Investigation Of The Protective Effect Of Recombinant Catalase Of Helicobacter Pylori On Intestinal Inflammation In Experimental Rat

Background & Objectives: Reactive oxygen species (ROS) including superoxide anion, hydrogen peroxide and hydoxyl radical are the natural products of aerobic metabolism of animal and human cells. It is involved in anti-infectious process and cleared by a series of constitutively expressed antioxidant enzymes, such as Mn-SOD, Cu, Mn-SOD, CAT and GSH-Px, physiologically to keep homeostasis . However, the over-accumulation of ROS are able to damage cells by injuring mtDNA, protein and lipids pathologically.Studies have shown that oxidative injury involved in the genesis of inflammation, cancer as well as some degenerative diseases, while the effect of antioxidants on the prevention and therapy of many diseases has been verified by a large volume of reports.Intestinal inflammation is accompanied by the increase of oxidative stress. Some antioxidants including catalase have displayed good anti-inflammatory effect in animal model. We have prepared recombinant4catalase of Helicobacter pylori (H. pylori) (rffpCAT) with high activity previously. We investigate its effect on the prevention and therapy of experimental rat colitis and the putative mechanisms in the current study. Our works focused on the following aspects: 1. To evaluate the protective effects of r/^籆AT on the intestinal inflammation of TNBS-induced colitis of rat. 2. To investigate apoptosis of rat intestinal epithelial cells with or without the treatment of rHpCAT. 3. To examine the effects of rflpCAT on cyclooxygenase (COX-2) expression and NF-KB activation in intestinal epithelial cells of rat.Methods: 1. Sprague-Dawley(SD) rat colitis was induced by enema inoculation of TNBS (trinitrobenzene sulfonic acid). All rats were randomly divided into the following five groups: (1) Control: routinely fed without any treatment. (2) Model only: TNBS treatment alone. (3) 5-ASA (5-Aminosalicvlic acid) treatment: TNBS treatment followed by the administration of 5-ASA intra-anally. (4) rHpCAT treatment: TNBS treatment followed by the administration of rHpCAI intra-anally.(S) r/fpCAT prevention: intra-anal administration of rHpCAT followed by the administration of TNBS orally. Histological changes of the rat colonic mucosa were observed under light as well as the electro-microscope. 2. Apoptosis of intestinal mucosa was determined by in situ TUNEL assay and Hoechst 33258 fluorescent staining. 3. The expression of COX-2 by rat colonic mucosal tissue was detected by immunohistochemical staining, RT-PCR as well as western blotting methods. 4. The activation of NF-icB in the intestinal epithelial cells was evaluated by electrophoretic mobility shift assay (EMSA). 5. The generation of intracellular reactive oxygen species(ROS) of intestinal epithelial cells was detected by flow cytometry after staining with 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA).Results: 1. One week after the administration of TNBS, all rats developed severe intestinal mucosa injury, including elucidation, erosion and ulceration. Both rHpCAI and 5-ASA treatments inhibited intestinal inflammation significantly (PO.01), while rHpCAI prevention generated most significant suppression. 2. TNBS induced obvious apoptosis in intestinal epithelial cells. However, both rHpCAT and 5-ASA treatments suppress TNBS-induced apoptosis considerably. The apoptotic index of rats with rHpCAI prevention was significantly lower than that of rHpCAI and 5-ASA treatments (P<0.05). 3. TNBS-induced inflammation up-regulated the expression of COX-2 in intestinal mucosa. Administration with rHpCAI and 5-ASA were down-regulated the induced COX-2 expression. Moreover, the expression of COX-2 mRNA and protein in rHpCAT prevention rats was the lowest among all groups, wherever no difference was found between 5-ASA and rHpCAI treatments. 4. NF-DB binding activity to COX-2 promoter sequence was up-regulated by TNBs and inhibited by the treatment with rHpCAI considerably. 5. ROS was stimulated in intestinal epithelial cell line (LOVO and SW480) after the exposure to prooxidantsO and -HO). It is suppressed by rHpCAI in...