Effects Of Mechanical Stretch On The Function Of Tendon Cells And The Mechanosensitive Ion Channel PIEZO1

Objective:Tendon cells are subjected to a variety of mechanical loads in vivo,of which stretch load is the most important.Tendon cells can regulate cellular metabolism by responding to mechanical load,which plays an important role in the process of tendon injury and repair.However,there are few studies on how tendon cells respond to mechanical load.This article simulates the stretching load of tendon cell in vivo using the cell stretching system Flexcell 5000 T to study the effect of mechanical stretching on tendon cell function and the role of PIEZO1 in mechanical load response.Method:In our experiment,10 14-week-old male SD rats were selected to extract Achilles tendon tissue for primary culture of tendon cells,and third-generation cells were used for the experiment.Tendon cells were divided into static control group,1Hz4%,1Hz8%,and 1Hz12% stretching strength groups.Mechanical stretching stimulation of different intensities was applied to tendon cells in each stretching group using the FX-5000 T cell stretching stress system for 4 hours.Experimental instruments such as microscopy and cell counting instruments,as well as Western blot,RT-PCR,and flow cytometry were used to observe changes in tendon cell morphology,function,and inflammation.Subsequently,the PIEZO1 inhibitor GsMTx4 was applied to inhibit the function of PIEZO1,and the role of PIEZO1 in the response of tendon cells to mechanical stretching stimulation was preliminarily explored.Result:1.High strength mechanical stretching can reduce the proliferation of tendon cells.After 1 day of mechanical stretching,compared with the static control group,there was no significant difference in cell proliferation between the4% and 8% groups,while the 12% group showed a decrease in cell proliferation.After 3 days of mechanical stretching,compared with the static control group,the tendon cells in each stretching group showed long spindle growth,with the growth direction was consistent with the direction of the pulling axis.Compared with the static control group,there was no significant difference in cell density in 4% and 8% groups(P>0.05),while cell proliferation was significantly decreased in 12% group(P<0.05).2.Mechanical stretch can enhance the expression of genes related to tendon cell function.After 1 day of mechanical stretching,the PCR results showed that compared with the static control group,the stretching groups showed a significant increase in mRNA expression levels of SCX,MKX,DCN and Collagen Ⅲ(P<0.05).Although there was an increasing trend in mRNA expression of FMOD,PRG4,and TNMD,there was no statistical difference.Collagen I mRNA expression was significantly reduced(P<0.05).3.High strength mechanical stretching can induce inflammatory response in tendon cells.After 1 day mechanical stretching,PCR results showed that compared to the control groups,the inflammatory factors IL-6,IL-13,and TNF-α Gene expression is too low to detected in the 4% groups;the mRNA expression of IL-6,IL-13,IL-1β is no significant difference in 8% groups;the expression level of IL-6,IL-13,IL-1β,TNF-α and MCP-1 was significantly increased in the 12% groups;compared with the resting group,there was no significant difference in PGE2 mRNA expression among the 4%,8%,and 12% groups.4.Mechanical stretching can increase the expression of PIEZO1 gene in tendon cells and promote calcium ion influx.Western Blot results showed that tendon cells can express PIEZO1 protein.The PCR results showed that after 1 day of mechanical stretching,the PIEZO1 mRNA expression levels in the 4%,8%,and 12% strength stretching groups significantly increased compared to the control group;The results of intracellular calcium ion concentration detection showed that compared with the static group,4%,8%,and 12% stretching could increase the intracellular calcium ion concentration of tendon cells,but there was no statistical difference among the stretching groups.5.Inhibiting PIEZO1 can reduce the expression of functional related genes in tendon cells after mechanical stretchingAfter 1 day of mechanical stretching,PCR results showed that:(1)compared with the group without the addition of PIEZO1 inhibitor GsMTx4,the mRNA expression levels of SCX,FMOD,PRG4,and TNMD were significantly reduced in the resting group(P<0.05);(2)4% strength stretching group: Compared with the group without the addition of PIEZO1 inhibitor GsMTx4,the mRNA expression levels of SCX,MKX,FMOD,NFATC1,PRG4,TNMD,Collagen I,and Collagen Ⅲ were significantly reduced after the addition of PIEZO1 inhibitor GsMTx4(P<0.05);(3)8% strength stretching group: Compared with the group without the addition of PIEZO1 inhibitor GsMTx4,the expression levels of MKX,FMOD,PRG4,Collagen Ⅲ mRNA were significantly reduced after the addition of PIEZO1 inhibitor GsMTx4(P<0.05);(4)12% strength stretching group: Compared with the group without inhibitors,the addition of PIEZO1 inhibitor GsMTx4 significantly reduced the expression levels of PRG4 and Collagen Ⅲ mRNA(P<0.05).6.Inhibiting PIEZO1 can enhance the inflammatory response of tendon cells after moderate intensity mechanical stretching.After 1 day of mechanical stretching,the PCR results showed:(1)In the resting groups: compared with the groups without inhibitors,the mRNA expression levels of IL-6,IL-13,IL-1 β and TNF-α were observed after adding PIEZO1 inhibitor GsMTx4 were significantly reduced(P<0.05);(2)4% stretching groups: Compared with the groups without inhibitors,the mRNA expression levels of IL-1β and MCP-1 after adding GsMTx4 were observed significantly reduced(P<0.05);(3)8% stretching groups: Compared with the groups without inhibitors,after adding GsMTx4,the expression of mRNA of IL-6,IL-13,TNF-α was significantly increased(P<0.05);(4)12%stretching groups: Compared with the group without inhibitors,there was no significant change in the mRNA expression of related inflammatory factors after adding GsMTx4.7.Inhibiting PIEZO1 can reduce calcium influx in tendon cells after mechanical stretching.After 1 day of mechanical stretching,PCR results showed that compared to the group without the addition of inhibitors,there was no significant difference in PIEZO1 mRNA expression between the 4%,8%,and 12%strength stretching groups after the addition of PIEZO1 inhibitor GsMTx4.The calcium ion influx results showed that after adding the PIEZO1 inhibitor GsMTx4,there was no significant change in the calcium ion concentration in the resting group,while the intracellular calcium ion concentration in each stretching group significantly decreased.Research conclusion:1.Mechanical stretching can enhance the function of tendon cells by increasing the expression of PIEZO1 in tendon cells.High strength mechanical stretching can induce inflammation in tendon cells,leading to reduced proliferation of tendon cells.2.Inhibiting the function of PIEZO1 may reduce the calcium ion influx of tendon cells,reduce their response to mechanical stretching stimulation,and thereby inhibit the function of tendon cells,enhancing the inflammatory response of tendon cells caused by moderate mechanical stretching.