Systemic osteoprotective effects of Epimedii Folium and Ligustri Lucidi Fructus in senile osteoporosis rats by promoting the osteoblastogenesis and osteoclastogenesis based on MLP-ANN model

Osteoporosis (OP) is a systemic disease characterized by low bone mass and disruption of bone microarchitecture, resulting in increased bone fragility and prone to fracture [1]. Fractures resulting from OP become increasingly common in women after age 55 years and men after age 65 years, resulting in the increase in bone-associated morbidity, mortality and health-care costs [2]. With the aging of the population structure, the incidence of senile osteoporosis (SOP) is on the rise, which has become an increasingly serious health problem in the world [3]. The inhibition of both osteoblastic bone formation and osteoclastic bone resorption with age, and the prevalent bone resorption that overpasses bone formation is the fundamental pathogenesis in SOP [4, 5]. At present, the treatment of SOP, as same as OP, mainly relies on calcium supplementation, inhibition of bone resorption and promotion of bone formation [6]. However, several issues can interfere with the effectiveness of anti-SOP drugs in clinical practice, such as poor adherence to therapy and safety in long-term treatment, especially for elderly patients with liver and kidney dysfunction [7]. More importantly, these drugs target osteoblastic bone formation or osteoclastic bone resorption only and nearly no drugs targeting the both [8], which will effectively reduce the bone loss with fewer side effects. Therefore, it is necessary to find new effective drugs to overcome these problems in pursuit of better effect.

Herbal medicines have been prescribed in treating SOP for a long time, and numerous investigations confirmed some herbal medicines with bone protective effects (including natural compounds and marketed prescriptions). These findings indicate that herbal medicines can affect both bone formation and bone resorption [4]. Traditional Chinese medicine (TCM) believes that SOP is mainly caused by Shen (Kidney) deficiency. With the increasing of age, weak body, Shen deficiency, bone marrow loss and bone dystrophy are the main pathogenesis of SOP. Thus, the treatment of SOP accordingly follows the rule of tonifying Shen. Many herbs have been proved the function of Shen-tonify activities in TCM formulas for preventing and treating OP with lower toxicity compared with chemical synthetic drugs [9]. Among them, Epimedii Folium (EF) with the function of replenishing Shen-yang and Ligustri Lucidi Fructus (LLF) of replenishing Shen-yin are commonly used in the clinical treatment of SOP [10]. The combined formula of EF and LLF (EF&LLF) has been applied to treat OP for almost 50 years by Professor Shizeng Li and authorized by the State Patent Office of China (Patent No. Zl201410037992.5). Our previous researches demonstrated that EF&LLF had an anti-osteoporosis actions on ovariectomized rats [11], glucocorticoid-induced osteoporosis (GIOP) rats [12] and retinoic acid-induced osteoporosis rats [13]. Moreover, following investigations with effective components of EF&LLF, icariin and oleanolic acid have shown the effects on bone formation and bone resorption [14, 15]. However, both the direct and indirect effects of EF&LLF on SOP as well as its underlying mechanism are rarely investigated.

The balance of bone remodeling is important for maintaining bone structure and its functional activities [16], similar to the balance of yin and yang in TCM. Thus, the treatment principle of EF&LLF for SOP, restoring the balance between yin and yang to invigorate Shen and strengthen bone, is similar to rebalancing the bone remodeling. Under normal physiological conditions, bone tissue is always in the dynamic balance of osteoclasts absorbing old bone and osteoblasts forming new bone [17, 18]. With the increase of age, the activity and quantity of osteoblasts and osteoclasts decrease gradually, and the function of osteoclasts is relatively enhanced [19]. Thus an ideal drug for the treatment of SOP could stimulate both bone formation and bone resorption; more importantly, its stimulation for bone formation should be superior to that of bone resorption. The purpose of this study was to investigate whether EF&LLF could restore the bone remodeling through stimulating osteoblastogenesis and osteoclastogenesis in SOP and explore the mechanism of rebalancing bone remolding.

SOP is a systemic metabolic bone disease, and its aging process is accompanied by a variety of factors related to the homeostasis of bone remodeling. A single indicator cannot comprehensively and accurately assess the progression of SOP. Therefore, it is necessary to establish an appropriate SOP prediction model for assessing the efficacy of anti-SOP drugs. Artificial neural networks (ANNs) are complex mathematical models inspired from biological neural networks, which are capable to learn and adapt their predictive algorithms after the introduction of new information [20]. Multi-layer perception (MLP) is an important nonlinear multi-factor analysis method and one of the most widely used ANN models. In our previous study [21], a credible MLP-ANN model has been designed according to the dynamic changes of osteoporosis parameters in SOP rats aged from 6 to 17 months. In the present study, we focused on exploring the changes of the parameters related to bone remodeling in SOP rats, used the constructed MLP-ANN model to evaluate and compare the therapeutic effect of EF and LLF in single use or in combination, and provide experimental basis for the treatment of clinical SOP.

