Collagen Peptides Promote Osteogenesis and Angiogenesis by Activating the PI3K/AKT Signaling Pathway in Mice
Osteoporosis, characterized by reduced bone mass and disrupted bone microarchitecture, increases fracture risk and compromises skeletal integrity. Bone tissue comprises collagen fibers, vascular networks, and specialized cells whose behaviors are regulated by the extracellular matrix (ECM). Collagen hydrolysates (CH), derived from the ECM, have shown promise in mitigating osteoporosis by enhancing osteogenesis and angiogenesis. However, the structural complexity of CH—comprising diverse peptide sequences with varying biological activities—has hindered the identification of specific bioactive components and their mechanisms of action. This study elucidates the osteogenic and angiogenic peptides within CH, their molecular interactions, and their therapeutic effects in a murine osteoporosis model, while uncovering the underlying gene regulatory pathways.
In Vitro Osteogenic and Angiogenic Activities of CH
The osteogenic potential of CH was evaluated using MC3T3-E1 pre-osteoblasts. Cell proliferation, assessed via the CCK-8 assay, demonstrated a dose-dependent increase in viability upon CH treatment. Alkaline phosphatase (ALP) activity, a marker of early osteogenic differentiation, rose significantly in CH-treated cells compared to controls. Mineralization, visualized through alizarin red staining, revealed enhanced calcium deposition in CH groups, confirming late-stage osteoblast maturation.
Parallel experiments on EA.hy926 endothelial cells highlighted CH’s angiogenic properties. A cell migration assay showed that CH stimulated endothelial cell movement in a scratch wound model, while Matrigel tube formation assays revealed improved vascular network complexity. These findings indicate that CH contains peptides capable of dual stimulation—promoting both bone formation and blood vessel development.
Identification of Key Bioactive Peptides
To pinpoint active peptides, CH was analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), identifying 38 candidate peptides. Molecular docking studies focused on interactions between these peptides and epidermal growth factor receptor (EGFR), a key regulator of osteogenesis and angiogenesis. Two peptides—Pep-1 (RGPPGPMGPPGLAGPPGE) and Pep-2 (AGPPGPTGPAGPPGFPGAV)—exhibited strong binding affinity to EGFR, mimicking the interaction of natural EGFR ligands like epidermal growth factor (EGF). Critical residues (Leu382, Gln384, His409, Phe412, Val417, and Ile438) involved in EGF-EGFR binding were also engaged by Pep-1 and Pep-2, suggesting shared activation mechanisms.
CH Reverses Osteoporosis in Ovariectomized Mice
An ovariectomy (OVX)-induced osteoporosis model was employed to evaluate CH’s therapeutic efficacy. OVX mice exhibited elevated serum ALP levels (32.4 ± 3.1 U/L vs. sham 18.7 ± 2.5 U/L), reflecting aberrant bone turnover. CH treatment reduced ALP activity dose-dependently (25.6 ± 2.8 U/L at 100 mg/kg; 21.3 ± 2.4 U/L at 200 mg/kg), indicating normalized bone metabolism. Micro-CT analysis of femurs revealed that CH restored trabecular bone microarchitecture: bone surface area (BS) increased by 35%, bone volume/tissue volume (BV/TV) by 28%, and trabecular number (Tb.N) by 22%, while trabecular spacing (Tb.Sp) decreased by 18% compared to untreated OVX mice. Hematoxylin-eosin (H&E) staining corroborated these findings, showing reduced marrow adiposity and improved trabecular connectivity in CH groups.
Genomic Insights into CH’s Mechanism
PCR array analysis of murine serum identified 32 differentially expressed genes (DEGs) in OVX mice, 18 of which were normalized by CH. Key upregulated genes included EFNA5 (ephrin-A5), which promotes hematopoietic stem cell migration; HGF (hepatocyte growth factor), involved in tissue repair and angiogenesis; and FGF (fibroblast growth factor), regulating chondrocyte activity. Downregulated genes like TGF-α (transforming growth factor-alpha), a bone resorption stimulator, were suppressed by CH. Additionally, COL4A (collagen IV alpha chain) and OPN (osteopontin), critical for ECM synthesis, were upregulated, enhancing collagen deposition and matrix stability.
Network analysis linked these genes to the PI3K/AKT pathway, a central regulator of cell survival, proliferation, and angiogenesis. CH increased phosphorylation of PI3K and AKT in bone tissue, confirming pathway activation. This signaling cascade likely mediates CH’s dual effects: promoting osteoblast differentiation via RUNX2 and OSX upregulation while stimulating endothelial cell migration through VEGF and Ang-1 expression.
Molecular Mechanisms of CH-Derived Peptides
The EGFR-PI3K/AKT axis emerged as a pivotal mechanism. Pep-1 and Pep-2 binding to EGFR triggers receptor dimerization and autophosphorylation, activating downstream PI3K/AKT signaling. This pathway enhances osteoblast proliferation (via cyclin D1 upregulation) and inhibits apoptosis (by suppressing caspase-3). Concurrently, PI3K/AKT activation in endothelial cells elevates nitric oxide synthase (eNOS) activity, fostering vasodilation and angiogenesis.
Therapeutic Implications and Future Directions
This study positions CH as a multifaceted therapeutic agent for osteoporosis, addressing both bone loss and vascular insufficiency. The identification of Pep-1 and Pep-2 provides a foundation for developing peptide-based nutraceuticals or drugs. However, challenges remain, including optimizing peptide bioavailability and confirming efficacy in human trials. Future work should explore synergistic effects of CH with existing therapies, such as bisphosphonates, and investigate long-term safety profiles.
Conclusion
Collagen hydrolysates mitigate osteoporosis by delivering bioactive peptides that activate EGFR and downstream PI3K/AKT signaling. These peptides enhance osteoblast function, restore bone microarchitecture, and promote angiogenesis, addressing the multifactorial nature of osteoporosis. Genomic and molecular insights underscore the potential of CH as a novel therapeutic strategy, bridging ECM biology with targeted pathway modulation.
doi.org/10.1097/CM9.0000000000003458
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