Collagen peptides promote osteogenesis and angiogenesis by activating the PI3K/AKT signaling pathway in mice

To the Editor: Common symptoms of osteoporosis include low bone mass and deteriorating bone microarchitecture, leading to bone fragility and fractures. Indeed, bone is a complex tissue composed of collagen fibers, vasculature, and numerous specialized cells. Both cellular metabolism and cellular behavior in bone are regulated by the extracellular matrix (ECM). As the main ECM in the skeletal system, collagen hydrolysates have great potential in alleviating osteoporosis by promoting osteogenesis and angiogenesis. However, the composition and structure of collagen hydrolysate are complex. The peptides in collagen hydrolysates have different sequences and spatial structures, resulting in diverse biological activities.[1] Furthermore, recent studies have focused primarily on alterations in bone metabolism and the validation of specific gene pathways. Gene regulatory mechanism analysis is still scarce.[2] This work investigated the key peptide sequences in the prepared collagen hydrolysate (CH) responsible for osteogenic and angiogenic activities and their molecular mechanisms. Additionally, the protective or restorative effects of CH in osteoporotic mice and the regulation of the CH on osteoporosis-related genes were systematically studied. To characterize the in vitro osteogenic activity of the CH, we assessed the proliferation, differentiation, and mineralization potential of MC3T3-E1 cells using cell counting kit-8 (CCK-8) assay, alkaline phosphatase activity kit, and alizarin red staining assay, respectively. The impact of CH on EA.hy926 cells was characterized using cell migration assays and Matrigel culture assays. Subsequently, we identified the peptide sequences within the CH and utilized molecular docking analysis to elucidate the molecular mechanisms of action of the key peptides. Furthermore, we conducted animal experiments and used the micro-computed tomography (micro-CT) and hematoxylin-eosin staining to verify the impact of the CH on osteoporotic mice. The animal experimental protocols were approved by the Institutional Animal Care and Use Committee at Beijing TianTan Hospital, Capital Medical University (No. 202102002). Finally, the polymerase chain reaction (PCR) array chips were used to characterize gene expression in the serum of mice. Detailed information for all methods is provided in the Supplementary File, https://links.lww.com/CM9/C290. The CH simultaneously stimulated osteoblast proliferation, differentiation, and mineralization [Supplementary Figure 1, https://links.lww.com/CM9/C290] as well as endothelial cell proliferation and migration [Supplementary Figure 2, https://links.lww.com/CM9/C290]. These findings suggest that some active peptides in the CH might have osteogenic and angiogenic activities. Investigating the molecular mechanism of these peptides via molecular docking technology is crucial. HpepDock software (http://huanglab.phys.hust.edu.cn/hpepdock) excels in protein–peptide interactions.[3] AlphaFold2 (https://alphafold.com/) can accurately calculate the 3D structure of peptides, enhancing the accuracy of docking results. The activation of the epidermal growth factor receptor (EGFR) pathway via combination with bioactive peptides might be considered to enhance bone regeneration by promoting osteogenesis and angiogenesis. So we docked the major active peptides in the CH with EGFR using the above software, and 38 peptides in the CH were identified [Supplementary Table 1, https://links.lww.com/CM9/C290]. The analysis of these peptide sequences revealed that the 38 peptides could be condensed into 19 peptides, disregarding modifications such as hydroxylation [Supplementary Table 2, https://links.lww.com/CM9/C290], and their antiosteoporosis activity potential was also screened via molecular docking [Supplementary Table 3, https://links.lww.com/CM9/C290]. Two peptides, Pep-1 (RGPPGPMGPPGLAGPPGE) and Pep-2 (AGPPGPTGPAGPPGFPGAV), strongly bind to EGFR, indicating their potential role in antiosteoporosis activity. The key amino acid residues (Leu382, Gln384, His409, Phe412, Val417, and Ile438[4]) involved in these interactions were consistent with those known for epidermal growth factor [EGF] and EGFR [Supplementary Figure 3, https://links.lww.com/CM9/C290]. The results demonstrated that the molecular mechanism by which Pep-1 and Pep-2 promoted osteoblast proliferation might be similar to that of EGF. Despite technical limitations in fully restoring natural peptide and protein conformations, peptides in the CH obtained by molecular docking that have good interactions with EGFR may be potential anti-osteoporosis peptides. We further investigated whether the CH affects osteoporosis in mice. An ovariectomy (OVX)-induced osteoporosis model revealed that osteoporosis caused fat accumulation and weight gain in mice. The CH tended to alleviate the weight gain of the mice caused by osteoporosis to a certain extent [Supplementary Figure 4A, https://links.lww.com/CM9/C290]. Compared with the sham group, the model group experienced a substantial increase in alkaline phosphatase (ALP) serum levels. Bone synthesis and bone destruction are abnormally activated in mice after OVX, leading to the overexpression of ALP. As expected, a