EF and LLF documented in Chinese ancient medicinal literature have strong actions of replenishing Kidney-yang and Kidney-yin, respectively. They have been used to strengthen bone and treat osteoporosis for a long time in China. Osteoporosis is a chronic and complex disease that needs long-term medication intervention. However, neither Yang-tonifying prescription with the property of warm dryness nor yin-tonifying prescription with the sticky and greasy property is suitable for long-term clinical application. According to TCM theories, the combination of EF and LLF, a Yang-tonifying herb matched with adequate a Yin-tonifying herb, can make living things freely grow, flourish, and eliminate the side effects of each other [12]. EF is a famous Chinese edible herb, and the safety associated with this herb has attracted a great deal of attention due to its potential hepatotoxic effects [38]. Liver injury caused by Chinese patent medicine preparations containing EF has been frequently reported in recent years [39]. LLF is a well-known invigorator in TCM with hepatoprotective effect. The hepatoprotective effect of oleanolic acid (mainly active ingredient of LLF) was first reported in China and it has been used to treat liver disease in humans [40]. Several recent studies have supported the association between low BMD and alcoholic or nonalcoholic fatty liver disease, confirming a low negative correlation between liver fat content and lumbar BMD in middle-aged and elderly people [41, 42], and fatty liver and BMD may be linked by IGF-1, OPG, OCN, inflammatory cytokines and other factors [43]. The production of cytokines such as IL-1, IL-6, TNF-α, and RANKL during the onset of liver injury promotes osteoclast absorption, reduces BMD, and increases the risk of fracture. Alcohol has direct and indirect effects on the number and activity of osteoblasts and osteoclasts. Alcoholic fatty liver leads to imbalance of bone remodeling and reduction of bone formation. AST is normally present in tissues and cells, but low in serum. When liver cells degenerate, the permeability of cell membrane will increase, resulting in the increase of serum AST. The content of ALT is the highest in liver, which can reflect the damage of hepatocyte parenchyma. GGT is a kind of microsomal enzyme, which mainly exists in the intrahepatic bile duct epithelium and the cytoplasm of hepatocytes. High level of liver enzymes GGT, ALT and AST are important indicators to reflect liver injury [44]. In this study, an increase in serum AST, ALT and GGT, high blood lipids and hepatic lipid accumulation had been presented in 17 m rats compared with 6 m rats, suggesting that taking drugs for SOP should prevent liver injury. LLF relieved liver injury shown by the significant decrease in serum AST and GGT, while EF regulated lipid metabolism shown by the decreased TC, TG, LDL-C and area of liver adipocyte compared with 17 m rats. With the above two functions of preventing liver injury and regulating lipid metabolism, EF&LLF is more suitable for long-term use than EF and LLF.

The negative balance of bone remodeling is the basis pathogenesis of osteoporosis. Bone metabolism in the human body is a dynamic equilibrium process that is primarily regulated by bone resorption and bone formation [37]. During bone remodeling, bone resorption initiated by osteoclasts marks the beginning of the entire bone remodeling cycle, and bone formation led by osteoblast marks the end of the cycle. ALP is used commonly and clinically as a sensitive marker to reflect bone turnover rate because it can be stable and specific to represent the activity of osteoblasts [45]. As the most characteristic enzyme of osteoclasts, TRACP can reflect the activity of osteoclasts and bone resorption in vivo by detecting TRACP in serum and bone, and play a role in predicting bone quality and reducing fracture occurrence [46, 47]. ALP/TRACP can reflect the bone remolding rate. Our findings confirmed the ALP activity, ALP/TRACP and TRAP-positive osteoclasts were decreased in 17 m aged rats, which were largely increased by EF&LLF treatment. It can be inferred that both the osteoclast activity and osteoblast activity were suppressed, and the activity of osteoblasts are more weakened than that of osteoclasts in aged rats. Thus, the uncompensated bone formation to bone resorption could lead to bone loss. The protective action of EF&LLF was related to stimulating the activity of osteoblasts and osteoclasts and maintaining the balance of bone remolding in SOP rats.

