Background

The retraction and compression of gingival tissue have a significant impact on the efficiency and stability of orthodontic treatment, but the underlying molecular mechanism has not been fully elucidated. The aim of the current study was to investigate the effects of mechanical forces on the expression level of calreticulin (CRT), the activity of the calcineurin (CaN)/nuclear factor of activated T cells (NFAT) 3 signalling pathway, and extracellular matrix (ECM) synthesis in human gingival fibroblasts (HGFs) cultured on three-dimensional (3D) poly(lactic-co-glycolic acid) (PLGA) scaffolds and to further explore the mechanical transduction pathways that may be involved.

Materials and methods

A mechanical force of 25 g/cm² was applied to HGFs for 0, 6, 24, 48, or 72 h. The expression of CRT, CaN, NFAT3, phosphorylated NFAT3 (p-NFAT3) and type I collagen (COL-I) were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting. Subsequently, small interfering RNA (siRNA) was used to knock down the expression of CRT in HGFs, and the impacts of the applied force on the expression levels of CaN, NFAT3, p-NFAT3, and COL-I were also evaluated by RT-qPCR and western blotting.

Results

The application of mechanical force on HGFs cultured on 3D PLGA scaffolds led to a significant increases in CRT, CaN, and COL-I expression as well as a decrease in p-NFAT3 expression. However, the effects of mechanical force on CaN, p-NFAT3, and COL-I expression were reversed following downregulation of CRT and displayed a significant decrease in CaN/NFAT3 activity and COL-I synthesis.

Conclusion

This study showed that the CaN/NFAT3 signalling pathway and CRT appear to be involved in the mechanotransduction of HGFs, and downregulation of CRT inhibits COL-I synthesis potentially via the CaN/NFAT3 signalling pathway. Taken together, these findings ultimately provide novel insight into the mechanisms underlying mechanical force-induced ECM synthesis, which may be conducive to the development of targeted therapeutics to treat fibrotic diseases, including gingival fibrosis caused by orthodontic treatment.

中文翻译：

---

钙网蛋白沉默通过抑制活化 T 细胞 3 信号通路的钙调神经磷酸酶/核因子抑制三维聚乳酸-乙醇酸共聚物支架上培养的人牙龈成纤维细胞的细胞外基质合成

背景

牙龈组织的收缩和压缩对正畸治疗的效率和稳定性有显着影响，但其潜在的分子机制尚未完全阐明。本研究旨在探讨机械力对钙网蛋白（CRT）表达水平、钙调神经磷酸酶（CaN）/活化T细胞核因子（NFAT）3信号通路活性和细胞外基质的影响。在三维 (3D) 聚乳酸-乙醇酸共聚物 (PLGA) 支架上培养的人牙龈成纤维细胞 (HGF) 中的 ECM) 合成，并进一步探索可能涉及的机械转导途径。

材料和方法

^{对 HGF 施加 25 g/cm 2}的机械力0、6、24、48 或 72 小时。通过逆转录-定量聚合酶链反应 (RT-qPCR) 和蛋白质印迹法检测 CRT、CaN、NFAT3、磷酸化 NFAT3 (p-NFAT3) 和 I 型胶原蛋白 (COL-I) 的表达。随后，使用小干扰RNA（siRNA）敲除HGFs中CRT的表达，并通过RT-评估了外加力对CaN、NFAT3、p-NFAT3和COL-I表达水平的影响。 qPCR 和蛋白质印迹。

结果

对在 3D PLGA 支架上培养的 HGF 施加机械力导致 CRT、CaN 和 COL-I 表达显着增加以及 p-NFAT3 表达减少。然而，在 CRT 下调后，机械力对 CaN、p​​-NFAT3 和 COL-I 表达的影响被逆转，并且 CaN/NFAT3 活性和 COL-I 合成显着降低。

结论

该研究表明，CaN/NFAT3 信号通路和 CRT 似乎参与了 HGF 的机械转导，而 CRT 的下调可能通过 CaN/NFAT3 信号通路抑制 COL-I 的合成。总之，这些发现最终为机械力诱导的 ECM 合成机制提供了新的见解，这可能有助于开发治疗纤维化疾病的靶向疗法，包括由正畸治疗引起的牙龈纤维化。