Department of Orthopaedics, The Third Affiliated Hospital of Soochow University, Changzhou, Jiangsu, China.
Department of Orthopaedics, The Third Affiliated Hospital of Soochow University, Changzhou, Jiangsu, China.
J Chin Med Assoc. 2018 Jul;81(7):611-618. doi: 10.1016/j.jcma.2017.12.005. Epub 2018 Apr 9.
Osteoporosis is one of the bone-metabolic diseases associated with decreased bone renewal and bone mineral density. β-aminoisobutyric acid (BAIBA), a natural thymine catabolite, can reduce inflammation in skeletal muscle and alleviate hepatic endoplasmic reticulum stress. However, the roles of BAIBA in osteoblast proliferation and differentiation remain largely unknown.
The cultured MC3T3-E1 cells received various treatments in this study, including BAIBA alone, HO alone, BAIBA plus N-acetyl-l-cysteine and BAIBA plus apocynin. Cell proliferation was determined by CCK-8 assay and H-Thymidine incorporation. Cell differentiation was evaluated by determining mRNA level of differentiation makers and ALP, and ALP activity. Reactive oxygen species (ROS) were determined by DHE staining while superoxide anion level and NAD(P)H oxidase activity were determined by the lucigenin-derived chemiluminescence method. The content of hydrogen peroxide (HO) was detected using a commercial kit. The level of NOX1, NOX2 and NOX4 was determined by Western-blot or qRT-PCR.
We show that treatment of BAIBA stimulated the proliferation of MC3T3-E1 osteoprogenitor cells and enhanced the gene expression of osteoblast differentiation markers. Incubation of MC3T3-E1 cells with BAIBA evoked increases in NAD(P)H oxidase-derived reactive oxygen species (ROS). Scavenging of reactive oxygen species (N-acetyl-l-cysteine) or inhibition of NAD(P)H oxidase (apocynin) abolished the BAIBA-elicited proliferation and differentiation of MC3T3-E1 cells.
Our results provide the first evidence that BAIBA stimulates proliferation and differentiation of osteoprogenitor cells via activation of NAD(P)H oxidase/ROS signaling.
骨质疏松症是一种与骨更新和骨矿物质密度降低有关的骨代谢疾病。β-氨基异丁酸（BAIBA）是一种天然胸腺嘧啶分解产物，可减少骨骼肌炎症并减轻肝内质网应激。然而，BAIBA 在成骨细胞增殖和分化中的作用在很大程度上尚不清楚。
在这项研究中，培养的 MC3T3-E1 细胞接受了各种处理，包括单独的 BAIBA、HO、BAIBA 加 N-乙酰半胱氨酸和 BAIBA 加 apocynin。通过 CCK-8 测定和 H-胸苷掺入测定细胞增殖。通过测定分化标志物和碱性磷酸酶（ALP）的 mRNA 水平以及 ALP 活性来评估细胞分化。通过 DHE 染色测定活性氧（ROS），通过荧光素酶衍生的化学发光法测定超氧阴离子水平和 NAD（P）H 氧化酶活性。使用商业试剂盒检测过氧化氢（HO）的含量。通过 Western-blot 或 qRT-PCR 测定 NOX1、NOX2 和 NOX4 的水平。
我们表明，BAIBA 处理刺激 MC3T3-E1 成骨前体细胞的增殖，并增强成骨细胞分化标志物的基因表达。BAIBA 孵育可引起 NAD（P）H 氧化酶衍生的活性氧（ROS）增加。清除活性氧（N-乙酰半胱氨酸）或抑制 NAD（P）H 氧化酶（apocynin）可消除 BAIBA 诱导的 MC3T3-E1 细胞增殖和分化。
我们的结果首次提供了证据，表明 BAIBA 通过激活 NAD（P）H 氧化酶/ROS 信号通路刺激成骨前体细胞的增殖和分化。
