Environmental Factors In The Intestine Regulate The Intestinal Mucosal Barrier Through Vitamin D Receptors And Related Mechanisms

Part One:Primary study of the impacts of intestinal inner environmental factors on Mucosal Barrier in colonic cell lines through Vitamin D ReceptorObjectives:To investigate the impacts of Vitamin D Receptor(VDR)on mucosal barrier function in colonic epithelial cell lines and the influences and mechanisms of intestinal inner environmental factors(hypoxia environment and butyrate).Methods:1.VDR knock-down cell lines experiments:1).We screened the expression of VDR in several colonic epithelial cell lines by Western blot and RT-PCR,then we chose DLD-1 and Caco2.2).We established stable VDR knock-down(Small Hairpin-VDR,Sh-VDR)cell line and control(Small Hairpin-Normal Control,Sh-NC)cell line by lentivirus package technology in DLD-1 and Caco2.3),We used transepithelial electric resistance(TEER)measurement to evaluate the integrity of single layer cell in DLD-1 Sh-VDR cell line.4).We detected the expression of tight junction(TJ)protein Zo-1,Occludin and adhesion junction(AJ)protein E-cadherin by Western blot in Sh-VDR cell lines and Sh-NC cell lines of DLD-1 and Caco2.2.Cell experiments on VDR knock-down cell lines under hypoxia in vitro:1).DLD-1 cells were treated separatedly with hypoxia(1%02),Vitamin D(100 nM)or Vitamin D plus hypoxia for 48 h.Then we detected the expression of tight junction proteins(Zo-1,Occludin and Claudin-1),adherent junction protein(E-cadherin)and VDR by Western blot.2).We also used DLD-1 Sh-VDR and Sh-NC cell lines and detected those proteins expressions after hypoxia treated for 48h by Western blot.3.Cell experiments on VDR knock-down cell lines under butyrate treatment in vitro:1).DLD-1 cells were treated with butyrate(0.3mM-3mM)for 48h,then the mRNA and protein expressions of Zo-1,Occludin,E-cadherin and VDR were detected by RT-PCR and Western blot.2).Those proteins were also detected in Sh-VDR and Sh-NC cell lines of DLD-1 with 0.3mM butyrate treated for 24h by RT-PCR.Results:1.VDR knock-down cell lines experiments:1).Compared with Sh-NC control group,the expressions of TJ proteins Zo-1,Occludin and AJ protein E-cadherin decreased significantly in DLD-1 and Caco2 cell lines after VDR knock out(n=5,P<0.001);2).Transepithelial electric resistance was decreased significantly in DLD-1 Sh-VDR cell(n=3,22.0±3.28 vs.4.33± 1.73,P=0.009;n=3,21.0± 1.86 vs.9.33±0.58,P=0.004)after 7-day and 11-day continuous cultivation.2.Cell experiments on VDR knock-down cell lines under hypoxia in vitro:1).Compared with normoxia control group,the expressions of Occludin,Claudin-1 and VDR increased significantly after hypoxia treated for 48h(n=3,P<0.001)in DLD-1 cell.Besides the proteins mentioned above,the expressions of ZO-1 and E-cadherin were also obviously higher in Vitamin D plus hypoxia group than single Vitamin D treatment group(n=3,P<0.001).2).After hypoxia treatment,Sh-VDR cell line showed significantly decreased expressions of all those mucosal barrier proteins as well as VDR when compared with untreated Sh-VDR cell line(n=3,P<0.001).3.Cell experiments on VDR knock-down cell lines under butyrate treatment in vitro:1).Compared with control DLD-1,the mRNA and protein expressions of tight junction proteins Zo-1,Occludin,adherent junction protein E-cadherin and VDR were enhanced obviously after 0.3mM butyrate treatment(n=3,P<0.001,P<0.001,P<0.001,P<0.001);2).Compared with DLD-1 Sh-NC and Sh-VDR control group,the mRNA levels of Zo-1,Occludin,E-cadherin in DLD-1 Sh-VDR were raised significantly after 0.3mM butyrate treatment in DLD-1 Sh-NC and Sh-VDR separatedly.