The Role And Mechanism Of Excessive Mechanical Stretch In Mediating Apoptosis Of Tendon Cells Via Piezo1

Objective:Tendinopathy is a disease caused by structural damage and dysfunction of tendon tissue,characterized by persistent pain during or after exercise,limited joint function and reduced exercise tolerance.The diagnosis of tendinopathy currently relies on clinical symptoms,medical history and imaging,and the common treatments are surgery,physical therapy and NSAIDs,but the combined treatment is not effective.Studies have shown that tendinopathy is closely related to a variety of risk factors,including mechanical stimuli,sports injuries,and genetic factors,among which overuse and mechanical tensile stress are considered to be important factors contributing to tendinopathy.However,how mechanical tensile stress mediates apoptosis in tendon cells is not yet known.Piezo Type Mechanosensitive Ion Channel Component 1(Piezo1)is a mechanosensitive ion channel protein discovered in recent years.Piezo1 is mainly involved in sensing,conducting mechanical signals and converting them into biological signals,and plays an important role in regulating apoptosis,cell proliferation,cell differentiation It plays an important role in the regulation of various cellular physiological activities such as apoptosis,cell proliferation and cell differentiation.Previous studies have demonstrated that Piezo1-mediated cellular Ca2+inward flow under excessive mechanical stretch stress is associated with apoptosis of intervertebral disc cells and chondrocytes,and Piezo1 can participate in apoptosis by regulating mitochondrial dysfunction and endoplasmic reticulum stress signaling pathways,but there are no studies related to piezo1 and tendon cell apoptosis.The aim of this study was to explore the role and mechanism of apoptosis induced by the mechanosensitive ion channel protein Piezo1 in the presence of excessive mechanical stretch stress in tendon cells.Methods:Tendon cells of the index,middle and ring finger deep flexor muscles of both hind paws of mice were isolated and cultured,and the Flexcell 5000 tension system was used to establish a cell model of mechanical stretch-induced apoptosis in tendon cells with a frequency of 0.5 Hz and 0,5%,10%,15% and 20% stretch shape variables as parameters.Mitochondrial membrane potential assay and flow cytometry were used to detect apoptosis in tendon cells,and western blot and calcium fluorescence probe were used to detect the expression level and biological activity of Piezo1 protein channel in tendon cells to verify the role of Piezo1 in tendon cell apoptosis.The tendon cells were divided into control group,chemical agonist Yoda1 group,inhibitor Gs MTx4 group,Piezo1 knockdown lentivirus group(Lv-Piezo1)and control lentivirus group(Lv-Ctrl)Mitochondrial membrane potential assay and flow cytometry were used to detect apoptosis in tendon cells,and western blot was used to detect the activation of the Piezo1 downstream signal Calpain2/BAX/Caspase3 axis in tendon cells.The tendon cells were divided into control group,siRNA-mediated Calpain2 knockdown group(si-Calpain2),and control siRNA group(si-Ctrl)to detect the expression levels of downstream BAX and Caspase3 axes.Results:Excessive mechanical stretch stress induced apoptosis in tendon cells,and the degree of apoptosis in tendon cells was upregulated with increasing stretch deformation(p<0.05),and the process was accompanied by increased Piezo1 protein levels and Piezo1activation(p<0.05).Apoptosis levels were higher in the chemical agonist Yoda1 group under excessive mechanical stretch stress than in the control group(p < 0.05),the inhibitor Gs MTx4group(p < 0.05),the Piezo1 knockdown lentivirus group(Lv-Piezo1)(p < 0.05)and the control lenti virus group(Lv-Ctrl)(p < 0.05),Yoda1 further promoted apoptosis induced by excessive mechanical stretch stress,while Gs MTx4 and Lv-Piezo1 could inhibit apoptosis induced by excessive mechanical stretch stress(p < 0.05).The expression levels of Calpain2,BAX and cleaved-Caspase3 were elevated in tendon cells under excessive mechanical stretch stress(p < 0.05).siRNA-mediated Calpain2 knockdown group(si-Calpain2)had reduced Calpain2 expression levels(p < 0.05),and lower expression levels of BAX and cleaved-Caspase3 than the control group(p < 0.05),and the control siRNA group(si-Ctrl)(p< 0.05).Conclusions:In this study,we investigated for the first time the role and mechanism of Piezo1 in the induction of apoptosis in tendon cells by excessive mechanical tensile stress.In vitro cultured tendon cells were subjected to mechanical tensile stress with different morphological variables,and the Piezo1 agonist Yoda1,Piezo1 inhibitor Gs MTx4 and Lv-Piezo1 knockdown virus were used to intervene in tendon cells.probe assay,confirmed the role of mechanical tensile stress in mediating apoptosis of tendon cells through Piezo1.Subsequently,we used si-Calpain to intervene in tendon cells and analyzed Calpain2/BAX/Caspase3 and Piezo1 protein expression by Western Blot to verify that mechanical tensile stress can initiate tendon cell through Piezo1 activation of downstream Calpain2/BAX/Caspase3 signaling pathway to apoptosis.Therefore,we suggest that Piezo1 is a potential therapeutic target for the treatment of tendinopathies.