Apoptosis and Inflammation Involved with Fluoride-Induced Bone Injuries.

Fluoride affects bone metabolism by intervening with osteoblast and osteoclast activity, and fluoride exposure also triggers chondrocyte degradation [109,110]. To shed light on the specific mechanisms of fluoride-induced bone injuries, we discovered that the apoptosis of osteoblasts and the inflammation of osteoclasts and chondrocytes were involved in fluoride-induced bone injuries. Additionally, the Col1a1, Bcl2, Fgfr1, Mmp9, Mmp13, Bmp2, and Bmp7 genes were identified as the key regulatory factors of fluoride-induced bone injuries (Figure 6).

In this study, a total of 112, 35, and 41 DEGs were obtained in the identified 63 osteoblast studies, 16 osteoclast studies, and 14 chondrocyte studies, respectively. According to the KEGG enrichment analysis in osteoblasts, the PI3K-Akt signaling pathway was significantly enriched. The PI3K-Akt signaling pathway was closely associated with bone metabolism [111,112,113]. Studies demonstrated that the PI3K-Akt signaling pathway participated in the excessive proliferation and differentiation of osteoblasts in rats [86]. Combined with the TF–mRNA–miRNA regulatory network, the Col1a1, Bcl2, and Fgfr1 genes were identified as the key genes for bone formation, which was regulated by the PI3K-Akt signaling pathway (Figure S1). Col1a1 is a hydrophilic protein that belongs to the collagen family and is closely associated with osteogenesis imperfecta, osteoporosis, and other skeletal injuries [114,115,116]. The LINC00313/miR-218-5p/COL1A1 axis contributed to osimertinib resistance through the PI3K-Akt signaling pathway, confirming that Col1a1 is likely to be the upstream gene for the PI3K-Akt signaling pathway [117]. Additionally, the overexpression of Fgfr1 promoted the activation of the PI3K-Akt pathway [118]. Fgfr1 plays essential roles in osteocytes during bone remodeling, and this gene is suggested to be a potential therapeutic target for the prevention of bone loss [119]. Moreover, the low expression of Akt increased the level of the pro-apoptotic protein Bax, which failed to form heterodimers with the anti-apoptotic protein Bcl2, resulting in osteoblast apoptosis [120]. Based on these studies, fluoride may activate Col1a1, Bcl2, and Fgfr1 and regulate osteoblast apoptosis through the PI3K-Akt signaling pathway. Furthermore, selenium methyl cysteine (CTD 00000103) was predicted to be a potential drug according to the DSigDB database. Supplementation with selenium methyl cysteine increased bone mineral density [121]. Studies have demonstrated that selenium methyl cysteine protects against liver injuries by inhibiting apoptosis [122]. In the future, it could be applied for treating fluoride-induced bone injuries based on these findings.

Osteoclasts are a type of multinucleated bone-resorbing cells that originate from the myeloid lineage of hematopoietic stem cells in bone marrow [123]. In our study, GO analysis indicated that osteoclasts were responsible for bone resorption. Moreover, the Mmp13 and Mmp9 genes were mainly enriched in the IL17 signaling pathway (Figure S2). IL-17 is a proinflammatory cytokine. Recent studies have shown that IL-17 promotes osteoclast-induced bone loss by regulating glutamine-dependent energy metabolism. Moreover, IL-17 treatment increases the expression of osteoclast marker genes Mmp9 and Mmp13 [124]. The Mmp9 and Mmp13 genes are involved in the process of bone resorption through the activation and differentiation of osteoclasts [125,126]. In this study, we found that fluoride exposure activated the Mmp9 and Mmp13 genes and regulated bone resorption through the IL-17 signaling pathway. Among the top 5 candidate drugs in osteoclasts, CGS-27023A was identified as a potent matrix metalloproteinase inhibitor. It can be utilized to regulate the process of bone resorption through Mmp13 and Mmp9 [127].

Furthermore, we focused on the enrichment analysis of chondrocyte-related DEGs. GO analysis suggested that chondrocytes were significantly important for the development of the skeletal system. KEGG enrichment analysis showed that the Bmp2 and Bmp7 genes were mainly enriched in the TGF-beta signaling pathway (Figure S3). BMPs are a type of extracellular multifunctional signaling cytokine that belongs to the TGF-beta family [128]. Bmp2 and Bmp7 have been shown to induce endochondral bone formation and subsequently form long bones [129,130]. The high expression of Bmp2, along with TGF-beta1, promotes the expansion of Treg cells, leading to significant inflammation [7,131]. A large number of studies has also confirmed that fluoride exposure leads to an inflammatory response [132,133,134]. Moreover, the PPI analysis identified several hub proteins related to inflammation, such as Akt, Il6, Runx2, Bcl2, and Beclin1 [135,136,137,138]. Studies have shown that TGF-beta degrades the extracellular matrix, induces chondrocyte differentiation, and even leads to bone necrosis [139,140]. According to the analysis of candidate drugs, calcium phosphate is supposed to be used for inhibiting bone necrosis. Studies have indicated that calcium phosphate could be used in bone grafts due to its composition, supporting our findings [141].

Overall, the above findings provide promising insights for skeletal fluorosis. However, our study still has certain limitations. The specific regulatory mechanisms of the PI3K-Akt signaling pathway, IL-17 signaling pathway, and TGF-beta signaling pathway in bone disorders’ exposure to fluoride are needed to be further verified via in vivo and in vitro experiments.

In conclusion, the PI3K-Akt signaling pathway is involved in the apoptosis of osteoblasts and the IL-17 and TGF-beta signaling pathways are involved in the inflammation of osteoclasts and chondrocytes, playing a part in the process of fluoride-induced bone injuries. The Col1a1, Bcl2, Fgfr1, Mmp9, Mmp13, Bmp2, and Bmp7 genes are the key regulatory factors in fluorosis bone metabolism.