Mechanical Stimulation Mediated By Piezo1 Protein Regulates The Intestinal Mucosal Mucous Barrier

Intestinal mucosal barrier is an important physiological barrier existing on the surface of the intestinal skin cavity,which plays an important role in protecting the intestinal mucosa from the invasion of harmful substances in the intestine.The pathogenesis of a variety of intestinal diseases involves intestinal mucosal barrier dysfunction,but there are many factors affecting the intestinal mucosal barrier,and the specific regulatory mechanism is still not fully clarified.The Piezo1 protein is a recently discovered mechanically sensitive ion channel in the true sense,which can directly sense mechanical stimulation,open the non-selective cation channel,convert the perceived mechanical signal into a biological electrical signal,and play an important role in the regulation of cellular functions such as morphology,proliferation,differentiation,secretion,etc.In intestinal epithelial cell,that abundant Piezo1 is expressed,while the intestinal microenvironment also has a variety of mechanical signal,such as tensile stress,shear stress and pressure in the intestinal cavity.Whether these signal can be perceived by the Piezo1,thus affect the intestinal mucus barrier is undetermined at present.Part 1: the expression level of Piezo1 affects the mucin formation of LS174 T cellsObjective: to explore whether the expression level of Piezo1 is related to the mucin formation of LS174 T cellsMethods: the expression of Piezo1 protein in LS174 T cells was determined by immunofluorescence.The changes of mucin2 and mucin5 AC protein levels were detected by ELISA,and the changes of mucin2 and mucin5 AC mRNA levels were detected by RT-pcr.Results: Piezo1 protein was expressed abundantly in LS174 T cells.The mRNA levels of mucin2 and mucin5 AC in LS174 T cells decreased to less than 50% of the normal group after LS174 T cells Piezo1 expression was significantly down-regulated(p < 0.05,p < 0.05).The corresponding protein level also decreased significantly(p < 0.05,p < 0.05).After LS174 T cells Piezo1 expression was up-regulated,mucin2 and mucin5 AC protein levels significantly increased(p < 0.05,p < 0.05),and mRNA levels showed an upward trend,but no significant.Conclusion: the expression of Piezo1 in LS174 T cells significantly affected the formation of mucin2 and mucin5 AC.Part 2: the activation state of the Piezo1 channel affects LS174 T cells mucin expressionObjective: to investigate the effect of the activation state of Piezo1 channel on the LS174 T cells mucin expressionMethods: LS174 T cells were treated with piezo1-channel specific agonist Yoda1 and specific inhibitor GsMTx4.MRNA levels of mucin2 and mucin5 AC were detected by RT-pcr.Changes in mucin2 and mucin5 AC protein levels were detected by ELISA.Results: mRNA and protein levels of mucin2 and mucin5 AC in LS174 T cells were significantly increased after Yoda1 excited activation of Piezo1 channel(p < 0.01,p < 0.01).After the opening of Piezo1 channel inhibited by GsMTx4,the mRNA levels of mucin2 and mucin5 AC in LS174 T cells were significantly decreased(p < 0.01),but the protein levels were not significantly decreased.Conclusion: activation of Piezo1 pathway significantly affects the expression of mucin2 and mucin5 AC in LS174 T cells.Part 3: the activated state of the Piezo1 channel affects in vitro intestinal mucosal mucus secretionObjective: to investigate the effect of Piezo1 channel activation on intestinal mucosal mucus secretionMethods: colonic mucosa were isolated from healthy mice,and the mucosal mucus.thickness was measured by a self-made extracorporeal perfusion device and a mucosal mucus.thickness measuring device.Then intestinal mucosa of mice was treated with piezo1-channel specific agonist Yoda1 and inhibitor ruthenium red(RuR).Results: the colonic mucosal mucus thickness of mice was detected by using the self-made in vitro perfusion maintenance device and the intestinal mucosal mucus thickness detection device.The thickness of the proximal colonic mucosal layer was 222.3±65.6 m,and that of the distal colonic mucosal layer was 435.5±54.4 m.After the intervention of Yoda1 on the colonic mucosa of mice,the rise of colonic mucosal mucin thickness was significantly increased(p < 0.01).Under the condition of RuR intervention,there was no significant rise in colonic mucosal mucin thickness(p=0.291).Conclusion: Yoda1 that stimulates the opening of Piezo1 channel can promote the secretion of colonic mucin in mice,and RuR that inhibites the opening of Piezo1 channel can reduce the secretion of colonic mucin in mice.Part 4: different forms of mechanical stimulation promote the expression of LS174 T cell mucin expressionObjective: to investigate the effect of different forms of mechanical stimulation on the expression of LS174 T mucinMethods: LS174 T cells were stimulated with force of constant frequency and strength via using Flexcell's cell mechanics application devices and speical culture plates.LS174 T cells inoculated on ordinary plates were cultured on a miniature warped plate shaker,and the cells within were stimulated by shear force.LS174 T cells were cultured with different concentrations of gelatin to form plates with different bottom hardness.The expressions of mucin2 and mucin5 AC in LS174 T cells under mechanical stimulation were detected by RT-pcr and ELISA.Results: the expression of mucin2 and mucin5 AC in LS174 T cells was significantly promoted by mechanical stimulation.Shear stress stimulation can promote the expression of mucin2 and mucin5 AC in LS174 T cells,the effect of which is related to the stimulation time.The expression of mucin2 and mucin5 AC in LS174 T cells can be down-regulated by stress stimulation under certain conditions.Conclusion: mechanical stimulation has a significant effect on the expression of mucin in LS174 T cells,and the effect is related to the form and time of mechanical stimulation.Part 5: the catalytic effect of mechanical stretch stimulation on LS174 T cell mucoprotein mediated by Piezo1Objective: to investigate the mechanism of Piezo1 in the process of regulating mucus secretion with mechanical tension stimulationMethods: LS174 T cells were cultured under mechanical traction stimulation with piezo1-channel specific inhibitor GsMTx4,and their effects on the expression of Mucin2 and Mucin5 AC were detected by ELISA.Changes of Erk signaling pathway in LS174 T cells under mechanical stretch stimulation and Piezo1 agonist and inhibitor intervention conditions were detected by RT-pcr and Westernblot.LS174 T cells treated with Erk pathway inhibitor PD98059 and Piezo1 agonist were cultured to detect the expression of mucin2 and mucin5 AC.Results: Piezo1 channel specific inhibitor GsMTx4 significantly inhibited the expression of Mucin2 and Mucin5 AC in LS174 T cells stimulated by mechanical tension.The expression of phosphorylated Erk in LS174 T cells was significantly increased under mechanical stimulation and Piezo1 agonist.Piezo1 inhibitor could down-regulate the expression of phosphorylated Erk under mechanical stimulation.Erk pathway inhibitor can down-regulate the expression of mucin2 and mucin5 AC in LS174 T cells under mechanical stimulation,and reduce the promoting effect of Piezo1 agonist on the expression of mucin in LS174 T cells.Conclusion: Piezo1 may mediate the promoting effect of mechanical tension stimulation on the expression of mucin2 and mucin5 AC in LS174 T cells through Erk pathway.