The Effects Of Curcumin On Intestinal Mucosal Barrier Function Of Rat Enteritis

Background/ObjectiveThe intestinal barrier functions a shielding band which can prevent the invasion of causative agents and toxin.Under the conditions of wound,opration,chemotherapy, radiotherapy,severe pancreatitis or long-term parenteral nutrition,the structure and the barrier function of intestinal mucous membrane may be seriously damaged,which may cause the intestinal mucosal barrier dysfunction,then causes the intestinal bacteria translocation,even induces systemic inflammation response syndrome (SIRS),at last result in multiple organs dysfunction syndrome(MODS ).But it is still difficult to detect the gut barrier function directly.The D-lactate,which may be produced by many germs,is metabolic end product of bacteria in gastrointestinal tract.Without any D-lactate dehydrogenase in the body of mammalian,the D-lactate from enterbacterium will go into blood and the level of D-lactate will increase when intestinal mucosa damages or mucosa permeability changes.Therafore,the examination of D-lactate in peripheral blood may reflect the damage and permeability of intestinal mucosa.DAO is a kind of endocellular enzyme with high activitiy,which exists in the plasm of chorionic villi in intestinal stratum supravasculare in human and all other mammals.When intestinal epithelial cells are injured,the release of endocellular DAO increases and to enters the intestinal intercellular space,the lymph vessel and the blood stream,and finally causes DAO in blood plasma to elevate.The activitiy of DAO in peripheral blood is stable.When small intestinal mucosal barrier nonfunction,the intestinal mucosa membrane cell falls off into the enteric cavity,then DAO enters lymph vessel and the blood stream in the intestines intercellular space,causes blood DAO to elevate. The activity of blood DAO may reflect the extent of damage and repair in intestinal tract.Leukocytic's over-activation is a very important bit of the IBD patho process. Leukocytic staying and exosmoseing on inflammation position depend on ICAM,s expressin and function in the surface of WBC and endothelial cell.ICAMS are glycoproteinwhich are synthesized by the cell and then assembled in the cell surface or secrete to the extracellular matrix,involving in cell-cell or cell-extracellular matrix interaction.And ICAM-1,belonging to the immunoglobulin superfamily, received the attention in recent years in the process of IBD.The expression of ICAM-1 in the normal intestine tissue are usually low in the vascular endothelial cell,in the intestines mucosa lamina propria and in the monocyte-macrophages in the lymph nodes.Myeloperoxidase(MPO) is a major presence in the neutrophil of an enzyme,its level of activity reflects the extent of neutrophil infiltration.Superoxide dismutase(SOD) is endogenous superoxide radical scavengers,their activity level can be reflected in part the ability of the body eliminate free radicals.Nowadays study show,the mitogen-activated protein kinases(MAPKs) cascade,especially p38 play a critical role in intestinal damage such as ischemic, inflammation and apoptosis,At present,it is still short of effective way to protect intestines mucosal barrier function.So it is a global topic today to search for natural medicines effective for various diseases,curcumin,as a low-toxitity natural meidcinal herb,has recently been documented at home and abroad to have anti-infection, anti-inflammation,anti-oxidization and free-radical removal effect.Curcumin is ratified as food additive by World Health Organization(WHO),Food and Drug Administration(FDA).Up-to-date study found that the widely used food additive curcumin is able to attenuate experimental colitis through a mechanism correlated with the inhibition of the activation of NF-κB and effects a reduction in the activity of p38 MAPK.Curcumin could prevent the increase in Caco-2 permeability,indicating that NF-κB activation was required for the TNF-α-induced increase in Caco-2 permeability.Curcumin decreases binding of Shiga-like toxin-1B on human intestinal epithelial cell line HT29 stimulated with TNF-αand IL-1β:suppression of p38, JNK and NF-κB p65 as potential targets.Curcumin significantly attenuated Stx-1 induced cell death.In this study,by developing rat models of enteritis and of intestinal epithelial damage,we assessed the protective role of curcumin on intestinal mucosal barrier function,the activation of epithelial intra- and extra-cellular MAKP signaling transduction and nuclear transcription factors,and the release of the inflammation and chemical mediators,with the objecitve of clarifying the molecular mechanism of curcumin's role in the intestinal barrier function,through in vivo and in vitra experiment systemsMethodsⅠThe protecitve role of curcumin on the intestinal mucosa of rats with methotrexate-induced enteritisEnteritis was induced in rats through peritoneal injection of methotrexate(20 mg/kg).The rats were randomly divided into 4 groups:normal control group (peritoneal injection of normal saline