Osteoporosis (OP), a condition characterized by thin and brittle bones, compromises bone strength and predisposes bones to fractures, especially the bones in the hip, spine, and wrist (Cummings and Melton, 2002; Compston et al., 2019). The prevalence of OP is on the rise, owing to the aging population, with millions of people worldwide either already having OP or being at high risk due to low bone mass (Black and Rosen, 2016; Compston et al., 2019). Studies have suggested that approximately one in two women and up to one in four men aged 50 and older will suffer a bone fracture due to OP (Sambrook and Cooper, 2006; Rachner et al., 2011; Compston et al., 2019). Although immense strides have been made in drug development, the incidence of OP is growing exponentially (Rachner et al., 2011; Compston et al., 2019). Design and development of effective drugs that can delay the pathological progress of OP have the potential to revolutionize healthcare provision.

Owing to their low toxicity, natural active compounds of a plant origin are attracting attention (Zhu et al., 2018; Suroowan and Mahomoodally, 2019). Polydatin (POL, 3, 4, 5-trihydroxystibene-3-β-mono-D-glucoside), a stilbenoid compound obtained from the root of Polygonum cuspidatum, has a long history of use as a Chinese traditional medicine in a wide array of diseases (Chen et al., 2013; Jiang et al., 2013; Huang et al., 2015; Mele et al., 2018). Polydatin (POL) has been reported to enhance the anti-oxidant ability of bone marrow stromal cells (BMSCs) and to induce bone remodeling (Chen et al., 2019). A recent study reported that POL has anti-OP activity in ovariectomized mice (Shen et al., 2020). However, to the best of our knowledge, the mechanism of POL’s anti-OP activity remains elusive and requires further investigation.

Bioinformatic analyses have been widely applied in the elucidation of potential molecular mechanisms underlying diseases (Agarwal and Searls, 2009; Zampieri et al., 2017). In the current study, we employ a set of bioinformatic tools to identify the target genes and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways involved in POL’s mechanism of anti-OP activity. Our primary aim was to identify the potential molecular and cellular mechanism of POL in OP. We analyzed the shared KEGG pathways between POL-targeted genes and OP, and performed in vitro assays to validate our hypothesis.

Polydatin, a stilbenoid compound obtained from the root of P. cuspidatum, is believed to possess anti-osteoporotic activity (Chen et al., 2016, 2019; Zhou et al., 2016). BMSCs have the ability of self-renewal and multidirectional differentiation. They can potentially differentiate into adipocytes, osteoblasts, and chondrocytes (Yim et al., 2014; Ruiz et al., 2016). Therefore, BMSCs play a key role in the treatment of OP. As shown in previous study, POL can protect from oxidative stress and promote BMSCs migration (Chen et al., 2019). POL has also been shown to possess notable anti-OP activity via regulation of OPG, RANKL, and β-catenin (Zhou et al., 2016). In addition, a study suggested that POL may promote BMSC migration via the ERK 1/2 signaling pathways (Chen et al., 2016). However, the precise mechanism of POL’s anti-OP activity has yet to be investigated.

In this study, we identified 27 KEGG pathway shared between POL-targeted genes and OP. The top five KEGG pathways with the smallest q-values were Chemokine signaling pathway, Renal cell carcinoma, MAPK signaling pathway, Neurotrophin signaling pathway, and Pathways in cancer. The hub genes of the five signaling pathway were MAPK1, MAPK3, and MAP2K1.

By mapping the KEGG pathways related to target genes, we found that POL exerts its biological effects through regulating Classical MAP kinase pathway, JNK, and p38 MAP kinase pathway. POL-targeted genes ERK (MAPK1, MAPK3), MEK1 (MAP2K1) were involved in the above pathways. And the identified POL-targeted genes are associated with proliferation, differentiation, inflammation, cellular growth and differentiation, and cytokine production.

As is known, there are three major subfamilies of MAPK: the extracellular-signal-regulated kinases (ERK MAPK, Ras/Raf1/MEK/ERK), the c-Jun N-terminal or stress-activated protein kinases (JNK, SAPK), and p38 (Fang and Richardson, 2005; Cargnello and Roux, 2011). JNK and p38 have similar functions and are related to inflammation, apoptosis, and growth (Wagner and Nebreda, 2009). ERK is responsible for basic cell processes, including cell proliferation and differentiation (Guo et al., 2020). Several studies have suggested that the ERK-MAPK pathway can positively regulate bone development (Radio et al., 2006; Ge et al., 2007; Shim et al., 2013). At the same time, studies have shown that the p38 MAPK pathway is essential for bone production and bone homeostasis (Greenblatt et al., 2010; Weske et al., 2018). In addition, osteoclast formation and survival can be inhibited through the attenuation of JNK/c-jun and NFκB signaling (Abe et al., 2003; Krum et al., 2010).

Protein phosphorylation (PP) is a common regulatory mode in organism and plays an important role in the process of cell signal transduction (Kummer and Ban, 2021). It was widely demonstrated that PP is the most basic, universal and important mechanism for regulating and controlling protein activity and function (Jiang et al., 2021). To validate our bioinformatic results, the phosphorylation level of MAPK signaling pathway was detected. Our results indicated that POL reduced the phosphorylation levels of ERK1/2, p38α and JNK in MC3T3-E1, suggesting MAPK signaling pathway involved in the regulation of POL, which is high incidence with the bioinformatic results. In the current study, we proved that POL induces osteoblastic differentiation via suppressing the MAPK signaling pathway, and further signaling pathways involved in the protective functions of POL on OP will be verified in future studies.

Like other bioinformatic analysis, some limitations could be found in the study. On the one hand, the effect of activation or suppression of MAPK signaling on osteoblasitc differentiation was not explored in the present research. On the other hand, animal osteoporotic model was not constructed and the beneficial effect of POL on OP was nor demonstrated in vivo.

However, it is worth noting, that previous researches has identified POL as a potential activator of the Sirtuin family, which is involved in specific biological functions, including regulation of transcription, cell cycle, cell differentiation, apoptosis, anti-oxidation, and genomic stabilization (Chen and Lan, 2017; Sun et al., 2021). Sirtuins, which is highly conserved NAD+ dependent deacetylases, exist in most organisms and play a key role in promoting the health and survival (Sinclair and Guarente, 2006; Haigis and Sinclair, 2010). According to previous studies, sirtuins can regulate the lifespan of lower organisms and age-related diseases in mammals (Imai and Guarente, 2014). A study has shown that sirtuin might play an important role in the treatment of mitochondrial dysfunction, aging, and metabolic diseases (Westphal et al., 2007). As an activator of sirtuin, resveratrol can reduce oxidative stress and inflammation by acting on Akt and MAPK signaling pathways (Shin et al., 2012). A related study has reported that sirituin affects the MAPK pathway by regulating the phosphorylation of p38, JNK, and ERK (Becatti et al., 2012). Therefore, this evidence taken together with our results, allows for speculation that polydatin might alleviates osteoporosis by acting on the Sirtuin family and regulating biological processes and MAPK signaling. This highlights a potential path for subsequent research.

Our study determined that POL exhibited protective effects in OP, as evidenced by a suppression of MAPK signaling in vitro. This study identifies a promising potential candidate for the treatment of OP.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.

GL conceived and designed the study. BM and YS supervised the study. ZL, YX, and YH performed the bioinformatics analysis and experiments and wrote the manuscript. LC, WZ, and HX analyzed the data. LH and AP provided advice and technical assistance. CY and XX revised the figures and tables. All authors approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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