Macrophage Autophagy Links Corticotropin Releasing Hormone To IBD Deterioration

Objective:The incidence of inflammatory bowel disease in China is increasing year by year.It brings great burden to the patients.The pathogenesis is unclear.At present,classic immunosuppressive agents and biological therapy cannot really cure the disease.There is a lack of efficacy for some patients with recurrent conditions,which needs further research and exploration.Psychosocial stress has been proved to be a critical factor in the occurrence and development of inflammatory bowel disease.Severe patients usually show higher stress level,obvious emotional and psychological problems as well as the disease activity,intestinal mucosal ulcer and inflammatory infiltration.The downstream mechanism has not been fully elucidated.Previous studies of our group have proved that stress factors can aggravate IBD via promoting macrophage autophagy.The purpose of this study is to further explore the ways in which psychosocial stress factors play a role in IBD and the effect of autophagy on inflammatory bowel disease model.Method:1.Clinical data and sample analysis and testingAmong the patients,6 mild/moderate patients and 6 severe patients were selected to compare their CPSS score and modified Mayo score.The mucosal lesions,histological staining,M1 macrophage infiltration and autophagy markers in patients with different CPSS scores were compared and analyzed.The infiltration of macrophages and the expression of CRH receptor in intestinal biopsy specimens of normal tissues and patients with ulcerative colitis were detected.2.DSS-induced mouse IBD model related experimentsCorticotropin-releasing hormone was used to simulate the role of psychosocial stress factors.In vivo,we established the mouse IBD model by DSS.The mice were randomly divided into groups.Certain groups were injected intraperitoneally with CRH every day to make the mice in a state of stress.The changes of body weight and disease activity index in each batch of mice were recorded.The intestinal epithelial damage and HE score were observed by terminal colon histomorphology staining.At first experimental mice were used to verify the aggravating effect of CRH on DSS mouse model.They were divided into four groups:CON,CRH,DSS and DSS+CRH.The expression of intestinal inflammatory factors and the content of autophagy markers were detected.The levels of CRH receptor CRHR1and CRHR2 in intestinal tissue were measured.Then the mice were injected with CRH receptor 1 antagonist antalarmin and CRH receptor 2 antagonist astressin₂-B respectively to observe the effect of blocking CRH in the intestinal tract of inflammatory mice.After that mice were set up with the experimental group injected with the autophagy inducer rapamycin.Afterwards mice were injected with autophagy inhibitor chloroquine.Finally mice were treated with another autophagy inhibitor 3-MA.The changes of intestinal autophagy level,mucosal barrier and inflammatory factor m RNA were detected after blocking the autophagy pathway.3.Mouse macrophages and colonic organoidsBone marrow derived macrophages and colonic organoid were isolated and cultured.The inflammatory model of BMDMs in vitro was established by LPS stimulation.The intestinal barrier and cell proliferation of colonic organid were observed after co-culture with BMDMs.The autophagy level of macrophages and the expression of inflammatory cytokines m RNA in macrophages stimulated by LPS were detected after adding CRH.The levels of autophagy and inflammatory factors of macrophages were detected after adding chloroquine,an autophagy blocker.Its effect on the CRH treated groups was observed.Result:1.Psychological stress has a positive association with the severity and autophagy level of IBD patientsIn IBD patients,compared with mild/moderate patients,severe patients had higher CPSS score,intestinal macrophage infiltration and autophagy level.The autophagy level was positively correlated with CPSS score（P<0.01）.Compared with normal tissues,the content of CRH receptor in inflammatory sites of human intestine also increased,and it was partially co-located with macrophages.2.CRH aggravates the severity of disease and inflammation in IBD experimental modelsAs for the mice,compared with the con group,the condition of the mice did not change significantly when CRH was used alone.The weight of the mice in the DSS group decreased,the DAI score increased,and the colon length shortened significantly（P<0.01）.Histological staining showed that the intestinal epithelial structure was destroyed and the inflammatory cell infiltration increased.The expression of intestinal inflammatory factors increased（P<0.01）,and the expression of intestinal CRHR1 and CRHR2 also increased（P<0.01）.Compared with DSS group,the weight of DSS+CRH group decreased further while the DAI score increased further（P<0.01）,and the length of colon shortened further（P<0.01）.The damage of tissue structure was more serious and the histological inflammation score increased（P<0.05）.The level of inflammatory factors increased,but the CRH receptor did not change significantly.Compared with DSS+CRH group,the disease phenotype and histological inflammation score of mice injected with CRHR1 blocker antalarmin were reduced（P<0.01）,but there was no significant change in mice injected with CRHR2blocker astressin₂-B.In DSS+CRH+rapamycin group,there was no significant effect on body weight of DSS+CRH group,DAI score and colon length.But the inflammation score increased slightly（P>0.05）.DSS+rapamycin group,compared with DSS group,body weight decreased（P<0.05）compared,DAI score increased（P<0.05）,colon shortened（P<0.01）,and histological inflammatory infiltration increased（P<0.05）.Using the autophagy blocker chloroquine,the weight loss of mice decreased（P<0.01）,the DAI score decreased（P<0.01）,the inflammation alleviated（P<0.05）,and the level of intestinal autophagy decreased.But the length of colon was not significantly affected.After using another autophagy blocker 3-MA,the weight loss and DAI score were also reduced（P<0.01）.At the histological level,the integrity of epithelium was also increased and the inflammation score was reduced（P<0.05）.But it still had no significant effect on the length of colon.3.CRH promotes autophagy and inflammatory level of macrophagesIn vitro,after adding CRH,the autophagy level of macrophages stimulated by LPS and the expression of inflammatory factors increased.After inhibiting autophagy with chloroquine,the increase of autophagy and inflammatory factors caused by CRH were weakened.LPS stimulated macrophages could destroy the intestinal barrier function and restrict the proliferation.And the expression of CRH receptor also increased（P<0.05）.Conclusion:CRH can promote the exacerbation of IBD patients and mouse models by acting on CRHR1 widely distributed in the intestine.Its downstream mechanism may be to promote intestinal macrophage autophagy.