| Background and aims:IBD,including UC and CD,is a kind of chronic and recurrent inflammatory disease occurred in gastrointestinal tracts and characterized by recurrent abdominal pain,diarrhea,hematochezia,weight loss,and fatigue.Patients suffered from IBD in their work and daily life and Too many medical and financial resources have been spent on the management of IBD.The specific pathogenesis of IBD remains unclear,several hypotheses have been proposed.Genetics,diet,gut microbiota and psychosocial factors all play important roles in the pathogenesis of IBD.IBD was once mainly found in people in developed countries such as Europe and USA.However,in recent years,the incidence of IBD is increasing in Asian countries such as China and in these countries,people suffer from psychological stress with the the development of economy.Previous studies have confirmed that negative psychological factors such as anxiety can increase the risk of IBD.In addition,anxiety is also the most common psychological comorbidity in IBD patients.About 30%of IBD patients suffered from anxiety or depression,and these patients have more aggressive disease progression,poorer treatment effect and longer hospital stay.Activation of the HPA axis is one of the main markers of the body’s response to anxiety,and previous studies have shown that serum glucocorticoids levels(cortisol in humans,corticosterone in mice)are significantly increased in IBD patients and animal models,and are significantly correlated with the degree of disease activity.Cortisol(corticosterone in mice)secretion is primarily dependent on HPA axis activation and is a marker of HPA axis activation.The role of anxiety in the development of IBD remains unclear.Therefore,this study aims to clarify the relationship between the focal level of UC patients and the degree of disease activity and treatment,and to clarify the role of anxiety on the occurrence and development of colitis using animal models.Methods1.Statistics and analysis of clinical information(1)Patients diagnosed with UC in the Department of Gastroenterology and endoscopy room of Qilu Hospital Affiliated to Shandong University were enrolled.Clinical information such as age,gender and disease severity were collected.Mayo score and self-rating Anxiety Scale(SAS)were scored to evaluate the anxiety level of UC patients and its correlation with disease severity.(2)Main information such as the frequency of defecation,blood in the stool,and endoscopic mucosal manifestations were collected through consultation and visits to the outpatient and inpatient electronic medical record system.The IBD physician who had visited the patient in the past scored the patient’s overall condition,and finally,the Mayo score was used to judge the degree of disease activity and grade the patient.(3)After fully explaining the test content to the patient,the anxiety of the patient was assessed with the SAS self-test Anxiety Scale.The patients answered the questions independently in a quiet environment.After answering the questions,the scores were summarized to get the original SAS score.The original score was multiplied by 1.25 and rounded to the whole number as the standard score,according to which the anxiety of the patients was graded.(4)Spearman rank test was used to analyze the correlation between patients’ anxiety grade and disease activity degree to determine whether there was a significant correlation between them.2.Construction of animal models(1)The construction of anxiety model.Anxiety was induced using a wrap-restraint stress(WRS)procedure,which was performed by placing mice into 50-ml tubes with a hole for ventilation.All immobilization operations were performed for consecutive 7 days at fixed periods(10:00 am to 1:00 pm)to eliminate the effects of circadian rhythms.After immobilization,the mice were placed in their home cages.(2)Colitis was induced by OXA enema.Anesthesia was induced by intraperitoneal injection of 1%pentobarbital sodium at a dose of 50mg/Kg.100ul 1%OXA(dissolved in 50%ethanol).After sedation,mice in the control group and the restraint stress group were given 100ul of 1%OXA(dissolved in 50%ethanol)for enema,and the mice were held in a head-down vertical position for 20 min before returning to their cages to ensure that the oxazolone was retained in the colon.Heating pad was used during enema procedure.