Intestinal Alkaline Phosphatase Can Regulate Intestinal Epithelial Tight Junctions

Intestinal barrier function is closely linked with host health.The integrity of the intestinal barrier in the intestinal lumen can effectively prevent translocation of bacteria and endotoxin and other harmful substances from passing through intestinal mucosa to circulation system.Normal human intestinal mucosal barrier composed of four parts,including the mechanical barrier,chemical barrier,immune barrier and biological barrier.The mechanical barrier refers to the combination of intestinal epithelial cells and the integrity of structural intestinal epithelial tight junction between each other.This structure composed of intestinal mucosal epithelial cells and tight junctions.When the tight junction protein complex was comprimsed,intestinal permeability would increased and leading to mechanical barrier dysfunction,which makes bacteria and other harmful substances can transfer into circulation system easily.Studies have shown that impaired intestinal barrier function is related with many intestinal diseases and systemic diseases,such as inflammatory bowel disease,infection,sepsis,metabolic syndrome and obesity.Intestinal alkaline phosphatase(IAP)is one of the four subtype isoenzyme of alkaline phosphatase(AKP).It is a kind of glycoprotein which anchored on intestinal epithelial cell membrane through glycosylphosphatidylinositol.It mainly distributed in the duodenum to ileum and can be released into the intestinal lumen.Previous studies showed that IAP can help maintaining intestinal homeostasis,regulating lipid absorption,detoxifying LPS and nucleotides in the intestinal lumen,inhibiting intestinal inflammation,regulating of intestinal flora,and even transferring into the circulation system.Until now research regarding the impact of IAP on intestinal mucosal barrier remain limited.The previous work of our lab found that IAP activity decreased when mice after fasting.Fedding mice with IAP can improve the intestinal permeability comprimsed by high fat diet.Hence we speculate that IAP may be involved in the maintenance of intestinal mucosal barrier function,that it may has a protective effect on integrity and functionality of tight junctions directly or indirectly.In this paper,we designed animal experiments and cell biology experiments respectively to investigate the effects of knocking out IAP gene on mice intestinal permeability and tight junction transformation.We feed mice with exogenous calf IAP to observe the transformation of tight junction protein and related gene expression level;We created stable IAP knock out mouse embryonic cell line and Caco2 cell line;We transfected Caco2 cells with DsRed plasmid which loaded with IAP gene to creat a stable and highlyexpressing-IAP gene cell lines;Then gene expression/protein translation and imagic examnation were done on all those different cell lines.Cells were further used to develop single cell barrier model in transwell.Using different stimulation ways and different signal pathway inhibitors to investigate the related mechanism.This study aim to discover and comfirm that whether IAP has protective effect on intestinal epithelial tight junction and if yes,then what is the molecular mechanism involved.Part Ⅰ Using Animal Experiment to Investigate the Relationship Between IAP and Tight JunctionsObjective:To evaluate the effect of knocking out IAP gene or feeding with exogenous IAP on mouse intestinal permeability and tight junction.Methods:Eight-week old wild-type and IAP knockout(Akp3-/-)female C57BL/6 mouse were selected.Mice were gavaged with FITC-dextran(4kD)in a dosage of 0.6mg/g,or injection into a segment of intestinal loops with the length of 4 cm.Four hours later blood samples were collected to test the concentration of FITC-dextran,which can be conversed into intestinal permeability.Intestinal tissue sample were further investigated by Westernblot,qPCR,HE staining,immunofluorescence staining and transmission electron microscopy scanning.Also IAP-KO mice were feeded with exogenous IAP to see the change of tight junctions among intestinal mucous.Results:Compared to that of wild-type mice,IAP-KO mice showed a significantly increase of intestinal permeability and a decreased tight junction protein expression level(ZO-1,ZO-2 and Occludin).HE staining showed that the intestinal epithelial structure of IAP-KO mice were comprised compared with that of wide type mice,Electron microscopy and