Therapeutic Strategy For Inflammatory Bowel Diesase Based On Blocking The Recruitment And Activation Of Colonic Immune Cells

Emerging evidence suggests that microRNA (miRNA)-mediated gene regulation influences a variety of autoimmune disease processes, including inflammatory bowel diseases (IBD), but the biological function of miRNAs in IBD remains unclear.In the first part of the experimental section, we examined whether miRNAs were differentially expressed in2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced and IL-10knockout (KO) chronic colitis and studied the potential functions of miRNAs that regulated pathological genes during the inflammation process. miRNA levels were assessed in TNBS-induced and IL-10KO colitic colon by microarray and qRT-PCR. The influence of miR-141on its putative target genes, CXCL12P, and inflammatory cell migration was investigated in cultures of colonic epithelia cells and colitic models. The role of miR-141was further studied in experimental colitis by intracolonic administration of miR-141precursors or inhibitors. An inverse correlation between miR-141and CXCL120was observed in the colons from colitic mice and CD patients. Further study demonstrated that miR-141directly regulated CXCL12P expression and CXCL12P-mediated leukocyte migration. Upregulation or downregulation of miR-141in the TNBS-colitic or IL-10KO colon regulated leukocyte infiltration and alleviated or aggravated experimental colitis, respectively. Additionally, colonic overexpression of CXCL12P abolished the therapeutic effect of miR-141in TNBS-induced colitis. The first part of the experimental section showed that miR-141targeting the CXCL12pathway is a possible mechanism underlying inflammatory cell trafficking during both experimental colitis and Crohn’s diseases (CD). Inhibiting colonic CXCL12expression and blocking colonic immune cell recruitment by using miRNA precursors represents a promising approach that may be valuable for CD treatment.In the second part of the experimental section, we investigated whether miR-16can bind to the AU-rich elements (ARE) of TNF-α and IL-12p40to regulate their expressions. The influence of miR-16on its putative target genes, TNF-α and IL-12p40was apporved in cultures of peritoneal macrophages. Based on the fact that colon macrophages are the one of main sources of TNF-α and IL-12p40and the colonic macrophage targeted delivery system which were previously established, miR-16precursor was delivered into the colonic macrophages by galatose modified low molecular weight chitosan (G-LMWC) to study the therapeutic effects of miR-16in TNBS induced colitis model. The levels of TNF-α and IL-12p40were significantly decreased in colon tissues of mouse colitis model after intracolonic administration of miR-16precursor/G-LMWC complex. Body weight change, histological score and cytokines determination indicated that the symptom of colinic inflammation was greatly alleviated. All the results taken together demonstrate that miR-16precursor with macrophage targetting property will be potentially applied in the clinical therapy of IBD in the future.Reactive oxygen species (ROS) exhibit a key role in the pathogenesis of IBD.3,3’-Diindolylmethane (DIM) can protect against oxidative stress via a breast cancer susceptibility genel (BRCA1)-dependent manner. The aim of the third part of the experimental section was to examine the therapeutic effects of DIM in experimental colitis and investigate the possible mechanisms underlying its effects on intestinal inflammation. The therapeutic effects of DIM were studied in TNBS-induced colitis. Pathological markers of colitis severity, antioxidant activity and ROS generation in colonic tissue were measured. The impact of DIM on ROS-induced endothelial vascular cell adhesion molecule1(VCAM-1) expression and leukocyte-endothelial cell interaction was further investigated in cultures of endothelial cells and TNBS-induced colitis model. Administration of DIM was demonstrated to attenuate experimental colitis, as judged by pathological indices. DIM could effectively stimulate the expression of BRCA1in vitro and in vivo, reduce ROS generation, leading to the inhibition of VCAM-1expression and leukocyte-endothelial cell adhesion, and finally resulted in an alleviation of experimental colitis. DIM has shown anti-IBD activity in animal models by inhibiting ROS-induced VCAM-1expression and leukocyte recruitment via a BRCA1-dependent antioxidant pathway, and thus may offer potential treatments for IBD patients.