Background We previously confirmed that mechanical stimulation is an important factor in the repair of tendon–bone insertion (TBI) injuries and that mechanoreceptors such as transient receptor potential ion-channel subfamily V member 4 (TRPV4; also known as transient receptor potential vanilloid 4) are key to transforming mechanical stimulation into intracellular biochemical signals. This study aims to elucidate the mechanism of mechanical stimulation regulating TRPV4. Methods Immunohistochemical staining and western blotting were used to evaluate cartilage repair at the TBI after injury. The RNA expression and protein expression of mechanoreceptors and key pathway molecules regulating cartilage proliferation were analyzed. TBI samples were collected for transcriptome sequencing to detect gene expression. Calcium-ion imaging and flow cytometry were used to evaluate the function of TPRV4 and cellular communication network factor 2 (CCN2) after the administration of siRNA, recombinant adenovirus and agonists. Results We found that treadmill training improved the quality of TBI healing and enhanced fibrochondrocyte proliferation. The transcriptome sequencing results suggested that the elevated expression of the mechanistically stimulated regulator CCN2 and the exogenous administration of recombinant human CCN2 significantly promoted TRPV4 protein expression and fibrochondrocyte proliferation. In vitro, under mechanical stimulation conditions, small interfering RNA (siRNA)-CCN2 not only inhibited the proliferation of primary fibrochondrocytes but also suppressed TRPV4 protein expression and activity. Subsequently, primary fibrochondrocytes were treated with the TRPV4 agonist GSK1016790A and the recombinant adenovirus TRPV4 (Ad-TRPV4), and GSK1016790A partially reversed the inhibitory effect of siRNA-CCN2. The phosphoinositide 3-kinase/ protein kinase B (PI3K/AKT) signaling pathway participated in the above process. Conclusions Mechanical stimulation promoted fibrochondrocyte proliferation and TBI healing by activating TRPV4 channels and the PI3K/AKT signaling pathway, and CCN2 may be a key regulatory protein in maintaining TRPV4 activation.

中文翻译：

---

机械刺激通过在肌腱-骨插入愈合过程中激活 TRPV4 信号通路促进纤维软骨细胞增殖：CCN2 起重要调节作用


背景我们之前证实，机械刺激是修复肌腱-骨插入 （TBI） 损伤的重要因素，并且瞬时受体电位离子通道亚家族 V 成员 4 （TRPV4;也称为瞬时受体电位香草醛 4） 等机械感受器是将机械刺激转化为细胞内生化信号的关键。本研究旨在阐明机械刺激调节 TRPV4 的机制。方法 采用免疫组化染色和 western blotting 评估损伤后 TBI 软骨修复情况。分析机械感受器和调节软骨增殖的关键通路分子的 RNA 表达和蛋白表达。收集 TBI 样本进行转录组测序以检测基因表达。钙离子成像和流式细胞术用于评估 siRNA 、重组腺病毒和激动剂给药后 TPRV4 和细胞通讯网络因子 2 （CCN2） 的功能。结果 我们发现跑步机训练提高了 TBI 愈合的质量并增强了纤维软骨细胞增殖。转录组测序结果表明，机械刺激调节因子 CCN2 的表达升高和重组人 CCN2 的外源给药显着促进了 TRPV4 蛋白表达和纤维软骨细胞增殖。在体外，在机械刺激条件下，小干扰 RNA （siRNA）-CCN2 不仅抑制原代纤维软骨细胞的增殖，还抑制 TRPV4 蛋白的表达和活性。 随后，用 TRPV4 激动剂 GSK1016790A 和重组腺病毒 TRPV4 （Ad-TRPV4） 处理原代纤维软骨细胞，GSK1016790A部分逆转了 siRNA-CCN2 的抑制作用。磷酸肌醇 3-激酶/蛋白激酶 B （PI3K/AKT） 信号通路参与了上述过程。结论 机械刺激通过激活 TRPV4 通道和 PI3K/AKT 信号通路促进纤维软骨细胞增殖和 TBI 愈合，CCN2 可能是维持 TRPV4 激活的关键调节蛋白。