significant decrease in ALP activity occurred in the presence of the CH [Supplementary Figure 4B, https://links.lww.com/CM9/C290]. The CH alleviated the high level of ALP in the serum of mice caused by osteoporosis. Bone mass and bone microarchitecture reflect the global quality of bone. Micro-CT scanning and hematoxylin–eosin staining results demonstrated that the CH could reverse the OVX-induced deterioration of bone microarchitecture, as shown by the increase in the bone surface area, percent bone volume, and trabecular number and the decrease in trabecular separation [Figure 1]. Notably, the antistrophic nature of bone tissue and the mechanical qualities of bone tissue samples from small animals severely reduce the precision of the findings. We confirmed the dose-dependent antiosteoporotic activity of the CH in osteoporotic mice on the basis of valid femoral and tibial bone microarchitecture data. Detailed description of results is provided in the Supplementary File, https://links.lww.com/CM9/C290.Figure 1: Microarchitecture of right femur of OVX mice with various treatments. (A–E) Representative 3D images of micro-CT based trabecular bone of femur at 6 weeks after CH induction. Quantitative analysis of the (F) BS, (G) percent bone volume (BV/TV), (H) trabecular number (Tb. N), and the (I) trabecular spacing (Tb. Sp) in each group. * P <0.05. (J) Representative H&E staining images showed osteoporosis. BV/TV: Percent bone volume; BS: Bone surface; CH: Collagen hydrolysate; H&E: Hematoxylin–eosin; micro-CT: Micro-computed tomography; OVX: Ovariectomy; Tb.N: Trabecular number; Tb.Sp: Trabecular separation; Tb.Th: Trabecular thickness.To elucidate the anti-osteoporosis mechanisms of the CH, we sought to study the gene profiles [Supplementary Table 4, https://links.lww.com/CM9/C290]. In-depth analysis of the differentially expressed genes in osteoporotic mice after the CH intervention helps to elucidate the underlying mechanism. The therapeutic effect of the CH on OVX-induced osteoporosis in mice was supported by the finding that the CH treatment restored abnormal gene expression in osteoporotic mice [Supplementary Figure 5, https://links.lww.com/CM9/C290]. The network schematic resulting from the summary of these related pathways is shown in Supplementary Figure 5C, https://links.lww.com/CM9/C290. The key growth factors involved include ephrin-A5 (EFNA5), hepatocyte growth factor (HGF), fibroblast growth factor (FGF), and transforming growth factor-α (TGF-α). EFNA5 enhances hematopoietic stem/progenitor cell migration and adhesion. HGF, which is integral to embryogenesis, wound repair, vasculogenesis, and tissue/organ regeneration and morphogenesis, exerts its effects through paracrine/autocrine signaling and epithelial–mesenchymal interactions. Therefore, EFNA5 and HGF are implicated in promoting angiogenesis. FGF can regulate chondrocyte proliferation and differentiation. TGF-α can stimulate osteoclast formation and bone resorption. Type IV collagen alpha chain (COL4A) and osteopontin (OPN) are two genes capable of synthesizing the ECM. COL4A can promote collagen synthesis. OPN inhibits the development of osteoporosis by regulating the expression of hyaluronic acid, type II collagen, proteoglycans, and other cartilage matrix components. Focal adhesion kinase (FAK) is essential for cell adhesion, migration, proliferation, and differentiation. Studies have shown that reduced FAK activity results in H-type angiodysplasia and high susceptibility to osteoporosis.[5] All of the above genes can directly or indirectly activate the phosphatidylinositol-3 kinase (PI3K)/protein kinase B (AKT) pathway, promoting bone repair and bone angiogenesis.[6] These results suggested that the CH could alter osteoporosis by activating the PI3K/AKT pathway in OVX mice through multiple pathways. Oral administration of the CH to mice results in digestion, absorption, and first-pass metabolism, but the specific bioactive peptides involved in osteogenesis and angiogenesis are unknown. Despite reports that some long peptides are directly absorbed,[7] identifying these peptides in mouse serum is crucial. In summary, the present study demonstrated that the CH could attenuate osteoporosis by enhancing bone quality, reducing osteoporotic bone loss, and promoting angiogenesis. The two novel peptides with osteogenic activity and angiogenesis in the CH were screened according to their interaction with EGFR, and the bioactivities of the peptides were demonstrated. The genomics data suggest that the possible mechanism of the anti-osteoporosis effect of the CH containing the above active peptides may involve the activation of the PI3K/AKT signaling pathway through the regulation of genes related to different growth factors and ECM synthesis, thereby promoting bone formation and angiogenesis. Further investigations of the CH, incorporating the identified bioactive peptides as potential nutraceuticals for osteopathic conditions, are warranted. Consequently, additional rigorous clinical trials are necessary to substantiate the findings and validate the hypotheses presented herein. Funding This work was supported by grants from the Fundamental Research Funds for the Central Universities (No. JD2119) and the National Natural Science Foundation of China (No. 82102038). Conflicts of interest None.