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  • The Wnt signaling pathway has been demonstrated to be


    The Wnt signaling pathway has been demonstrated to be responsible for a variety of biological processes. The three best-characterized Wnt signaling pathways are the canonical Wnt pathway, the noncanonical planar cell polarity pathway, and the noncanonical Wnt/calcium pathway. The canonical Wnt/β-catenin signaling pathway is a vital factor in regulating osteoblast proliferation, differentiation, and ultimately bone formation [38,39]. Cytosolic β-catenin protein is the principal mediator of canonical Wnt signaling. In Wnt/β-catenin signaling pathway activation, β-catenin no longer degrades and accumulates in the cytoplasm; eventually, it is translocated into the nucleus to act as a transcriptional coactivator. The expression of some β-catenin targets, including runt-related transcription factor 2 (RUNX2), bone morphogenetic protein 2 (BMP-2), and BMP-4, is important for osteoblast differentiation [30]. Several animal studies have shown that Pb negatively affected bone mass and fracture repair through inhibiting the canonical Wnt pathway [4,5]. These effects may be mediated by an increase in the secretion of the Wnt signaling antagonist sclerostin in osteocytes [5]. However, the effect of Pb on Wnt signaling in osteoblast differentiation still needs to be verified. Icaritin (ICT) is a prenylflavonoid derivative from the genus Epimedium, a traditional Chinese herbal medicine that has long been used as a bone-protecting agent. ICT has a variety of beneficial biological activities, alzheimer\ including growth inhibition or apoptosis induction in a variety of cancer alzheimer\'s disease [23,32,35]; LPS-induced inflammation attenuation [34]; and neuroprotective effects [20]. Regarding bone metabolism, ICT can enhance the differentiation of osteoblasts while suppressing osteoclastic differentiation in vitro [19], and stimulate osteoblastic differentiation and inhibit adipogenesis in mesenchymal stem cells [29]. Our recent research showed that ICT can induce osteoblastic differentiation through MAPK pathways, including ERK1/2 and p38 in MC3T3 E1 subclone 14 cells [36].
    Materials and methods
    Discussion Pb is a well-known toxic environmental and air pollutant derived from anthropogenic activities, such as fuel combustion, industrial production and waste incineration. When Pb is absorbed, it is primarily accumulated in the bone and kidneys. Pb exposure has been associated with retarded skeletal growth [8], decreased trabecular bone volume and thinner growth plate cartilage in growing animals [14], impaired bone biomechanics and structure in ovariectomized rats [22], and increased bone resorption during pregnancy and lactation [18]. Despite numerous studies indicating that Pb exposure has negative effects on the bones, the underlying molecular mechanism remains poorly understood. Pb can enter cells through simple diffusion, membrane carriers or ion channels and can, in turn, accumulate in tissues and cells [12], and affect the function of a variety of cells, including those of the nervous system [27], the microvascular endothelium [31], the kidney [6], and the immune system [15]. In vitro studies showed Pb could cause apoptosis in osteoblasts [25]. Our results show that Pb that has not yet reached toxic doses has been able to inhibit the differentiation and maturation of MC3T3-E1 subclone 14 cells. The Wnt/β-catenin signaling is a critical anabolic pathway for osteoblastic bone formation. Wnt3a, an important member of the Wnt protein family, can activate the Wnt/β-catenin pathway, and its expression is associated with osteoblast differentiation [3]. Decreased Wnt3a gene expression is associated with suppression of the Wnt/β-catenin pathway and osteoblastic differentiation [24]. Dickkopf-1 (Dkk-1) is an upstream antagonist of Wnt components [26]. Increased Dkk-1 levels inactivate the Wnt/β-catenin pathway and inhibit osteoblastic differentiation [16]. Glycogen synthase kinase 3 (GSK3) plays a central role in the canonical Wnt signaling pathway and has multiple functions in cell growth and differentiation [9]. In fact, decreased levels of pGSK3β/GSK3β can induce the Wnt/β-catenin pathway inactivation [24]. β-catenin is the central molecule of the canonical Wnt pathway; it is a transcriptional activator of the T cell factor/lymphoid enhancer factor 1 (TCF/LEF-1) family of DNA binding proteins in the Wnt pathway [17]. In the absence of Wnt ligands, β-catenin is phosphorylated by GSK3β and is ubiquitylated and targeted for proteasome degradation, whereas pGSK3β can increase the cytoplasmic stabilization of β-catenin [37]. Decreased expression and translocation of β-catenin leads to decreased expression of osteoblastic genes, such as ALP, COL1 and RUNX2, in osteoblasts [24]. Several studies have also shown that Pb negatively affected bone mass and fracture repair through inhibiting the canonical Wnt pathway in animal experiments [4,5]. We also explored the Pb modulation of the Wnt/β-catenin signaling in MC3T3-E1 subclone 14 cells. Pb downregulated Wnt3a, pGSK3β and RUNX2 expression, increased Dkk-1 expression, and inhibited β-catenin expression and translocation. Our findings suggest that Pb inhibited osteoblast differentiation and inactivated canonical Wnt signaling. In another study, an environmental pollutant, aluminum trichloride, was found to have the similar effect on canonical Wnt signaling in primary rat osteoblasts [13].