Investigation On The Protective Mechanism Of Catalase On Intestinal Epitheial Cells

The epithelial cell is the most important components of intestinal barrier .The destroy of intestinal barrier is accompanied by the increase of reactive oxygen species (ROS).As the second messenger for cellular, ROS can activate many cell signal pathways, which modulate the secretion of pro-inflammation cytokines and chemokines that can destroy the intestinal barrier. Catalase(CAT) is a hydrogen peroxide scavenger, and can break down hydrogen peroxide to water and oxygen. During the process of inflammation CAT can scavenge ROS, then protect the cell. We cloned and purified the Helicobacter pylori(Hp) catalase during the study on Hp , and found the activation of our cloned CAT was better than the bovine hepatic catalase which was bought from Sigma company. In the present study, we want to investigate the protection mechanism of CAT (cloned from Hp and bought from Sigma company) on the intestinal barrier in vivo and in vitro, including the activation of Extracellular Signal Regulated Kniase (ERK) and Nuclear Factor kappa B (NF-kB) ,the secretion of pro-inflammation cytokines and chemokines, the apoptosis of intestinal epithelial cells.The protective mechanism of CAT on SW480 cells stimulated by lipopolysaccharideSW480 cells were devided into 6 groups at random and serum-starved over night prior to treatment , then treated with LPS (100ng/ml )for 30 minutes, The bovinehepatic CAT (200U/ml) was added prior to 30minutes or at the moment of the stimulation of LPS. The Hp-CAT was collected and added according to the activation same as the bovine hepatic CAT. The stimulation was finished 30 minutes later. Effection of CAT on apoptosisUsing flow cytometry to detect apoptosis of cells , apoptosis rate of cells which treated with LPS alone was increased significantly (P<0.01), but the rate was decrease after pretreatment with CAT (P<0.05), there were no differences between the LPS group and the CAT treat group. The changes of mitochondria and apopotosis of cells were observed by transmission electron microscopy, there were remar(?)ble mitochondrial swelling and apoptosis in cells which treated with LPS , the mitochondrial swelling and apoptosis were not observed in cells which pre-treated with CAT, but could be observed in cells treated with CAT. The above results would suggest that CAT could protect the mitochondrion and decrease the apoptosis. Influence of CAT on the expression of TNF-α,IL-1β,IL-8 and COX-2.The expressions of mRNA were detected by a semi-quantatitive assay RT-PCR. The expressions of TNF-α,IL-1β and IL-8 mRNA were significantly higher in the I PS treated group than in the control group (P<0.01). After pretreatment of CAT, the proinflammatory cytokines mRNA expressions were significantly decreased(P< 0.05). The COX-2 protein expression was determined by immunohistochemical staining and Western-blot . Normal cells were stained very gently positive ,becaise COX-2 expresses in the colonic cancer cells. Having been stimulated by LPS for 30 min, the cells shrinked and were significantly positive stained. After pretreatment of CAT, cells were stained positive,but was much gentle than LPS group , there were no differences between LPS group and CAT treated group.These data suggested that CAT could inhibit the expression of TNF-α IL-1β, IL-8 and COX-2. Modulation of cell signal pathway by CAT in vitroThe translocation of ERK was observed by using laser scaning confocal microscopy (LSCM), many translocations were observed after the stimulation of LI'S , but the translocations were forbidden by the pre-treatment of CAT.The phosphorylation of ERK 1/2 was determined with antiphosphospecific antiboday by using western blot, phosphorylation of ERK was much significant after the stimulation by LPS , but after pretreatment with CAT, the phosphorylation was decreased. The NF-kB expression, determined by immunohistochemical staining with a NF-kB antibody, was stronger in the cytoplasm than in the nuclei in the control. The NF-kB expression was significantly stronger in the nuclei of LPS treated cells. When CAT was pretreated, the number of positive cells was significantly decreased. The degaradation of IkB in the cytoplasm, detected by Western blot analysis, the expression of IkB was weaker in the LPS treated cells than in the control. After CAT was pretreated, the IkB expression in the cytoplasm was significantly stronger than that of in the LPS treated cells, but weaker than in the