Research On The Role Of PPARα In Ulcerative Colitis Induced By DSS

Object:With the continuous development of social economy,the incidence of Ulcerative Colitis（UC）in China has been increasing year by year.It has been reported that Peroxisome proliferator-activated receptorα（PPARα）is highly expressed in the colon and is involved in the development and differentiation of intestinal villi.The mechanism of action of intestinal PPARαin the pathophysiology of UC has not been reported yet.In this study,we intended to investigate its changes and possible effects in UC model induced by Dextran Sulfate Sodium Salt（DSS）using mice specifically deficient in intestinal PPARα.Methods:（1）UC model was induced by DSS in wild-type mice,and colon tissue damage was observed by HE staining.The expression levels of PPARαand MUC2 were detected by real-time quantitative RT-PCR and immunofluorescence.Intestinal Pparα^ΔIEC mice were established by mating PPARα-flox mice with Villin-Cre mice.（2）The body weight of Pparα^fl/fl and Pparα^ΔIEC mice in the same litter was monitored.Colon length was measured,and the number of colonic gastrocytes was observed by HE and PAS staining.Western blotting and immunofluorescence were used to detect the expression of MUC2 protein.The expression levels ofβ-catenin and Notch1proteins were detected by Western blotting.The nuclear localization ofβ-catenin was performed by immunofluorescence method.MUC2,anti-microbial proteins（Reg3β,Reg3γ）,Wnt/β-catenin signaling pathway related genes（Ephb3,Ephb2,C-Myc,Lrp6,Lrp5）and Notch signaling related genes（Notch1,Hes2,Hes6,Hey L,Hey1,Hey2）and expression levels of transcription factors（Math1,Klf4,Gfi1,Spdef）that control goblet cell differentiation and maturation were detected by real-time quantitative RT-PCR.（3）Male hominatal Pparα^ΔIEC and Pparα^fl/fl mice drank water containing 2%DSS（W/V）for 7 days.FITC was used for intestinal penetration test,colon length was measured and disease activity index score was performed.Colon tissue injury was observed by HE staining,and histopathological score was obtained.The expression levels of MUC2and transcription factors（Math1,Klf4,Gfi1 and Spdef）regulating goblet cell differentiation and maturation were detected by real-time quantitative RT-PCR.On the4th and 7th days after drinking DSS,the number of colonic goblet cells was observed by PAS staining.The expression of MUC2 protein was detected by immunofluorescence.（4）In the mixed antibiotic（ABx）model,the number and function of colon goblet cells were observed by HE and PAS staining.（5）After 16 weeks of western diet feeding,colon length was measured,colon goblet cells were observed by HE and PAS staining,and the expression level of MUC2 was detected by real-time quantitative RT-PCR.（6）Colonic organs were cultured and treated with PPARαinhibitor GW6471（10μM）and PPARαagonist WY14643（50μM）,and the expression of MUC2 protein was detected by immunofluorescence.Caco2 colon cancer cells were cultured and treated with PPARαinhibitor GW6471（10μM）and PPARαagonist WY14643（50μM）.The expression levels of MUC2,Math1,Klf4 and Gfi1 were detected by real-time quantitative RT-PCR.Results:（1）Compared with the control wild-type mice,the colon of DSS treated mice showed serious tissue damage,such as inflammatory infiltration and crypt reduction;PPARαm RNA and protein in colon tissue were up-regulated by 1.68 fold（P<0.05）and 1.57 fold（P<0.05）.MUC2 m RNA was down-regulated by 57%（P<0.001）,and protein was down-regulated by 56%（P<0.01）.