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.

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.

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.

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.

To investigate the role and mechanism of Piezo1, a mechanically-sensitive ion channel protein, in excessive mechanical stretch induced apoptosis of tendon cells.

Tendon cells of 8-week-old male C57 mice（n=20, body mass: 22~26 g）were isolated and cultured, and a cell model of mechanical stretch induced apoptosis of tendon cells was established using the Flexcell system. Tendon cells were divided into the control group, the 20% elongation group, the 20% elongation+Yoda1 group, the 20% elongation+GsMTx4 group, the 20% elongation+Piezo1 knockdown lentivirus group（Lv-Piezo1）and 20% elongation+control lentivirus group（Lv-Ctrl）. Mitochondrial membrane potential assay, flow cytometry assay, Western blot assay and calcium fluorescence probe were used to detect the role of Piezo1 in tendon cell apoptosis. Tendon cells were divided into the 20% elongation group, the 20% elongation+siRNA-mediated Calpain2 knockdown group（si-Calpain2）, and the 20% elongation+control siRNA group（si-Ctrl）. Western blot assay was used to detect the activation of Piezo1 downstream signal Calpain2/BAX/Caspase3 axis.

The level of apoptosis with excessive mechanical tensile stress was higher in the 20% elongation group than those in the control group（P<0.05）, and Yoda1 promoted mechanical tensile stress induced apoptosis in tendon cells（P<0.05）, while GsMTx4 and Lv-Piezo1 showed the opposite effect（P<0.05）. The expression of Calpain2, BAX and cleaved-Caspase3 in tendon cells was enhanced by excessive mechanical stretch stress（P<0.05）. The 20% elongation+si-Calpain2 group had lower expression levels of BAX and cleaved-Caspase3 than those in the 20% elongation and the 20% elongation+control siRNA group（P<0.05）. Knockdown of Calpain2 inhibited the expression of BAX and cleaved-Caspase3（P<0.05）and attenuated apoptosis in tendon cells（P<0.05）.

Excessive mechanical stretch stress can induce apoptosis of tendon cells via the activation of Piezo1 and the downstream Calpain2/BAX/Caspase3 pathways. Piezo1 holds promise as a potential therapeutic target for tendinopathy.