control. NF-kB DNA binding activity was evaluated by electrophoretic mobility shfit assay (EMSA). NF-kB was activited significantly in LPS treated group, After pretreatment of CAT, the activation of NF-kB was significantly weaker than that of LPS treated group, but stonger than that of control group.The above results would suggest that CAT could inhibit the activation of NF-kB and the translocation and phosporylation of ERK .protection of CAT on intestinal barrier with ulcerative colitis in ratsUlcerative colitis (UC) was induced in rats with the oral innoculation with dextran sulfate sodium (DSS) for seven days, For the study of CAT effects, the rats were randomly divided into groups of 6.For prophylactic treatment, mice were treated with CAT 1 week before the administration of DSS.The CAT was diluted in PBS and was given for 1x10 U/Kg body weights by intracolonic administration.For therapeutic treatment,CAT administration began on the day of treatment with DSS.Rats were sacrificed after drinking DSS for 7 days.The dose of Hp-CAT was according to the activation of bovine hepatic CAT. Effection of CAT on the stool and histologyThe symptoms were recorded daily, diarrhea was observed on the second day in rats treated with DSS , and hematochezia was found in all rats in the end. There was diarrhea on the third day in rats pre-treated with CAT, but hematochezia diarrhea was not found during this period .There were no differences between CAT treated group and DSS group. Histology was observed with light microscopy , there was infiltration of neutrocytophilia and lymphocythemia with loss of integrity of the colon mucosa in the gland in rats treated with DSS , after pretreatment with CAT, the inflammatory cell infiltration and loss of integrity were fewer than that of DSS group. Theobservation of scanning electromicroscopy revealed that the villi and epithelium were lost in rats treated with DSS , goblet cells and basement tissue were exposed, th;re were no significant loss of villi and epithelium after pretreatment with CAT , but he secretive granules were increased. Effection of CAT on the apoptosis of intestinal epithelia cells in ratsThe apoptosis of intestinal epithelial were detected by TUNEL probe , there w ;re many specific stained cells in rats treated with DSS , the numbers of specific staii ed cells were decreased in rats pretreated with CAT. This data suggest that CAT coild protect cells from apoptosis in vivo. Influence of CAT on the expression of TNF-a,IL-ip,IL-8 and COX-2.The expressions of mRNA were detected by a semi-quantatitive assay RT-PCR. The expressions of TNF-a,IL-1 Pand IL-8 mRNA were significantly higher in the DSS treated group than in the control(P < 0.01). After pretreatment of CAT, I he proinflammatory cytokines mRNA expressions were significantly decreased(P< 0.05). The COX-2 protein expression was determined by immunohistochemiral staining and Western-blot . The intestinal epithelia cells were significantly positive stained in rats treated with DSS .After pretreatment of CAT, cells were stain ;d positive,but was much gentle than DSS group , there were no differences between DSS group and CAT treated group.These data suggested that CAT could inhibit t le expression of TNF-a,IL-1 p, IL-8 and COX-2. Modulation of cell signal pathway by CAT in vitroThe phosphorylation of ERK1/2 was determined with antiphosphospecij ic antiboday by using western blot, but there were no results detected (data were n3t given). The NF-kB expression, determined by immunohistochemical staining with a NF-kB antibody, the NF-kB expression was significantly stronger in the nuclei )f DSS treated cells. When CAT was pretreated, the number of positive cells wis significantly decreased. The degaradation of IkB in the cytoplasm detected ty Western-blot analysis, the expression of IkB was weaker in the DSS treated cells thrn in the control. After CAT pretreatment, the IkB expression in the cytoplasm was significantly stronger than that of in the DSS treated cells, but weaker than that in ti.e control. NF-kB DNA binding activity was evaluated by electrophoretic mobility shiit assay (EMSA). NF-kB was activited significantly in DSS treated group, Aft<;rpretreatment of CAT, the activation of NF-kB was significantly weaker than that of DSS treated group, but stonger than that of control group.The above results would suggest that CAT could inhibit the activation of NF-kB in rats intestinal epithelial cells with UC.The phosphorylation of ERKl/2 is a early stage of stimulation.