The Expression And Function Of Piezo Mechanosensitive Ion Channel In Orthodontic Periodontal Tissue

Objective：Piezos ion channel family has been newly identified as a non-selective cation ionchannel that is abundantly expressed in bladder, lung, colon and dorsal root ganglion. Therapid mechanosensitive current was inhibited in N2A cells after its Piezo’s gene wasknocking down using siRNA, thus Piezo ion channel was considered to play an importantrole in force detecting of cells. Periodontal ligament fibroblast (PDLF) and osteoblast(OB) is thought to be mechanocytes that have biological response to mechanical force.However, the expression and function of Piezo ion channel in periodontal tissue is stillunclear at present. The purpose of this study was to investigate the expression, location and function in vivo and vitro in order to explore the role of Piezo in orthodonticperiodontal tissue and cells.Methods：1. To confirmed the expression of Piezos ion channel in periodontal tissue and cells,the characteristic and location of Piezos were detected by indirect immunofluorescenceand RT-PCR techniques. This would contribute to further explore the role of such ionchannel in periodontal tissue.2. To investigate the variation tendency of expression of Piezos ion channel inperiodontal tissue, an experimental animal model of orthodontic tooth movement in ratswas established using orthodontic spiral closing spring. The variation tendency of Piezosexpression in periodontal tissue and trigeminal ganglion was both been recorded. Hereby,we can probed into whether Piezos take part in the process of pain feeling andregeneration when orthodontic force act on periodontal tissue.3. The cyclical stress was loaded on PDLF, osteoblast (MC3T3) and Periodontalligament stem cell (PDLSCs) in vitro to simulated orthodontic environment by using acyclical stress loading system. The expression of Piezos ion channel was recorded onRNA and protein level to explore its variation tendency.4. For the sake of further understanding of Piezos’ function in PDLF that wastreated with cyclical stress, we applied GsMTx4—a specific blocker of Piezos ionchannel, to block Piezos channel during force loading process, and meanwhile quantifiedthe ATP release to get more information of Piezos in cell mechanical response and paintransduction process.5. To tested the synergistic effect of cyclical stress and GsMTx4, PDLSCs wereinduced to neuron-like cells by using hypoxic-ischemic brain tissue of rats. So that thefunction of Piezos ion channel in neuron injury-regeneration can be further identified.Results：1. Positive staining was observed in PDLF and OB by immunofluorescence stain. Piezos was mainly expressd in PDLF, but no positive staining was found in osteocyte.The expression distribution of Piezos in periodontal ligament was not uniform whichappeared to be mostly at the plasma membrane and a less in the cytoplasm.2. In vitro culturing of PDLF and MC3T3, Piezo1and Piezo2had approachedparity on RNA level. Its expression distribution was partly in the plasma membrane andcytoplasm， partly appeared to be dot gathered, while no nucleus positive expression wasobserved. There was no specific positive expression in primary cultured PDLSCs.3. In orthodontic tooth movement model of rats, the overall tendency of Piezo1showed to be increasing until it reached its peak in6d,9d group (P<0.05) and then turn tofalling; the overall tendency of Piezo2showed to be increasing until it reached its peak in9d,12d group (P<0.05).4. While orthodontic force was acted on periodontal tissue, Piezo1mRNA in TGincreased gradually and showed significant up-regulated in6d,9d group; meanwhile,Piezo2mRNA in TG changed less obviously and showed significant up-regulated in9dgroup. On protein level, Piezo1increased slowly until it reach its peak in9d group;Piezo2increased significantly in6d group, then it retain its rising trend untill to12dgroup.5. When mechanical force was applied on PDLF cultured in vitro, a significant riseof Piezos mRNA and protein was observed after30minutes by real-time PCR andwestern blot and this significance was continued to24h group (P<0.05); Piezo1mRNAreached its peak in4h group (P<0.05), then it began to decrease; Piezo2mRNA reach itspeak in2h group (P<0.05) and then decreased. In MC3T3, Piezos protein didn’t showany obvious changes during24h mechanical force loading except that Piezo1mRNAraised significantly in24h group (P<0.05). In PDLSCs, Piezos mRNA increasedsignificantly, and then decreased but the significance was continued in24h group(P<0.05); there was little protein expression in control group of PDLSCs, which hadgradually expressed this ion channel along with mechanical force.6. After24h mechanical force loading, ATP release increased significantly inPDLF (P<0.05), which can be down-regulated by Piezos’ inhibitor GsMTx4(P<0.05). However, the quantity of ATP release in PDLF blocked by GsMTx4was stillsignificantly higher than control group (P<0.05).7. The expression of Piezos at protein level which was observe by western blotafter24h neural inducing increased but was lower than the group treated with both neuralinduction and cyclical stress; there was no significant changes of expression for PDLSCswhen Piezos was blocked by GsMTx4. There was expression of NSE at protein level inneural induction treated group, but the expression was lower than that of the grouptreated with both neural induction and cyclical stress; the group treated with neuralinduction, cyclical stress and GsMTx4showed the highest expression quantity.Conclusions：1. Combined the abundant expression of Piezos ion channel in periodontal tissueand cells with the basis of existing research, it could be inferred that Piezos probably takepart in mechanical sense transduction of periodontal tissue. The variation tendency of thischannel during orthodontic tooth movement could be related to the increase of density ofnerve fiber. The accumulation of Piezo in cells which were cutured with force increasedwith time, which may be correspond to the injury-regeneration of periodontal nerveendings.2. Continuous orthodontic force induced up-regulated expression of Piezos ionchannel in periodontal tissue. A similar tendency of expression of such channel in TGwas observed. It implies that the mechanical stimulus is recieved by Piezos in periodontaltissue and than transmits to TG.3. With the effect of stress, Piezos mediates large quantities of ATP release, whichcould be speculating that Piezo may play a role in signal transduction of orthodontic pain.4. Inhibition of Piezos channel can up-regulate the neuron marker in PDLSCsduring neural induction, It was inferred that Piezo channel in periodontal tissue can sensemechanical stimulus and than have an effect on the outgrowth of neuron fiber. So thatPiezo may have an effect on injury-regeneration of neuron in orthodontic periodontal.