(3)Enzyme-linked Immunosorbent Assay(ELISA)was used to detect the effect of restraint stress on serum corticosterone level in mice,and to evaluate the activity of HPA axis in mice.The changes of body weight,colonic length,histopathological score,and the expression levels of inflammatory cytokines and intestinal barrier proteins were evaluated in the anxiety model and control mice after OXA enema.Results:1.A total of 79 patients between the ages of 18 and 71 were enrolled in the study.There were 41 males(51.9%)and 38 females(48.1%),6 patients(7.6%)had lesions confined to the rectum,24 patients(30.4%)had lesions involved the left colon,and 49 patients(62%)were extensive colon type.2.Mayo score was performed for each patient according to clinical symptoms,endoscopic manifestations,and the assessment of IBD physician.The score showed that there were 11 patients in remission and 68 patients in active phase,including 19 patients with mild activity,31 patients with moderate activity,and 18 patients with severe activity.The anxiety of each patient was evaluated according to the SAS standard score:75.9%UC patients suffered different levels of anxiety,19 patients had no anxiety,10 patients had mild anxiety,27 patients had moderate anxiety,and 23 patients had severe anxiety.3.Among the patients without anxiety,42.1%were in remission,47.4%were in mild activity,5.3%were in moderate activity,and 5.3%were in severe activity.Among the patients with mild anxiety,20%were in remission stage,33.3%were in mild activity stage,40%were in moderate activity stage and 6.7%were in severe activity stage.Among the patients with moderate anxiety,11.5%had mild activity,65.4%had moderate activity and 23.1%had severe activity.Among the patients with severe anxiety,10.5%had mild activity,36.8%had moderate activity and 52.6%had severe activity.Correlation analysis between SAS score and Mayo score of disease activity showed that SAS score was positively correlated with Mayo score.4.Among the 30 patients with poor response to glucocorticoid treatment,90%had SAS scores greater than or equal to 50,and the proportion was 66.7%in the group with fair response to glucocorticoid treatment.The rate of poor response to glucocorticoid therapy was 38.5%among patients with SAS scores of 50 or greater and 18.8%among those with SAS scores of less than 50.After stratified analysis of patients with SAS score greater than or equal to 50,the proportion of poor effect of hormone therapy was 20%in patients with mild anxiety,52.2%in patients with moderate anxiety,and 68.4%in patients with severe anxiety.5.Compared with the control group,the wrap restraint stress group had higher serum corticosterone level.More weight loss,shorter colonic length,higher pathological score,higher inflammatory cytokines level and lower tight junction protein expression level were observed among anxiety model mouse after induced enteritis.Conclusion:1.About 75.9%of UC patients have anxiety symptoms,and the incidence of anxiety in active UC patients is significantly higher than that in remission patients.2.Patients’ anxiety score was positively correlated with disease activity score.Patients with higher anxiety score had higher degree of disease activity and higher probability of poor effect of glucocorticoid treatment.3.The HPA axis of anxiety model mice was significantly activated,and the degree of intestinal inflammation was more severe after OXA enema.Background and aims:The hypothalamic-pituitary-adrenal(HPA)axis,as the center of neuroendocrine regulation when the body is exposed to stress and other stimuli,has been shown to be overactivated in negative emotions such as anxiety.Cortisol(corticosterone in mice),also known as "stress hormone",is recognized as a marker of HPA axis activation and mediated the regulation of metabolism and immun system to cope with changes in external environment during conditions such as stress.The levels of cortisol in serum and saliva of patients with inflammatory bowel disease are significantly increased and positively correlated with the degree of disease activity.Previous studies have showed the immune disorder in UC patients is mainly characterized by overactivation of typeⅡ immunity.Previous studies have confirmed that the levels of IL-5/IL-13 in the mucosa and serum of UC patients are significantly higher.In addition,patients with higher levels of IL-13 in the mucosal tissues showed more aggressive disease progression,poor treatment effect and poorer prognosis.In addition to adaptive immunity,innate lymphoid cells(ILC)are also important components of homeostasis,among which Group 2 innate lymphoid cells(ILC),ILC2 is mainly distributed in the lung,intestine and other mucosal tissues and organs.It expresses specific transcription factor GATA3 and can continuously secrete IL-5,IL-13 and other type Ⅱimmune-related cytokines.In Oxazolone(OXA)-induced type Ⅱ immune-dependent enteritis model,overactivation of ILC2 has been shown to exacerbate OXA-induced intestinal inflammation.Glucocorticoids have been