循环拉伸和 TNF-α 联合作用对 MC3T3-E1 细胞成骨分化的影响。

Combined effects of cyclic stretch and TNF-α on the osteogenic differentiation in MC3T3-E1 cells.

机构信息

Shanxi Medical University School and Hospital of Stomatology, Shanxi Province Key Laboratory of Oral Diseases Prevention and New Materials, Taiyuan 030001, China.

出版信息

Arch Oral Biol. 2021 Oct;130:105222. doi: 10.1016/j.archoralbio.2021.105222. Epub 2021 Jul 31.

DOI:10.1016/j.archoralbio.2021.105222

PMID:34358809

Abstract

OBJECTIVE

The study aimed to investigate the combined effects of cyclic stretch and tumor necrosis factor-alpha (TNF-α) on the osteogenic differentiation of MC3T3-E1 cells and the role of the nuclear factor-kappaB (NF-κB) signaling pathway in this process.

DESIGN

MC3T3-E1 cells were treated with TNF-α (0.5 and 10 ng/mL) and cyclically stretched using the Flexcell tension system 4000 with 12 % elongation for 12 h. Furthermore, to explore which cytokines might be regulated by the NF-κB signaling pathway in osteogenic differentiation, the cells were pre-treated with NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), and then cyclically stretched for 12 h in the presence of 10 ng/mL of TNF-α. RT-PCR and western blot were utilized to detect the expression of type Ⅰ collagen (COL1), osteocalcin (OCN), runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), osteoprotegerin (OPG), receptor activator of NF-κB ligand (RANKL), NF-κB, and phosphorylated NF-κB (p-NF-κB) at gene and protein levels.

RESULTS

Cyclic stretch alone increased the expression of COL1, OCN, Runx2, ALP, and OPG, decreasing the expression of RANKL and the RANKL/OPG ratio. The upregulation or downregulation induced by cyclic stretch were restrained in the presence of TNF-α. The p-NF-κB/NF-κB ratio increased at any stimulation. Inhibition of NF-κB signaling pathway restrained the expression variations of COL1, OCN, ALP, OPG, and RANKL induced by TNF-α combined with cyclic stretch.

CONCLUSION

The results indicated that TNF-α inhibited the osteogenic differentiation of MC3T3-E1 cells induced by cyclic stretch and NF-κB signaling pathway might play a role in this process.

摘要

目的

本研究旨在探讨循环拉伸和肿瘤坏死因子-α（TNF-α）联合作用对 MC3T3-E1 细胞成骨分化的影响，以及核因子-κB（NF-κB）信号通路在此过程中的作用。

设计

采用 Flexcell 张力系统 4000 以 12%的伸长率对 MC3T3-E1 细胞进行 12 h 的循环拉伸，并分别用 0.5 和 10ng/mL TNF-α处理；同时，为了探讨 NF-κB 信号通路在成骨分化过程中可能调节哪些细胞因子，先用 NF-κB 抑制剂吡咯烷二硫代氨基甲酸盐（PDTC）预处理细胞，然后在存在 10ng/mL TNF-α的情况下进行 12 h 的循环拉伸。采用 RT-PCR 和 Western blot 检测Ⅰ型胶原（COL1）、骨钙素（OCN）、 runt 相关转录因子 2（Runx2）、碱性磷酸酶（ALP）、骨保护素（OPG）、核因子-κB 受体激活剂配体（RANKL）、NF-κB 和磷酸化 NF-κB（p-NF-κB）在基因和蛋白水平的表达。

结果

单独的循环拉伸增加了 COL1、OCN、Runx2、ALP 和 OPG 的表达，降低了 RANKL 的表达和 RANKL/OPG 比值。TNF-α存在时，循环拉伸引起的上调或下调受到抑制。任何刺激下 p-NF-κB/NF-κB 比值均增加。NF-κB 信号通路的抑制抑制了 TNF-α联合循环拉伸诱导的 COL1、OCN、ALP、OPG 和 RANKL 表达的变化。