The disordered communication between osteoclasts and osteoblasts are critical mechanisms of the imbalance of bone remodeling and bone loss. The molecular mechanisms of cellular communication between osteoblast and osteoclast are one of the central issues in bone cell biology [48]. Secreted by osteoblasts, M-CSF is an important cytokine for survival, differentiation, cell migration and activity in macrophages and osteoclasts [49], can enhanced the differentiation of bone marrow precursors to osteoclast precursors [50]. Also secreted by osteoblasts, OPG can bind with RANKL and block its bind and activation with RANK to inhibit osteoclast differentiation and activation; RANKL can bind with its receptor RANK, which is expressed on osteoclastic progenitor cells, and activates the down signaling pathways related with osteoclast growth and differentiation. The ratio of OPG to RANKL critically influences the osteoclast formation [37]. Conversely, osteoclasts also influence bone formation by osteoblasts via ATP6v0d2. Secreted by osteoclasts, ATP6v0d2, as a regulator of osteoclast fusion and bone formation, is required for efficient preosteoclast fusion and mediates extracellular acidification in bone resorption [51, 52]. Wnt5a has opposing effects on bone remodeling that are dependent on the cell of origin: osteoblast-derived Wnt5a stimulate the development of osteoclasts by up-regulating the expression of RANK [53, 54]; osteoclast-derived Wnt5a promotes bone proliferation and bone formation [34]. In addition, cytokine released from the resorbed bone matrix, such as TGF-β1, BMP2 and IGF-1, also affects the activity of osteoblasts and osteoclasts. During bone formation, BMP2 exhibits strong osteosynthetic activity in vivo and in vitro [55], and conducts information between cells and interstitial cells through autocrine and paracrine regulation, regulating the differentiation and proliferation of bone cells [56]. TGF-β1 stimulates the proliferation and differentiation of mesenchymal cells, promotes the proliferation of osteoblasts, chondrocytes and the synthesis of extracellular matrix and inhibits the production and biological activity of osteoclasts [57, 58]. IGF-1, formed by paracrine and autocrine, exists widely in bone tissue. It, playing roles in bone cell function and bone metabolism, can promote osteoblast differentiation and proliferation and inhibit withering, which is beneficial to the mineralization and synthesis of bone matrix [59]. In summary, the molecular mechanisms of cellular communication between osteoblast and osteoclast are crucial for bone remodeling. The schematic presentation of the molecular mechanisms of osteoblast-osteoclast interaction was shown in Fig. 9.

In this study, we found that the protein and mRNA expression of OPG, RANKL, Wnt5a, M-CSF, ATP6v0d2, BMP2, IGF-1 and TGF-β1, and the ratio of OPG/RANKL were significantly decreased in 17 m aged rats compared with 6 m young rats, which further demonstrated both bone formation and bone resorption were suppressed, and the uncompensated bone formation to bone resorption led to bone loss in SOP. Therefore, ideal drugs of SOP can not only stimulate osteoblastogenesis and osteoclastogenesis, but also stimulate higher osteoblastogenesis than osteoclastogenesis. And these drugs can alleviate bone loss and promote the new bone formation through taking systematic effects on both bone formation and bone resorption. EF&LLF could upregulate the protein and mRNA expression of OPG, RANKL, Wnt5a, M-CSF, ATP6v0d2, BMP2, IGF-1 and TGF-β1 and increase OPG/RANKL compared with 17 m rats. These results provided reliable evidence that EF&LLF could prevent bone loss in SOP rats through systematic regulatory effects on osteoclastogenesis and osteoblastogensis so as to stimulate higher bone turnover rate. However, further research about how these molecules affect osteoblast-osteoclast interaction is needed.

Our previous study demonstrated that the degeneration of bone structure and bone metabolism in SOP rats during the aging process of rats aged from 3 to 17 months [21]. Based on the dynamic changes of the detected indexes in male rats of different ages, such as bone mass, bone biomechanics, bone microstructure, serum bone turnover markers and bone turnover rate, we has constructed a reliable and reasonable MLP model for evaluating and comparing the comprehensive effects of drugs on treating SOP. In this study, we input the relevant parameters of 6 m and 17 m group rats into the established MLP model, and found that the agreement between the experimental data and the predicted value of MLP is very high, suggesting that the model is credible. Then, we used the MLP model to evaluate the therapeutic effect of EF and LLF on SOP rats. 17 month old rats treated with EF, LLF or EF&LLF recovered to a mean value of 11.27, 11.60 and 9.77 months, respectively, confirming that EF and LLF in single use or combined use have the osteoprotective effect. In addition, EF&LLF had better effects on bone formation and bone resorption than EF or LLF.

In summary, our study provided reliable evidence that EF&LLF with fewer side effects prevented bone loss in SOP rats through taking systematic regulatory effects on osteoclastogenesis and osteoblastogensis. These findings suggested that herbal medicines might be potential resources for developing new drugs development for SOP.