METHODS: Preosteoblast cells (MC3T3-E1), bone marrow-derived stromal cells (ST2), and a direct 2D co-culture system were treated with BDNF. The effect of BDNF on cell proliferation was determined using the CCK-8 assay. Osteoblast differentiation was assessed based on alkaline phosphatase (ALP) activity and staining and the protein expression of multiple osteoblast markers. Calcium accumulation was examined by Alizarin red S staining. For the animal study, we used ovariectomized Sprague-Dawley rats and divided them into BDNF and normal saline injection groups. MicroCT, hematoxylin and eosin (H&E), and tartrate-resistant acid phosphatase (TRAP) stain were performed for analysis.
RESULTS: BDNF significantly increased ALP activity, calcium deposition, and the expression of osteoblast differentiation-related proteins, such as ALP, osteopontin, etc., in both ST-2 and the MC3T3-E1 and ST-2 co-culture systems. Moreover, the effect of BDNF on osteogenic differentiation was diminished by blocking tropomyosin receptor kinase B, as well as inhibiting c-Jun N-terminal kinase and p38 MAPK signals. Although the animal study results including bone density and histology showed increased osteoblastic and decreased osteoclastic activity, only a portion of parameters reached statistical significance.
CONCLUSIONS: Our study results showed that BDNF affects osteoblast differentiation through TrkB receptor, and JNK and p38 MAPK signal pathways. Although not statistically significant, the trend of such effects was observed in the animal experiment.
方法:前成骨细胞(MC3T3-E1),骨髓来源的基质细胞(ST2),和直接2D共培养系统用BDNF处理。使用CCK-8测定法测定BDNF对细胞增殖的影响。基于碱性磷酸酶(ALP)活性和染色以及多种成骨细胞标志物的蛋白表达来评估成骨细胞分化。通过茜素红S染色检查钙积累。对于动物研究,我们使用去卵巢的Sprague-Dawley大鼠,并将其分为BDNF和生理盐水注射组。MicroCT,苏木精和曙红(H&E),和抗酒石酸酸性磷酸酶(TRAP)染色进行分析。
结果:BDNF显著增加ALP活性,钙沉积,成骨细胞分化相关蛋白的表达,如ALP,骨桥蛋白,等。,在ST-2和MC3T3-E1和ST-2共培养系统中。此外,BDNF对成骨分化的影响通过阻断原肌球蛋白受体激酶B减弱,以及抑制c-JunN末端激酶和p38MAPK信号。尽管包括骨密度和组织学在内的动物研究结果显示成骨细胞活动增加,破骨细胞活动减少,只有一部分参数达到统计学意义。
结论:我们的研究结果表明,BDNF通过TrkB受体影响成骨细胞的分化,以及JNK和p38MAPK信号通路。虽然没有统计学意义,在动物实验中观察到这种效应的趋势。