Low-dose aspirin promotes osteogenic differentiation and osteogenic activity in osteoporotic rats by regulating Opg/Rankl/Rank Axis

Purpose: To investigate the effect of low-dose aspirin (ASP) on osteogenic differentiation and osteogenic effects in osteoporotic rats, and the associated mechanism. Methods: The bone marrow mesenchymal stem cells were randomly divided into 4 groups: control group and 3 drug groups treated with graded doses of ASP i.e. 0.5, 1 and 2 mmol/L, with 4 wells in each group. The OPG, RANKL protein expression levels, OPG/RANKL ratio, alkaline phosphatase (ALP) level, and ALP secretion at days 3, 5 and 7 were compared amongst the groups. Results: ALP secretion in BMSCs was markedly higher in the 1 mmol/L ASP group than in control (p < 0.05). The OPG protein concentration and OPG/RANKL ratio were markedly higher in ASP-treated BMSCs than in control, while RANKL protein expression level was significantly lower than that in control (p < 0.05). In BMSCs treated with ASP at doses of 0.5 and 2 mmol/L, OPG protein expression levels and ratio of OPG/RANKL were markedly lower than those in 1 mmol/L ASP group, while the mean level of RANKL protein expression was markedly higher than that in 1 mmol/L ASP group (p < 0.05). Conclusion: Low-dose aspirin increases the expression of ALP, and promotes calcification which relates to upregulation of OPG and inhibition of the OPG/RANKL/RANK axis. This provides new leads for the development of new anti-osteoporosis drugs.


INTRODUCTION
Postmenopausal osteoporosis has become a global public health problem, and its prevention and treatment have attracted a lot of attention from clinical researchers [1]. China is one of the countries with a high incidence of osteoporosis. A survey has shown markedly higher risk of osteoporotic hip fracture in women than the total risks of breast cancer, ovarian cancer and endometrial cancer [2]. In clinical practice, drugs such as hormone-stimulating calcitonin, and parathyroid hormone are used to treat postmenopausal osteoporosis. However, these treatment methods have certain disadvantages such as slow onset time, long treatment time, numerous adverse reactions, and high financial cost [3]. Therefore, it is important to evolve new, effective, and highly safe drugs for osteoporosis treatment. Osteoprotegerin (OPG) is formed in osteoblasts, the number of which can increase with cell differentiation, and is currently the only negative regulatory factor found in osteoclasts [4]. The function of receptor activator of nuclear factor-κB (RANK) is similar to that of OPG to a certain extent, and it partially blocks the ligand of RANKL, thereby significantly inhibiting osteoclast bone resorption [5].
Aspirin (ASP), a drug commonly used for middleaged and elderly people, is easily affordable, and it has very few adverse reactions. Moreover, it is used in a wide range of treatments. Ingestion of low-dose aspirin for more than 1 year resulted in significant increase in BMD, especially in elderly females and post-menopausal women who took aspirin daily for 4 years [6]. Some studies have used ASP to treat osteoporotic rat models after ovariectomy, and found that the drug significantly improved trabecular bone and cortical bone density in rats [7]. Therefore, in this study, it was hypothesized that ASP has an anti-osteoporosis effect. However, ASP has not been used clinically to treat osteoporotic lesions. This study was carried out to determine the influence of ASP on postmenopausal osteoporosis, and the process involved.

Materials
Twelve female Sprague Dawley rats aged 3 weeks (mean weight = 245 ± 25 g) were purchased from Guangzhou Focus Biotechnology Co. Ltd. The rats were raised in separate cages (4 in each cage), and were permitted ad libitum availability of feed and water which were changed once every 12 hours. Adaptive feeding was done 7 days before the start of the study. This research was approved by the Animal Ethical Committee of Yiwu Stomatological Hospital (approval no. 20190421), and conducted according to the guidelines of Principles of Laboratory Animal Care [8].

Treatments
The rats were anesthetized with intraperitoneal injection of 1% pentobarbital sodium, and their ovaries were removed so as to establish ovariectomized rat model. After 12 weeks, the ovariectomized rats were sacrificed via atlas dislocation, and BMSC osteoblast-inducing cells were isolated. The cells were subjected to primary culture and subculture.
Then, 3 to 6 generation BMSCs were seeded in a 24-well plate at a density of 1×10 6 cells/well. Four groups of cells were used: control group, and 3 groups of BMSCs treated with ASP at doses of 0.5, 1 and 2 mmol/L, with 4 wells for each group. The BMSCs in the control group were untreated.
Alkaline phosphatase (ALP) assay kit was used to assay ALP secretion in each group of BMSCs at days 3, 5 and 7; while ALP staining kit was used to detect ALP in each group of BMSCs on day 7. After 21 days, BMSCs in each group were stained with alizarin so as to observe the number of calcium nodules and calculate the percentage of calcified area. With respect to the latter, 3 specimens were randomly selected from each specimen under high magnification, and the mean area was obtained. Using the control group as the standard, the % of calcified area was calculated.
The expression levels of OPG and RANKL in BMSCs of each group were determined after 14 days using ELISA kits, and the OPG/RANKL ratio was calculated.

Statistical analysis
Data analysis was done using SPSS version 22.0. Measurement data for ALP secretion and calcium nodules in BMSCs in each group are expressed as mean ± standard deviation (SD). Student's t-test was used for two-group comparison, while one-way analysis of variance (ANOVA) was used for comparison amongst multiple groups. Values of p < 0.05 indicated significant differences.

