Bone is a kind of tissue remodeled constantly. Under normal circumstances, there is a balance between bone formation and bone resorption, which is essential for maintaining bone quality and compressive stress characteristic. This balance can be achieved by regulating the activity of osteoblasts and osteoclasts, which are responsible for bone formation and absorption, respectively. However, under certain pathological conditions, an imbalance between bone resorption and bone formation may occur, leading to abnormal bone remodeling and the development of bone diseases such as osteoporosis. Clinically, this manifests as age-related osteoporosis associated with bone loss and increased fat accumulation [24].
Mesenchymal stem cells which are capable to multi-directionally differentiate can be easily cultured and expanded. However, the ability of MSCs to differentiate into functional osteoblasts is still limited in vivo [25]. The main reason is the adipogenic tendency of the precursor cell can suppress the tendency of osteogenic differentiation then breaks this bidirectional balance to cause osteoporosis [26]. Once osteoblast differentiation is abnormal, it will have an important impact on bone metabolism balance. On the other hand, osteoclasts responsible for bone resorption evolve from the lineage of monocytes differentiated by hematopoietic stem cells, and their differentiation process is also regulated by a variety of factors. Multiple studies have shown that the expression of PPARγ, C/EBPα and C/EBPβ in hematopoietic cells are essential for activating osteoclast differentiation and maturation and can directly affect the osteoclastogenesis process of hematopoietic stem cells [27–29]. This indicates that adipogenic differentiation of MSCs can directly promote osteoclastogenesis and bone resorption by breaking the coupling balance between osteoblasts and osteoclasts, which in turn leads to the occurrence of osteoporosis. Therefore, promoting the osteogenic differentiation of MSCs and inhibiting their adipogenic differentiation to correct the imbalance of bone metabolism is one of the directions for the treatment of osteoporosis.
In this study, we demonstrated for the first time that HSYA enhance osteogenic effect of hBMSCs through KDM7A regulating histone demethylation in β-catenin in vitro. In vivo, HSYA can promote skeletal development and prevent the development of OVX-induced osteoporosis rats’ model in vivo.
In vitro, the results from current study showed that HSYA did not affect the viability of hBMSCs on a wide range of concentrations, indicating that HSYA had no toxicity to hBMSCs. In order to evaluate the effect of HSYA on osteoblastic differentiation of hBMSCs, as previously [30], we first investigated the role of HSYA on ALP activity, an early marker of osteoblastic differentiation. ALP hydrolyzes pyrophosphate to phosphate, which reacts with calcium to form hydroxyapatite to promote mineralization, suggesting that ALP has a huge positive impact on bone formation [31]. Our result indicated that HSYA significantly increased the activity of ALP in hBMSCs. As expected, HSYA also enhanced calcium nodule formation, a functional marker of mineralization [32]. These results showed that HSYA could promote the osteogenic ability of hBMSCs.
Next, we checked the changes of Runx2 and β-catenin in HSYA treated hBMSCs. The runt family transcription factor Runx2 is a key transcriptional regulator and plays a key role in osteoblast differentiation [33]. Osteoblast differentiation normally occurs in Runx2+OSX− mesenchymal cells, while in Runx2-deleted embryos, osteoblast differentiation is inhibited, indicating that Runx2 is a key transcription factor for osteogenesis [34]. Furthermore, Wnt/β-catenin signaling directly enhances Runx2 through both canonical and non-canonical pathways, leading to bone formation ultimately [35]. Western blotting showed HSYA increased the Runx2 and β-catenin, suggesting that HSYA might promote osteogenic differentiation of hBMSCs through mediating β-catenin. To verify that, we found luciferase activity of Wnt signaling reporter TOPflash and nucleus translocation of β-catenin were activated significantly by 10 µM HSYA, which meant β-catenin might be indicative of a key role of HSYA in promoting osteogenesis in hBMSCs.
