Role Of STIM1 On Intestinal Epithelial Restitution And Polyamines And Tryptophan Regulation In Porcine Intestinal Epithelium Cell

Mechanisms of intestinal epithelial repair（rapid repair and subsequent repair）are activated to protect the intestinal barrier when the intestinal mucosal barrier is destroyed.The rapid repair process mainly relies on cell migration,and subsequent repair mainly maintains the integrity of tight junctions by closing the intercellular space.The role and mechanism of stromal interaction molecule 1（STIM1）in regulating intestinal damage repair in intestinal porcine epithelial cell line（IPEC-J2）has not been reported at home and abroad.Whether polyamines and tryptophan can regulate restitution of wounded intestinal epithelial cells by regulating STIM1 remains to be further studied.The present study was conducted to explore the effect and the possible mechanisms of STIM1 on the cell migration and intestinal epithelial barrier function by an IPEC-J2 model.Moreover,this experiment was to investigate the role and mechanism of STIM1 on the cell migration and intestinal epithelial barrier function,and whether STIM1-mediated function was regulated by polyamines or tryptophan in IPEC-J2 cells by adding polyamines or tryptophan.Experiment 1:The effect of STIM1 on the migration of intestinal porcine epithelial cell line and the regulation of polyamines and tryptophanThis study was to explore the effect of STIM1 on cell migration,and whether STIM1-mediated function was regulated by polyamines and tryptophan in IPEC-J2 cells after scratch damage.The results were as follows:（1）STIM1-si RNA inhibited cell migration,reduced the protein concentration of STIM1 and transient receptor potential canonical 1（TRPC1）,and reduced the content of inositol trisphosphate（IP3）.However,overexpression of STIM1got opposite results.These results showed that upregulation of STIM1 promoted cell migration in intestinal porcine epithelial cell line.（2）1μM SKF96365（TRPC1 inhibitor）inhibited cell migration,reduced the protein concentration of STIM1 and TRPC1,and reduced the content of IP3,indicating that downregulation of TRPC1 reduced the cell migration of IPEC-J2 cells.（3）Overexpression of wild-type and mutant STIM1 can promote cell migration,increase the protein concentration of STIM1 and TRPC1,and increase the content of IP3.However,after treatment with 1μM SKF96365,cell migration,STIM1 and TRPC1 protein concentrations and IP3 content were decreased.These results indicated that overexpression of STIM1 enhanced the migration of IPEC-J2 cells through increasing the TRPC1 signaling pathway.（4）5m Mα-difluoromethylornithine（DFMO）can significantly inhibit cell migration,reduce the protein concentration of STIM1 and TRPC1 and reduce the content of IP3.Putrescine administration attenuated DFMO-induced downregulation of cell migration,the protein concentration of STIM1 and TRPC1 as well as the content of IP3.These findings revealed that polyamine deficiency led to the reduction of IPEC-J2 cells migration by reducing the STIM1/TRPC1 complex.（5）5m M DFMO can significantly reduce the IPEC-J2 cell migration,reduce the protein concentration of STIM1 and TRPC1,and reduce the content of IP3.Overexpression of wild-type and mutant STIM1 will alleviate the inhibitory effect of DFMO on cell migration,the protein concentration of STIM1 and TRPC1 as well as IP3 content.These results indicate that polyamines depletion can activate STIM1 and increase STIM1/TRPC1 complex to promote cell migration.（6）The administration of putrescine or tryptophan could promote the migration of IPEC-J2 cells,increase the protein concentration of STIM1 and TRPC1 and the content of IP3.And putrescine or tryptophan alleviated STIM1 silence-induced the reduction of cell migration,the decrease of the protein concentration of STIM1 and TRPC1 as well as the downregulation of the content of IP3.These results indicated that STIM1/TRPC1 signaling pathway was participated in the effect of polyamines or tryptophan on the migration of IPEC-J2 cells.Experiment 2:The effect of STIM1 on the intestinal epithelial barrier function of intestinal porcine epithelial cell line and the regulation of polyamines and tryptophanThis study explored the effect of STIM1 on intestinal epithelial barrier function in IPEC-J2 cells by constructing hydrogen peroxide（H₂O₂）-induced oxidative damage model.The results were as follows:（1）Compared with control group,H₂O₂（600μM）resulted in decreasing the protein concentration of STIM1,TRPC1,occludin,zonula occludens-1（ZO-1）and claudin-1,reducing the trans‐epithelial electrical resistance（TER）and increasing flux of fluorescein isothiocyanate dextran 4 k Da（FD4）（P<0.05）.And si RNA interference of the STIM1 significantly enhanced the effect of H₂O₂on the protein concentration of STIM1,TRPC1 and tight junction proteins,TER and FD4 flux（P<0.05）.These results revealed that upregulation of STIM1 could protect the barrier function by facilitating the expression of tight junctions,increasing TER and reducing intestinal epithelial permeability in IPEC-J2 cells.（2）Overexpression of wild-type STIM1 could significantly enhanced the protein concentration of occludin,ZO-1,claudin-1,STIM1 and TRPC1,improved the TER and reduced the FD4 flux in IPEC-J2 cells challenged with H₂O₂,and these effects of overexpression of STIM1were suppressed by repressing TRPC1（P<0.05）.These findings showed that overexpression of STIM1 upregulated the tight junction expression and reduced intestinal epithelial permeability by TRPC1 signaling in IPEC-J2 cells.（3）Compared with H₂O₂ group,H₂O₂+75μM putrescine group improved the protein concentration of occludin,ZO-1,claudin-1,STIM1 and TRPC1,enhanced IP3 content and TER as well as reduced FD4flux,these protective effects of putrescine were inhibited by knockdown of STIM1（P<0.05）.These findings showed that putrescine promoted the integrity of tight junctions and reduced intestinal epithelial permeability in IPEC-J2 cells through STIM1/TRPC1 signaling.（4）Compared with H₂O₂ group,700μM tryptophan enhanced the protein concentration of occludin,ZO-1,claudin-1,STIM1 and TRPC1,increased the TER and IP3 content as well as decreased the FD4 flux in IPEC-J2 cells during exposure to 600μM H₂O₂（P<0.05）.Compared with the tryptophan group,the tryptophan+STIM1-si RNA group significantly reduced the protein concentration of occludin,ZO-1,claudin-1,STIM1 and TRPC1,decreased the content of TER and IP3,and increased FD4 permeability in IPEC-J2 cells（P<0.05）.These results indicated that tryptophan contributed to the integrity of tight junctions and downregulating intestinal epithelial permeability via activating STIM1/TRPC1signaling pathway in IPEC-J2 cells.In summary,overexpression of STIM1 can mediate TRPC1 signal to increase cell migration,tight junction protein levels,and reduce intestinal epithelial permeability,thereby promoting the repair of porcine intestinal epithelial cells.Polyamines and tryptophan can improve the migration of porcine intestinal epithelial cells and the intestinal epithelial barrier function,thereby improving the repair of porcine intestinal epithelial damage.The STIM1/TRPC1 signaling pathway is involved in the repair of polyamines and tryptophan.