confirmed to promote the shift of Th1/Th2 balance to Th2 in vivo.Therefore,we hypothesized that anxiety may promote the occurrence and progression of enteritis by regulating the number of immune cells including Th2 and ILC2 and related immune dysfunction through cortisol.Methods:1.Anxiety model was established by a 7 days wrap restraint stress(WRS).The proportion and functional changes of colon lamina propria lymphocytes in mice with or without restraint stress was analyzed.Mice in the experimental group were subjected to bondage stress treatment for 7 consecutive days,and mice in the control group and experimental group were killed 7 days later.Lamina propria lymphocytes(LPL)were extracted by density gradient centrifugation.The effect of restraint stress on the proportion and function of various immune cell subsets in the lamina propria was analyzed by Flow Cytometry(FACS).2.Dexamethasone(DXMS,5mg/Kg)was injected intraperitoneally to mimic the activation state of HPA axis.After 7 days of continuous injection,mice were sacrificed and colon LPL was extracted by density gradient centrifugation.The effect of HPA axis activation on the proportion and function of various immune cell subsets in the lamina propria was analyzed by Flow Cytometry(FACS).3.Mice were given OXA to induce enteritis.After intraperitoneal injection for 7 days,mice in the control group and the DXMS injection group were given 100ul of 1%OXA(dissolved in 50%ethanol)for enema,and the head was kept vertically upright for 20 minutes,during which heating pad was used for heat preservation.After 20 minutes,the mice were placed horizontally in the cage and the cage temperature was maintained.One day later,the mice were sacrificed and the changes in body weight and colon length were evaluated.The colonic tissue was collected:the colonic tissue was fixed with 4%paraformaldehyde for 24 hours and then HE staining was performed to evaluate the severity of enteritis.RNA and protein were extracted from colonic frozen in liquid nitrogen to evaluate the levels of colonic inflammatory factors and barrier proteins.4.Glucocorticoid receptor(GR)inhibitor RU486 was intraperitoneally injected into mice in the simulation group of binding stress and HPA axis activation to verify whether the effects of binding stress and HPA axis activation on lymphocytes were mediated by GR.5.Colonic LPL was extracted from wild-type mice and cultured with different concentrations of DXMS.Flow cytometry was used to analyze the direct effect of DXMS on LPL.6.IL-13/SATA6 pathway inhibitor was administered.IL-13/SATA6 pathway inhibitor(RCM-1,1.7mg/Kg)was given intraperitoneally to WRS and DXMS injection mice for 7 consecutive days.After 7 days,the mice were given 1%OXA 100ul enema.After 1 day,the mice were sacrificed to evaluate the changes in body weight and colon length.The colonic tissue was collected:the colonic tissue was fixed with 4%paraformaldehyde for 24 hours and then HE staining was performed to evaluate the severity of enteritis.To evaluate the levels of colonic inflammatory factors and barrier proteins,RNA and protein were extracted from colonic frozen in liquid nitrogen.Results:1.Compared with the control group,the function of colonic ILC2 to secrete IL-5/IL-13 in the restraint stress group was significantly increased,while the secretory functions of Th2 and NKT cells were not significantly changed.2.Compared with control group,HPA axis activation can increase the function of colonic ILC2 to secret more IL-5/IL-13,and the function of Th2 and NKT cells were not significantly changed.3.Compared with control mice,HPA axis activation mice had more significant weight loss,shorter colonic length,higher colonic pathological score,higher levels of inflammatory factors TNF-α and IL-6,and lower expression levels of barrier proteins ZO-1 and Occludin after OXA enema.4.Intraperitoneal injection of RU486 could not eliminate the influence of restraint stress or HPA axis activation on ILC2 function in mice.5.In vitro,DXMS could inhibit IL-5/IL-13 secretion by ILC2s in a dose-dependent manner.6.Compared with mice without RCM-1 rejection,WRS and DXMS mice received RCM-1 injection had less weight loss,longer colon length,lower colonic pathological score,lower levels of inflammatory cytokines TNF-α and IL-6,and higher expression levels of barrier proteins ZO-1 and Occludin.Conclusion:1.HPA axis over-activation induced under anxiety can significantly increase IL-5/IL-13 secretion ability of colonic ILC2 in mice and aggravate OXA-induced colitis.2.Blocking IL-13/SATA6 signaling pathway can reverse the aggravation of enteritis caused by WRS and DXMS injection. |
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