only),model control group(MTX, 20mg/kg),curcumin group(100mg/kg)and N-Acetylcysteine(NAC,150mg/kg) positive control group(100mg/kg).From the first day that the models were made, the drugs were intragastrically administrated once a day for 7 days,the rats were perfused with the specified dosage,and the rats in the normal control group and the model control group were injected with saline.Every day for the whole duration of the model making,diarrhea and hematochezia diarrhea as observed with the naked eye were checked.On the 6^th day,the rats were killed,blood samples were collected from the hearts,intestinal segments were taken and immersed in paraffin for fixation,embedding,slicing and HE staining,and then underwent the general histopathological observation.Finally,30-50mg of the intestinal mucosa were collected for epithelial RNA,total proteins and ribonuclear proteins extraction.1.The disease activity index(DAI),colonic mucosal damage index(CMDI) and histological score(HS) of the rats were observed and evaluated.2.The activity of SOD and MPO in the tissues of the small intestines in the rats was determined by colorimetry.3.The levels of plasma D-lactate and DAO of the small intestinal were detected by UV-spectrophotometry.4.The levels of mRNA expressions of TNF-α,IL-1β,ICAM-1 were detected by a semi-quantatitive assay RT-PCR.5.The level of mRNA expressions of IL-10 was detected by ELISA(enzyme linked immunosorbent assay).6.The expressions of phosphorylated IκB,p38,ERK,JNK proteins were determined with antiphosphospecific antiboday by using western blot.ⅡStudy on the protective role of curcumin on LPS-induced stress reaction of IEC-6 cellsWith lipopolysaccharide as the stimulator,IEC-6 cells in the logarithmic phase were divided into 4 groups at random:the normal control group,LPS group, curcumin pretreatment group(curcumin pretreatment) and p38 inhibitor(SB203580) pretreatment group(SB pretreatment).LPS(100ng/ml) was added to all groups except normal control group which was no any treatment;Sixty minutes before LPS was added to the Pre-curcumin and Pre-SB group,curcumin(20μmmol/1) and SB (20μmol/1) were added to the groups,respectively.Then the LEC-6 cells were incubated for 30 minutes.1.The levels of mRNA expressions of IL-10 was detected by ELISA(enzyme linked immunosorbent assay).2.The levels of mRNA expressions of TNF-α,IL-1β,ICAM-1 were detected by a semi-quantatitive assay RT-PCR.3.NF-κB DNA binding activity was evaluated by NF-κB activation- tronslocation dectection kit.4.Degradation of IκB in the cytoplasm was detected by Western blot.5.The expressions of phosphorylated MKP-1 was determined with antiphosphospecific antiboday by using western blot.ResultsⅠThe protecitve role of curcumin in the intestinal mucosa of rats with methotrexate-induced enteritis1.Effects of curcumin on the rats' symptoms and histology With the rats in the MTX group,diarrhea was observed on the 2nd day after the injection of MTX; on the 3rd day,hematochezia diarrhea was observed with the naked eyes in some rats,and on the 6th day,in all of them.The group pretreated with curcumin and the NAC group exhibited no distinct differences:on the 3rd day,diarrhea was observed but hematochezia diarrhea was not.On the 6th day,hematochezia diarrhea was found in all the rats.Observation of the histological sections revealed a high expansion and hyperemia with the mucosal and submucosal blood vessels,loss of crypt foci shape of the colon,withering and depilation of the villus and a wide range of infiltration of inflammatory cells typical of inflammation,with the rats in MTX group.Compared with the model group,the inflammation,loss of crypt loci shape of the colon,withering and depilation of the villus were all markedly less intense, and the infiltration of inflammatory cells were fewer.No differences were observed with the normal control group.The results above illustrated that curcumin,helping stop the infiltration of inflammatory cells and alleviate the inflammation reaction and symptoms of enteritis,had a marked therapeutical effect on enteritis and a protective role for the intestinal mucosa of the rats.2.Determination of MPO and SOD activity of the intestinal mucosaIt was revealed that the SOD and MPO activity of the intestinal mucosa in model group were significantly higher than in normal group during the same period (P=0.000) and that for both the Curcumin group and the NAC group,MPO activity could be lowered and SOD activity could be increased.Furthermore,it was demonstrated that curcumin markedly increased the intestinal mucosal SOD expression of the rats and lowered the MPO activity.The results illustrated that curcumin had anti-oxidization effect and could lessen the infiltration of the inflammatory cells,thus having a protective role for experimental rat enteritis.3.The effect of curcumin on mucosal intestinal permeabilityIt was revealed that the two levels of the MTX group were distinctly higher than those of the normal group,and those of the Curcumin group and NAC group were markedly lower than those of