GART Mediates The Role Of Intestinal Epithelial Barrier Repair In The Occurrence And Development Of Crohn's Disease

Background and Purposes:Purine nucleotides play a very important role in energy supply,metabolism regulation and the formation of coenzymes.In the synthesis metabolism of purine nucleotides,simple nucleotides such as phosphoribosyl sugar,amino acids,one carbon unit and CO2 are used as raw materials to synthesis of purine nucleotides which is called the de novo synthesis pathway.Glycinamide ribonucleotide formyltransferase(GART)has been identified as a key enzyme in purine de novo synthesis and mediates apoptosis in many diseases such as liver cancer,non-small cell lung cancer and glioma.The purpose of this study is to explore the role of GART in the pathogenesis of Crohn's disease(CD)and its mechanism,and to provide a theoretical basis for a new diagnosis and treatment of CD.Methods Intestinal mucosa were collected from 6 patients with active CD and 6 healthy controls,and 2,4,6-trinitrobenzenesulphonic acid(TNBS)-induced acute colitis model was established.The colon tissues were collected after the mice were sacrificed.Human colon HCT-116 cancer cells and rat intestinal epithelial IEC-6 cells were cultured in vitro.The expression of GART in tissues and cells was detected by immunoblot and PCR.Flow cytometry,Hoechest,TUNEL,Caspase-3 activity assay and P53/PUMA assay were used to detect the effect of downregulation of GART expression on cellular apoptosis and its effect on the upstream and downstream of P38MAPK pathway and its mechanisms.Scratch assay was used to detect the migration capacity with decreased GART expression.Results GART expression was significantly increased in CD patients and TNBS-induced mouse colitis(P<0.01).Knockdown of GART level significantly increased the expression of p53/PUMA and phosphorylation of P38(P<0.01)in intestinal epithelial cells.Knockdown of GART expression induced more apoptosis than the control group by flow cytometry,Hoechest method,TUNEL assay,Caspase-3 activity assay(P<0.05).Tumor necrosis factor-a(TNF-?)stimulated of GART expression levels of intestinal epithelial cells in a time and dose-dependent manner,peaked at the time point of 20 ng/ml stimulated for 24 hours.The apoptosis of intestinal epithelial cells transfected with siGART with SB230580(P38MAPK inhibitor)pre-treated was significantly decreased compared with si GART treated group(P<0.05).The expression of MEKK3 and MKK3 in siGART transfected intestinal epithelial cells were upregulated(P<0.05),while the expressions of MKK3 and p-p38 in siMEKK3 transfected intestinal epithelial cells were significantly down-regulated(P<0.05).Conclusions GART plays a key role in inhibiting apoptosis of intestinal epithelial cell and promoting its migration,thus maintaining the integrity of the intestinal epithelial barrier and may be a new treatment target of CD.