Based on the above results, we concluded that 10 µM HSYA is the most suitable dose to promote the osteogenesis of hBMSCs. Therefore, RNA-sequencing analysis was used for the exploration of the mechanisms underlying the osteogenesis effects of HSYA. On the basis of previous report [12], we found HXTL capsule which contains Safflower mainly could promote osteogenesis of hBMSCs to ameliorate osteonecrosis of femoral head through histone demethylation in lncRNA-Miat. Therefore, we hypothesized the bone formation of HSYA is likely to be dependent on histone demethylation, and chose six KDMs orthologous genes with differential expression, coding their namesake proteins with the histone demethylase signature domain Jumonji C (JmjC), for further verification from the results of RNA-sequencing analysis. We found that KDM7A was the potential one to be targeted as its expression was significantly up-regulated in HSYA groups.
Histone lysine (K)-specific demethylase 7A (KDM7A) has been proved to remove di-methylation marks of histone H3K9 and H3K27 [36]. Previous research reported that KDM7A can suppress tumours through blocking tumours growth and angiogenesis and regulate neural differentiation and fibroblast growth factor-4 (FGF-4) expression. To clarify the precise role of KDM7A in osteogenesis of hBMSCs, we tested the effects of siRNA-KDM7A, the related markers including β-catenin were down-regulated in the absence of KDM7A through various experiments. Furthermore, HSYA can reverse this effect to some extent.
The effects of H3K27me2/H3K9me2 on regulating osteogenic differentiation are investigated in recent years, and H3K9me2 is supposed to be concluded that it has the impact on differentiation of hBMSCs [37, 38]. The cell immunofluorescence showed that HSYA could inhibit H3K27me2/H3K9me2 expression, which are the targets of KMD7A to be removed on the promoters of related genes [39]. And the CHIP assay showed that the binding ratio between H3K27me2 and promoter of β-catenin was partly blocked by HSYA interference, suggesting the osteogenesis effect of HSYA in hBMSCs is likely to be associated with histone dimethylation in promoter of β-catenin.
A recent study reports that KDM7A is a molecular regulator for adipogenic and osteogenic differentiation in stromal ST2 cell and mBMSCs line. The investigators suggest that KDM7A can inhibit osteogenesis through epigenetic control of C/EBPα and canonical Wnt signaling [40]. However, our results are in stark contrast because we provide evidence from knockdown of KDM7A that it is a positive factor on osteogenesis in vitro in hBMSCs. Under the condition that HSYA can enhance osteogenetic differentiation of hBMSCs, KDM7A was verified to be one potential mediator and contributor. Moreover, we showed that the H3K9m2 and H3K27m2 can be influenced by HSYA. There exist two main reasons can explain the differences: the KDM7A expression has not reached the peak until 8 days either in primary BMSCs-osteogenetic or ST2- osteogenetic, comparing with the peak expression of KDM7A in ST2-Adipogenic in 3 day. However, in subsequent experiments of that reports, the related genes and proteins markers were test within 3 days, which forms the contrast to ours (7 day). So, the timing of termination of culture and collection of cell samples may be one reason. In addition, the cell lines and vitro differentiation conditions are likely to be the causes of this opposite results as these two reports [41, 42].
Based on the vitro results, we further evaluated overall the biological function in accelerated mineralization of the bone and osteogenesis of HSYA through Chick Embryos. This was clearly demonstrated by the extent of calcification of the spine, radius, metatarsus and femur, which proved the promotion effect of HSYA on bone development, and in accordance with osteogenic ability of HSYA in hBMSCs basing on the theory of Cartilaginous Internalized Bone. Finally, we established an OVX rat model to further investigate whether HSYA has potential therapeutic effect in vivo. We can conclude that HSYA exhibit a remarkable protective effect on OVX-induced bone loss in a rat model as confirmed by micro-CT and histological and immunohistochemical analyses, and the relevant results were consistent with the in vitro study.