the MTX group in the same peirod(P=0.000).This proved that curcumin could improve the permeability of the intestinal mucosa and had a protective role in the rat intestinal barrier function.4.Influence of curcumin on the expression of TNF-α,IL-1β,ICAM-1 and IL-10.The levels of mRNA expressions of TNF-α,IL-1β,ICAM-1 were detected by a semi-quantatitive assay RT-PCR,and IL-10 were detected by ELISA(enzyme linked immunosorbent assay).The expressions of the above four were significantly higher in the MTX treated group than in the control group.The expression sof mRNA of TNF-α,IL-1β,ICAM- 1 of the curcumin group and NAC group were lower than those of the MTX group,but the expression of IL-10,higher.The expressions of all the four mentioned above exhibited no significant differences between the Curcumin group and NAC group.These data suggested that curcumin could inhibit the expression of TNF-α,IL-1βand ICAM-1,and up-regulate the expression of IL-10.5.Modulation of cell signalling pathway and IκB by curcumin in intestinal mucosaThe expressions of phosphorylated p38,ERK and JNK proteins were determined with antiphosphospecific antiboday by using Western blot.After MTX were gaven,marked phosphorylation of p38 was observed,while in the curcumin group and SB group,the phosphorylation was distinctly less,no distinct changes were observed in the phosphorylated JNK and ERK protein.Degradation of IκB in the cytoplasm was detected by Western blot.It was observed that the NF-κB was obviously activated,its expression was significantly stronger in the nuclei of MTX treated cells,and the IκB in the cytoplasm was markedly degraded.But the translocation of NF-κB and degradation of IκB were distinctly less in the curcumin group and NAC group than in the MTX group,and the activation of NF-κB in the curcumin group and NAC group,markedly less.ⅡStudy on the protective role and its mechanism of curcumin in LPS-induced stress reaction of IEC-6 cells1.Modulation of cell signalling pathway by curcumin in vitroThe expressions of phosphorylated MKP-1 proteins were determined with antiphosphospecific antiboday by using western blot.After LPS stimulation,marked phosphorylation of MKP-1 was observed,while in the curcumin pretreat group and SB pretreat group,the phosphorylation was distinctly increase.NF-κB DNA binding activity was evaluated by NF-κB activation-tronslocation dectection kit.Degradation of IκB in the cytoplasm was detected by Western blot.It was observed that the NF-κB was obviously activated,its expression was significantly stronger in the nuclei of LPS treated cells,and the IκB in the cytoplasm was markedly degraded.But the translocation of NF-κB and degradation of IκB were distinctly less in the curcumin pretreat group and SB pretreat group than in the LPS group,and the activation of NF-κB in the curcumin pretreat group and SB pretreat group,markedly less.2.Influence of curcumin on the expression of TNF-α,IL-1β,IL-10 and ICAM-1.The expressions of TNF-α,IL-1βand ICAM-1 were significantly higher in the LPS treated group than in the control group,and IL-10 was markedly lowerl.After pretreatment with curcumin and SB,the proinflammatory cytokines mRNA expressions were significantly decreased,and IL-10 was significantly increased.These data suggested that curcumin could inhibit the expression of TNF-α,IL-1βand ICAM-1,and markedly up-regulate the expression of IL-10.ⅢStatistical analysisAll data are presented as mean±S.D.of three independent experiments.Comparision of the effects of various treatments was performed using factor analysis,one-way ANOVA analysis of variance and a two-tailed t-test. Difference with a p value of＜0.05 were considered statistically significant. Conclusion1.Curcumin can protecte in the intestinal mucosa of rats with methotrexate-induced enteritis of small intestines,curcumin possessed the anti-oxidization effefct and could lessen the infiltration of the inflammatory cells.2.Curcumin could inhibit the expression of TNF-α,IL-1βand ICAM-1,and up-regulate the expression of IL-10 in methotrexate-induced enteritis of small intestines of rats and LPS-induced stress reaction of IEC-6 cells.3.Curcumin is able to inhibite the activation of NF-κB and effect a reduction in the activity of p38 MAPK in methotrexate-induced enteritis of small intestines of rats,but no effects on the expression of ERK and JNK protein.4.LPS alone enhances phosphorylation of MKP-1 and that curcumin in combination with LPS further enhances the levels of MKP-1 phosphorylation, compared to those observed in the presence of LPS alone.we also examined that curcumin could activate MAP-1 and subsequently diminish p38 phosphorylation and inhibits translocation of NF-κB in LPS -induced IEC-6.5.Curcumin is able to protecte the intestinal mucosa through a mechanism correlated with the enhances the levels of MKP-1 phosphorylation,compared and inhibition of the activation of NF-κB and effects a reduction in the activity of p38 MAPK,the subsequent reduction of the mRNA and ICAM-1 expressions of TNF-αand IL-1β,and the reinforcement of IL-10.