ALP secretion by BMSCs
On day 3, there was no significant difference in ALP levels between BMSC groups treated with ASP and untreated control group (p > 0.05). However, after 5 days, ALP secretion was markedly higher in BMSCs in the 1 mmol/L ASP group than in control. A similar result was obtained after 7 days in ALP groups, at which period the BMSCs in each group had differentiated and active osteoblasts with positive cytoplasmic reaction and small amounts of brown particles were present in the cytoplasm. These results are shown in Figure 1.

Number and area of BMSCs calcium nodules
The number and area of calcium nodules in BMSCs in ASP groups were significantly higher than those in control group. The number and area of calcium nodules in BMSCs treated with ASP at doses of 0.5 and 2 mmol/L were markedly lower than those in 1 mmol/L ASP group (p < 0.05). These results are presented in Table 2 and Figure 2.

OPG and RANKL proteins in BMSCs
The OPG protein levels and OPG/RANKL ratio were markedly increased in the ASP-treated BMSCs, relative to control, while the RANKL protein expression was markedly decreased, when compared to control (p < 0.05). The expression levels of OPG protein and OPG/RANKL ratio in BMSCs treated with ACP at doses of 0.5 and 2 mmol/L were significantly lower than those in BMSCs treated with ASP at a dose of 1 mmol/L, while RANKL protein expression was significantly higher (p < 0.05). These results are shown on Table 3.

DISCUSSION
Postmenopausal osteoporosis is a primary osteoporosis which manifests as systemic bone loss, degeneration of bone microstructure, increased bone fragility and increased risk of fracture [9]. Postmenopausal osteoporosis is a serious problem which deserves research attention. Thus, the focus of this research was to evolve a safe and more easily available drug for protecting against postmenopausal osteoporotic lesions, and for treating same. Clinical studies have shown that ASP increases the osteogenic gene opening and mineral accumulation of normal human BMSCs. Another study found that when BMSCs of mice were transplanted subcutaneously in nude mice, the bone formation ability of the ASP intervention group was significantly increased, when compared with the control group, indicating that ASP promotes bone formation and mitigates osteoporosis [10]. Alkaline phosphatase (ALP) is an important marker enzyme for differentiation of BMSCs. It is present in the cytoplasm of mature and active osteoblasts. The higher the activity of ALP, the greater the degree of osteogenic differentiation [11]. In this study, it was found that the secretion of ALP was markedly higher in BMSCs treated with ASP at a dose of 1 mmol/L than in control after 5 days, and after 7 days, the secretion of ALP in all ASP-exposed BMSCs was markedly higher than that of control. These results suggest that low-dose ASP increases ALP activity. Calcified nodule is used as an important indicator of osteogenic properties. Type I collagen is produced in osteoblasts and appears in the form of collagen. Type 1 collagen is rapidly cleaved in the matrix, resulting in the release of C-terminal and N-terminal peptides, thereby producing mature Type I collagen, which is one of the important components of bone organic matter. The results of this study revealed that the population and area of calcium nodules were markedly higher in the BMSCs of the ASP dose group than in control, and the expression was most significant when the ASP dose was 1 mmol/L. This suggests that low-dose ASP significantly promotes calcium salt deposition. Clinical studies have shown that OPG suppressed differentiation of osteoclast precursor cells and mature osteoclasts to form bone resorption pockets, and promoted osteoclast apoptosis. Moreover, it hindered osteoclastmediated bone resorption and improved cortical bone and cancellous bone density [12]. Animal studies have shown that after knocking out the OPG gene in mice, the mice suffered from severe osteoporosis, and that excessively increased levels of OPG expression in mice resulted in severe osteoporosis [13]. Therefore, many scholars believe that OPG can be used as a physiological inhibitor of osteoclast differentiation and function, which is beneficial for maintenance of balance between bone resorption and bone formation. Many studies have shown that the expression of OPG in bone tissue is closely related to OPG/RANKL/RANK pathways mediated by the maturation and activation of osteoblasts, and is key to the onset of postmenopausal osteoporosis [14]. Clinical studies have shown that after menopause, there are many RANKs on the surface of osteoclasts which can be combined with RANKL to activate osteoclasts, eventually leading to a "high absorption" state [15,16]. This study showed that the OPG protein expression level and OPG/RANKL ratio were markedly elevated in the BMSCs of the ASP dose group, relative to control, while the RANKL protein expression level was markedly lower. Moreover, ASP at a dose of 1 mmol/L ASP was more effective than at any other dose, suggesting that it can induce BMSCs to release OPG, competitively bind RANKL to block RANKL/RANK binding, and ultimately inhibit BMSCs activation and reduce bone resorption. This study has shown that lowdose aspirin significantly increased osteogenic differentiation and osteogenic effect in osteoporotic rats, increased the secretion of ALP, and promoted calcification, with the most significant effects produced at a dose of 1mmol/L. These effects were most likely exerted through a mechanism involving increased expression level of OPG, resulting in inhibition of the RANKL/RANK interaction, which in turn affected the OPG/RANKL/RANK axis. This research provides scientific basis for the innovative application of ASP in clinical prevention and treatment of postmenopausal osteoporosis.

DECLARATIONS
(no. Y201942167). The foundation is from the Ministry of Education in Zhejiang Province.