone microstructure analysis
The results of the previous preliminary experiment showed that the bone mineral density of the JGSQ-L group was not significantly improved compared with the model group, so only the JGSQ-H and JGSQ-M groups were tested. Compared with the Sham group, BMD decreased significantly in the OVX group but had a significant increase in the JGSQ-M group. Threedimensional imaging of the femur of rats in the Sham group showed the trabecular as being moderate in size, thickness, compact arrangement, and had a good bone connection, while the OVX group, had shorter, thinner, fewer, sparse, and fractured trabecular, with loose shape. After treatment with Jingui Shenqi pills, the bone microstructure improved significantly and had an intact trabecular structure (Fig.5).

Expression of mRNA in bone tissue
The expression of BMP-2, Smad1, and Runx2 mRNA in bone tissue was significantly decreased in the OVX group than the Sham group. However, the expression of BMP-2, Smad1 and Runx2 mRNA in bone tissue in the Jingui Shenqi pills treated group and E2 treated group was higher than in the OVX group (Fig.6A).

Protein expression in bone tissue
Western Blot analysis showed that the expression of BMP-2, Smad1, and Runx2 protein in bone tissue in the OVX group was significantly lower than in the Sham group, while the expression of BMP-2, Smad1, and Runx2 protein in bone tissue in Jingui Shenqi pills treated group and E2 treated group was higher than in the OVX group (P < 0.05). There was no significant difference between the treatment groups ( Fig.6B-6C).

Discussion
The majority of the middle-aged and elderly people have a Yang deficiency. They often experience symptoms such as sore waist and knees, fear of cold, tinnitus, and diarrhea. Kidney yang is the foundation of the whole body yang. A deficiency in kidney yang results in bones lacking normal qi and blood nourishment, failure of joints to flex and extend normally, and impaired support resulting in osteoporosis. Jingui Shenqi pills prescription is derived from the synopsis of the Golden Chamber and is known as "the first prescription tonic for kidney yang through the ages". Jingui Shenqi pills can invigorate the kidney yang, gasify and return to normal, replenish the kidney yang, smooth the blood flow, and nourish the muscles, veins, and bones [22].
Qi Jicong found that Jingui Shenqi pills can relieve the pain caused by hyperosteogeny of the calcaneus and effectively treat "bone impotence" [23]. Jingui Shenqi pills were also reported to increase bone mineral density [24]. Fructus Corni Neoglycoside I has also been shown to promote the proliferation and differentiation of osteoblasts [25], and significantly increase the mRNA expression levels of Wnt2, BMP2, β-catenin, OPG, and NOX4 in osteoblasts [26]. Additionally, dried Rehmannia glutinosa has been shown to significantly inhibit the effects of OVX on the femur and lumbar vertebrae in rats [27].
The decline of ovarian function in menopause and the sudden drop of estrogen levels leads to a fat metabolism disorder, hyper appetite, and decreased activity, which reduces the basal metabolic rate and increases the fat content resulting in obesity [28] [29]. Excessive differentiation of adipocytes reduces osteoblast differentiation and bone formation, resulting in reduced bone formation and increased bone resorption, thus a decrease in bone mineral content, and osteoporosis [30] [31]. This study revealed that Jingui Shenqi pills reduce the weight gain induced by ovariectomy, increases the trabecular volume, and thickness, reduces trabecular separation, increases bone mineral density, and prevent the occurrence of postmenopausal osteoporosis.
Changes in the levels of calcium and phosphorus are important indicators that reflect on bone metabolism and changes to the whole body [32] [33] [34]. ALP (an intracellular enzyme in osteoblasts), is an important marker of bone formation [35]. When bone metabolism is exuberant, osteoblasts are active, and the ALP activity is increased. Estrogen secretion is decreased in OVX rats, resulting in a high bone metabolic state, that is, bone formation is less than bone resorption, simulating the bone metabolism in postmenopausal women [36]. OPG is secreted by osteoblasts and vascular endothelial cells and inhibits the differentiation and maturation of osteoclast precursors. Therefore, the level of serum OPG plays an important role in the early diagnosis of osteoporosis [37] [38]. Jingui Shenqi pills regulate bone metabolism and balance bone remodeling by increasing the levels of serum calcium, phosphorus, and OPG, and reducing the level of serum ALP.
Bone morphogenetic protein (BMP) is a family of proteins with multiple biological functions, which binds receptors on the target cell membrane. This results in activation of the BMP/Smad signaling transduction pathway [39]. The BMP-2 ligand secreted by osteocytes binds to the receptors BMPRI and BMPRII on the cell membrane forming a complex. The phosphorylated type II receptor activates the type I receptor, which then activates and phosphorylates receptor-regulated Smads protein (Smad1/5/8) The regulated smads then bind to Smad4 and translocates to the nucleus. The complex binds to DNA binding protein and regulates the expression of downstream transcription factors Runx2 and OSX, thus regulating the transcription of genes related to different stages of osteogenic differentiation [16].
BMP/Smad signaling pathway is a classical pathway in promoting osteoblast proliferation and differentiation in bone metabolism [40]. BMPs are an important osteogenic factor in inducing BMSCs to differentiate into osteoblasts and may result in increased osteoblast ALP activity, collagen synthesis, nerve cell differentiation, and kidney development [41] [42]. Smad protein mainly regulates bone metabolism through TGF-β / BMP pathway, which directly or indirectly induces the proliferation, differentiation, and apoptosis of osteoblasts and osteoclasts [43] [44].
BMP/Smad signal pathway wholly regulates osteogenic differentiation, thus regulating osteogenic metabolism [45]. The study findings revealed a decrease in mRNA and protein levels of BMP-2,

Consent for publication
Not applicable.

Availability of data and materials
The data used to support the results of this study can be obtained from the frst author upon reasonable request.

Competing interests
The authors declare no conflict of interest.

Authors' contribution
Huaxin Wang is fully responsible for experimental design, paper drafting and finalization.