（2）Compared with Pparα^fl/fl mice,Pparα^ΔIEC mice showed no difference in body weight and colon length;In Pparα^ΔIEC mice,goblet cells were increased 1.95 fold（P<0.001）and MUC2 protein expression was increased 3.23 fold（P<0.001）.Pparα^fl/fl and Pparα^ΔIEC mouse goblet cellsβ-catenin did not enter the nucleus.Compared with Pparα^fl/fl mice,the expression of MUC2 protein in the colon of Pparα^ΔIEC mice was increased by 2.70 fold（P<0.001）,butβ-catenin and Notch1 protein levels were not significantly different.The expressions of MUC2,Reg3βand Reg3γin Pparα^ΔIEC mice were up-regulated 3.66 fold（P<0.001）,2.80 fold（P<0.001）,and 2.23 fold（P<0.001）,respectively.Compared with Pparα^fl/fl mice,the expression of Wnt/β-catenin signaling related genes（Ephb3,Ephb2,C-Myc,Lrp6,Lrp5）and Notch signaling related genes（Notch1,Hes2,Hes6,Hey L,Hey1,Hey2）were not significantly different.Compared with Pparα^fl/fl mice,the expression of Math1,Klf4,Gfi1 and Spdef in Pparα^ΔIEC mice were up-regulated by 3.56 fold（P<0.001）,4.97 fold（P<0.001）,5.16 fold（P<0.001）and 2.31 fold（P<0.001）,respectively.（3）After 7 days of DSS treatment,the body weight of Pparα^ΔIEC mice increased by26%（P<0.01）,the serum FITC concentration decreased by 74%（P<0.05）,and the colon length increased by 1.34 fold（P<0.001）,compared with Pparα^fl/fl mice.The disease activity index score was decreased by 37%（P<0.05）and the histopathological score was decreased by 50%（P<0.001）.Compared with Pparα^fl/fl mice,the expressions of MUC2,Math1,Klf4,Spdef and Gfi1 in Pparα^ΔIEC mice were upregulated 3.03 fold（P<0.001）,9.66 fold（P<0.001）,3.53 fold（P<0.001）,1.66 fold（P<0.01）and 8.42fold（P<0.01）,respectively.The number of colon goblet cells decreased in Pparα^fl/flmice after 4 and 7 days of DSS treatment.At 4 and 7 days of DSS treatment,the number of colonic goblet cells in Pparα^ΔIEC mice increased 1.27 fold（P<0.001）and 2.26 fold（P<0.001）,respectively,compared with Pparα^fl/fl mice.The expression level of MUC2protein was increased by 2.70 fold（P<0.001）and 2.12 fold（P<0.001）,respectively.（4）In ABx model,the number of colon goblet cells in Pparα^fl/fl and Pparα^ΔIEC mice was not significantly changed before and after bacteriolysis.Compared with Pparα^fl/flmice,the number of colonic goblet cells in Pparα^ΔIEC mice increased 1.70 fold（P<0.001）.（5）In western diet model,colon length of Pparα^fl/fl and Pparα^ΔIEC mice was reduced by 18%（P<0.001）and 13%（P<0.001）,respectively.Compared with Pparα^fl/fl mice,the expression of MUC2 m RNA in the colon of Pparα^ΔIEC mice was 2.16fold increased（P<0.001）.（6）Compared with the control group,the expression of MUC2 protein in colon organs treated with PPARαinhibitor GW6471 increased by 1.64 fold（P<0.001）.After administration of PPARαagonist WY14643,MUC2 protein expression was decreased by 51%（P<0.001）.Compared with the control group,the expression of MUC2 m RNA in Caco2 cells treated by GW6471 increased by 1.53 fold（P<0.001）,Math1,Klf4 and Gfi1 were up-regulated by 2.55 fold（P<0.001）,1.65 fold（P<0.001）and 2.10 fold（P<0.001）,respectively.MUC2 m RNA expression in WY14643 group was decreased by45%（P<0.001）,Math1,Klf4 and Gfi1 were decreased by 48%（P<0.001）,22%（P<0.001）and 55%（P<0.001）,respectively.Conclusions:The loss of intestinal PPARαresulted in increased colon goblet cells,increased expression of MUC2 and decreased sensitivity to DSS-induced UC.The increase of colon goblet cells induced by intestinal PPARαdeficiency may be related to the up-regulation of transcription factors Math1,Klf4,Spdef and Gfi1,which control goblet cell differentiation and maturation,but not related to intestinal microecology.Inhibition of intestinal PPARαmay reduce the risk of DSS-induced UC.