背景:尽管骨形态发生蛋白(BMP)相关的骨形成疗法具有很好的临床潜力,它们的副作用保证了对替代治疗肽的需求。BMP家族成员可以帮助骨修复;然而,来自BMP2/4的肽尚未被研究。
方法:在本研究中,鉴定了三种候选BMP2/4共有肽(BCP)1、2和3,并分析了它们在C2C12细胞中诱导成骨的能力。首先,进行碱性磷酸酶(ALP)染色测定以评估BCP的成骨作用。接下来,探讨了BCP对成骨标志物RNA表达水平和蛋白质丰度的影响。此外,通过BCP1和对BMPIA型受体(BRIA)的计算机分子对接模型访问了ALP的转录活性。
结果:BCP1-3诱导的RUNX2表达高于BMP2。有趣的是,其中,在ALP染色中,BCP1比BMP2显著促进成骨细胞分化,无细胞毒性。BCP1显著诱导成骨细胞标志物,与其他浓度相比,在100ng/mL时观察到最高的RUNX2表达。在转染实验中,BCP1通过RUNX2激活和Smad信号通路刺激成骨细胞分化。最后,计算机分子对接表明BCP1在BRIA上可能的结合位点。
结论:这些结果表明BCP1促进C2C12细胞中的成骨性。这项研究表明,BCP1是替代BMP2用于成骨细胞分化的最有希望的候选肽。
BACKGROUND: Despite the promising clinical potential of bone morphogenetic protein (BMP)-related therapies for bone formation, their side effects warrant the need for alternative therapeutic peptides. BMP family members can aid in bone repair; however, peptides derived from BMP2/ 4 have not yet been investigated.
METHODS: In this study, three candidates BMP2/4
consensus peptide (BCP) 1, 2, and 3 were identified and their ability to induce osteogenesis in C2C12 cells was analyzed. First, an alkaline phosphatase (ALP) staining assay was performed to evaluate the osteogenic effects of BCPs. Next, the effects of BCPs on RNA expression levels and protein abundances of osteogenic markers were explored. Furthermore, the transcriptional activity of ALP by BCP1 and in silico molecular docking model on BMP type IA receptor (BRIA) were performed.
RESULTS: BCP1-3 induced higher RUNX2 expression than BMP2. Interestingly, among them, BCP1 significantly promoted osteoblast differentiation more than BMP2 in ALP staining with no cytotoxicity. BCP1 significantly induced the osteoblast markers, and the highest RUNX2 expression was observed at 100 ng/mL compared to other concentrations. In transfection experiments, BCP1 stimulated osteoblast differentiation via RUNX2 activation and the Smad signaling pathway. Finally, in silico molecular docking suggested the possible binding sites of BCP1 on BRIA.
CONCLUSIONS: These results show that BCP1 promotes osteogenicity in C2C12 cells. This study suggests that BCP1 is the most promising candidate peptide to replace BMP2 for osteoblast differentiation.