immunofluorescence imaging indicated that IAP-KO mice have tight junctions structure shifting and abnormal distribution.Feeding the mice with exogenous IAP can effectively reduce intestinal epithelial damage and improve the expression of tight junction protein which resulted into the improvement of intestinal permeability.Conclusion:Compared with wild-type mice,IAP-KO mice showed a increased intestinal permeability,decreased tight junction protein expression level and comprimsed intestinal epithelial tight junction structural.Feeding the KO mice with exogenous IAP can effectively improve the intestinal permeability.Part Ⅱ Using Different Genetic Modified Cell Lines to Investigate the Relationship Between IAP and Tight JunctionsObjective:Using different genetic modified cell line to evaluate the relationship between IAP and tight junctions.Methods:We developed stable IAP knock out murine fibroblast cell line from mice embryo.Crispr/Cas9 technique was used to knock out the alpi gene in Caco2 cell line.IAP highly expression cell line were developed through IAP-DsRed plasmid transfection technology.All those cell lines were confirmed with Western-blot and qPCR.Transepithelial resistance(TEER)were measured to reflect the Caco2 cell barrier function.Furthermore,cell lines were growed in transwell to creat monolayer cell barrier model in vitro.Different dosage of LPS/EColi/TNF-α/IAP were added into cells to investigate their effect on barrier permeability of the cell barrier.Results:Gene expression level of ZO-1/ZO-2/Occludin in IAP-KO MEF cells were significant lower than that of wide type MEF cells.While Claudin expression levels showed no significant difference.Also as for the Caco2 cell line,gene expression level of ZO-1 was higher in the IAP plasmid-transfected Caco2 compared with that of regular Caco2,whicle no significant differences were observed in Occludin and Claudin.The expression level of ZO-1/ZO-2/Occludin decreased significantly in IAP-KO Caco2 cells compared with that of regular one.Immunofluorescence imaging showed that,compared with the control group Caco2,IAP-KO Caco2 is closely connected to ambiguity and there was destruction with multiple structures.A more clearly tight junctons structure was observed among IAP highly expression Caco2 cells.Stimulated by different doses of LP S/Ecoli/TNF-α/IAP showed that IAP can protect the damage caused by LPS/Ecoli/TNFα.IAP-KO Caco2 was more sensitive to the stimulation,and the addition of exogenous IAP can significantly improve the cell barrier and permeability.Conclusion:Human/mouse derived IAP-KO cells showed different level of compromised barrier function,which can be explained by the impact of IAP on tight junctions.Increased the IAP expression level in Caco2 cell line can effectively improve the expression of tight junction proteins;The process of forming Caco2 cell monolayer barrier in IAP knock out cell line was much lower than that of regular Caco2 cell line.Also they are more sensitive to LPS/Ecoli/TNF-α stimulation.Adding exogenous IAP can protect the cell barrier from the stimulation of LPS/Ecoli/TNF-α,which maybe realized through the regulation of tight junction.Part Ⅲ How Does IAP Impact Tight JunctionsObjective:To investigate the mechanism and signal pathway that may be involved in the regulation of tight junctions by IAP.Methods:IAP-KO cells were divided into two groups according to whether exogenous IAP was pre-incubated with the cells.Then different inhibitors of signaling pathways(NFKb/STAT3/ERK/JNK)were added to the cells for three consecutive days.Cells were collected in different timepoints to mease the tight junction gene expression or protein level.After the screening of signaling pathways,using siRNA technology to further analyze key molecules which may be involved in the regulation of IAP signaling pathway.Results:Incubating IAP-KO Caco2 cells with exogenous IAP can up-regulate the expression level of tight junction gene ZO-1/ZO-2/Occludin,while this effect can be blocked by inhibitors of ERK signaling pathway.NF-kB/STAT3/JNK pathway inhibitors showed no significant effect on the tight junctions.Cells were transfected with siRNA plasmid of PP2A or PKC.PP2A siRNA could effectively inhibit the upregulation effect of IAP on ZO protein,while no obvious difference was observed in the PKC siRNA group.Conclusion:IAP can regulate intestinal epithelial cells tight junction through ERK-PP2A signaling pathway.NF-kB,STAT3,JNK or WNT signal pathway were not involved.