Stress And Lubiprostone Affect The Intestinal Immune Function Of The Rats

Objective: Stress is a general body response to initially threateningexternal or internal demands, involving the mobilization of physiological andpsychological resources to deal with them. The major mediators of stresseffects are the sympathetic nervous system and corticotrophin releasinghormone, adrenocorticotropin, and cortisol, which arise following activationof the hypothalamic-pituitary-adrenal axis. It is a proven fact that stressincreases the susceptibility to inflammatory disorders, including those ofinfectious etiology. The magnitude and duration of stress-induced elevationsin catecholamine and glucocorticoid hormones can have significant effects onimmune cell distribution and function. More and more people focus on thei n f l u e n c e o n t h e i m m u n e s y s t e m b y t h e s t r e s s.The association of acute and chronic stress related with the susceptibilityto the functional gastrointestinal disease was proved. Gastrointestine not onlyworks as the place for the absorption of nourishment, but also the importanttarget organ at the stress situation, as well as the important immune organ. Theintestinal mucosal barrier which builds up with the intestinal epithelial cells,tight junction, adhesion junction and gap junction is work as a mechanicalbarrier. It acts as the first line to block the invasion from the pathogen. TheSecretory immunoglobulin A (S-IgA) is a member of the adaptive secretoryimmune system, which made the intestinal immune system works togetherwith the other immune system. Receptors of glucocorticoids andcatecholamines are found on almost all the immune cells, the increased plasmaand tissue concentrations of these hormones during stressful conditions alterthe function of these cells, at the same time; immune cells can affect then e u r o e n d o c r i n e s y s t e m b y r e l e a s i n g t h e c y t o k i n e.Lubiprostone is a novel drug for adult chronic idiopathic constipation Which investigated by Sucampo and Takeda pharmaceuticals company. Thisdrug can selective active the ClC-2channel which exist at the gastrointestinaltract epithelium tissue, increase the intestinal motive. It was found thatLubiprostone can prove the visceral hyperalgesia.Is the stress-induced change at the immune system depended on thetimeline? At the period of the gastrointestinal disease which induced by thechronic psychological stress, the mechanical barrier is damaged or not? Howabout the immune barriers? Can Lubiprostone reverse the dysfunction whichinduced by the chronic psychological stress?To answer these questions, we use SD rats to set up the animal model.The immune targets were examined at the different time point. Base of theresults, we try to find the target molecular during this change, and the possiblemechanism of the Lubiprostone.Methods：1Set up the stress-induced animal model which can affect thegastrointestinal function. The model would be used to study the changeswhich induced by stress. A time course should be draw from these results.Set up the animal models, observe the general condition of the rat whichaffect by stress. The observation about the fecal pellet output and the VMR toCRD can determine the model’s usage.The serum of rats was collected at different time point. The circulatingcytokine IL-1β、IL-2、INF-γ、IL-6、TNF-β level were detected by ELISA. Theresults used to setup a timeline about the stress-induced change at theintestinal immune system.SuperArray was used to detect the level of many kinds ofneurotransmitter, kinase and immune molecular which can be affected bychronic stress2the dynamic effect on the rats’ immune function by psychologicalstressReal-time PCR and Western Blot were used to examine the tissuecytokine IL-1β、IL-2、INF-γ、IL-6、TNF-β which gathered at different time. The SIgA of the intestinal juice was examined by ELISA3the effect of the enteric epithelium enteric epithelium protein whichinduced by chronic stressThe dose was determined according the paper from Takera and results.The rat were feed Lubiprostone everyday during the WAS.The enteric epithelium tight junction molecular of the rats was examinedby Real-time PCR.The enteric epithelium tight junction proteins of the rats were examinedby Western Blot and Immumofluorescence.The SIgA of the chronic–stress rat which treated by Lubiprostone wasexamined by ELISAResults:1Set up the stress-induced animal model which can affect thegastrointestinal function. The model would be used to study the changeswhich induced by stress. A time course should be draw from these results.Chronic stress inhibits the rat gain weight; the inhibitory action woulddisappear in several weeks.Compare with the control group, the serum level of IL-1β、IL-6、INF-γ at1h after1WAS were significantly increased, as well as the serum level ofIL-1β、IL-6at24h after1WAS were significantly increased, the serum level ofIL-1β、IL-2、IL-6、INF-γ、TNF-α at24h or72h after10WAS were nosignificantly increased. Compare with the control group.DNA assay shows some colonic molecular were upgrade, such asAdenosine A1receptor、BDNF、CCL12、CD200、Claudin2、CDK2、HDAC2、HDAC4、IL-6、Integrin-alpha M、P2RX3. Aurora kinase A、BAZ1b、Claudin1、Claudin6、CB2、CSF1、DMNT1、PRMT1and some others weresignificantly decreased after the chronic stress.2the dynamic effect on the rats’ immune function by psychologicalstressReal-time PCR shows the colonic level of IL-1β、IL-6were significantlyincreased after the acute or chronic stress, the colonic level of IL-2and IL-10 were significantly increased at24h after1WAS, the colonic level of TNF-αwere significantly increased at1h after1WAS and24h after1WAS, thecolonic levels of INF-γ were no significantly increased which compared withthe control group.Western Blot shows the colonic level of IL-1β、IL-6were significantlyincreased after the acute or chronic stress, the colonic level of IL-2weresignificantly increased at24h after1WAS and24h after1WAS, the colonlevel of TNF-α were significantly increased at1h after1WAS and24h after1WAS, the colonic level of INF-γ and IL-10were no significantly increasedafter the acute or chronic stress. Most of the results match the Real-time PCR.ELISA shows the level of SIgA was significantly increased at1h and24hafter1WAS, the level of SIgA was no significantly increased after the chronicstress treatment.3The effect of the enteric epithelium enteric epithelium protein whichinduced by chronic stressWestern Blot shows the level of the enteric epithelium tight junctionprotein ZO-1、Claudin-1、Occludin were significantly decreased after thechronic stress, the level of Claudin-2were significantly increased after thechronic stress. Lubiprostone may partly reverse the change of Claudin-1orOccludin by chronic stress treatment.ELISA shows the level of SIgA was no significantly change after thechronic stress and the Lubiprostone treatment.Conclusions:1Chronic psychological stress induces the rat’s visceral hyperalgesia.2Acute stress induces the serum level of IL-1β and IL-6weresignificantly increased, both acute and chronic stress induces the colon levelof IL-1βand IL-6were significantly increased.3The level of the enteric epithelium tight junction protein ZO-1,Claudin-1, Occludin were significantly decreased after the chronic stress, thelevel of Claudin-2were significantly increased after the chronic stress, whichlead to the damage of the mucosal barrier. Lubiprostone may partly reverse the change of Claudin-1and Occludin which caused by chronic stresstreatment.4Acute stress induces the level of SIgA was significantly increased; thelevel of SIgA was no significantly change after the chronic stress and theLubiprostone treatment.