(n=3,P<0.001,P<0.01,P<0.001)and VDR mRNA were raised significantly in Sh-VDR after 0.3mM butyrate treatment(P<0.001).Conclusions:1.The barrier function of single layer colon cell was decreased after VDR knock down in DLD-1 and Caco2.2.Hypoxia environment and microbial metabolism butyrate can regulate colonic epithelial barrier proteins' expression through VDR.3.VDR acts as a regulator for the expressions of intestinal mucosal barrier proteins under hypoxia environment in DLD—1 colon cell line,indicating that VDR pathway may be another crucially important protective methanism for gut barrier in low oxygen environment.Part Two:The effects and mechanisms of VDR on Intestinal Mucosal Barrier Function in VDR knock-out miceObjectives:To investigate the regulations and mechanisms of VDR on mucosal barrier function in VDR knock out(KO)mice.Methods:1.VDR KO mice:1).We used CRISPR/spCas9 techonology to establish C57BL/6 strain VDR knock-out founder mice and used 8-10 weeks aged VDR KO mice VDR(-/-)and their wild type(WT)siblings VDR(+/+)as control;2).We employed confocal fluorescence microscope to detect intestinal permeability of VDR KO mice and their WT control in vivo;3).We also detected the expression of those intestinal mucosal barrier proteins by Western blot in the colonic tissues of VDR KO mice and WT mice;4),Fecal flora 16s analysis from V3 to V4 region was also completed in VDR KO mice and WT mice.2.DSS Animal Model Methods on VDR KO mice:1).DSS model groups:Mice aged 8-10 weeks of WT control VDR(+/+)and VDR KO VDR(-/-)were divided into control group(Ctrl)and DSS-treated group(DSS),including Ctrl group(n=4),Ctrl+DSS group(n=4),K0 group(n=4)and KO+DSS group(n=4).1-5%DSS drinking water was provided for 5 days in DSS-treated group.During the time,body weight,stool property and occult blood were monitored everyday.We chose D7 as the endpoint of observation and recorded the colon length,general scores and pathological scores.2).We also used confocal fluorescence microscope to detect the intestinal permeability in vivo.3).We used electron microscope to observe intercellular junction structure.4).We detected the expression of tight junction protein Occludin by Western blot in mice colon.Results:1.VDR KO mice:1).Fluorescence intestinal permeability experiment of mice in vivo showed that compared with WT control group,the colonic permeability of KO mice increased with enlarged permeability scores(n=3,0±0 vs.1.1 ± 0.23,P<0.0001);2).Electron microscope(20000×)presented incomplete tight junction with indistinct borders in K0 mice,which indicated partly impaired barrier function.3).The expressions of TJ protein Zo-1,Occludin and AJ protein E-cadherin decreased significantly in KO mice.4).16S mice fecal flora analysis showed that two bacteria genus(Ruminiclostridium and Anaerotruncus),which were related to cellulose degradation and the productions of short chain fat acid(SCFAs)including butyrate and acetic acid,were decreased in VDR KO mice(n=5;P=0.023,P=0.029);Desulfovibrio and Odoribacter,which belonged to obligate anaerobes,were also decreased significantly in VDR K0 mice(n=5;P=0.041,P=0.003).2.VDR K0 DSS Model Results:1).On D7 after 1.5%DSS treatment,compared with K0 group,K0+DSS group showed increased disease activity index(0.25+0.50 vs.3.75+0.17,P=0.015),increased colon general scores(0±0 vs.3.75±0.25,P=0.011),significantly decreased colon length(7.72±0.17 vs.5.24±0.40,P<0.001).H&E staining of the whole colon showed completely destruction of distal colonic mucosal epithelium accompanied with increased pathological socres(0±0 vs.22.0±2.48,P=0.014);Confocal fluorescence microscope also showed almost complete loss of selective permeability function of colonic mucosa in K0+DSS group with obviously increased permeability scores(1.1±0.23 vs.1.8±0.13,P=0.022).Electron microscope showed severe destruction even absence of tight junction in K0+DSS group.Western blot showed decreased expression of TJ protein Occludin(P<0.001).2).Compared with Ctrl+DSS group,KO+DSS also presented increased general scores(2.50 ± 0.29 vs.3.75 ±0.25,P=0.032),significantly decreased colon length(6.83±0.23 vs.5.24±0.40,P=0.006),increased pathological socres(8.25± 1.80 vs.22.0 ± 2.48,P=0.021),increased permeability scores(1.2±0.13 vs.1.8±0.13,P=0.023).Electron microscope showed the similar results and Western blot results showed decreased expression of Occludin(P<0.001).Conclusions:1.After VDR knock-out in mice,the intestinal barrier function was seriously impaired and even lower concentration of DSS can induce severe colitis symptoms accompanied with repair dysfunction.2.The existence of VDR is important for colonic mucosal barrier and regeneration of intestinal mucosa.