| Objective:Ulcerative colitis(UC)is referred to the names of "diarrhea","intestinal wind","intestinal addiction","blood stool" in traditional Chinese medicine(TCM).The mainly pathogenesis of UC in TCM are turbid dampness,qi deficiency,qi depression,internal heat,yang deficiency,blood deficiency,blood stasis and zang-fu function disorder.UC is also known as a nonspecific ulcerative colitis,which is one kinds of inflammatory bowel disease(IBD).It is characterized of a chronic non-specific colitis,lesions mainly confined to the mucosa and submucosa of colon;the colonic mucosa would be showed inflammation and ulcers,and the first involving the distal colon and rectum,then to the proximal extension,and even the entire colon.It would be appeard the main clinical manifestations of diarrhea,abdominal pain,pus and blood stool with a chronic and recurrent course.The idea of holism,syndrome differentiation and internal-external therapy in TCM are taken to diagnosis and treating UC.Lingnan region is located in subtropical regions,the climate is hot,and the weather is wet.Local people like to have herbal tea,which easily hurts the stomach yang,and the function of spleen fails,then the pathological endogenous heat would be produced.So the UC patients in Lingnan region are showed the deficient root and excessive superficial symptom in TCM.There are many factors impact IBD,including immune response,genetic susceptibility,environmental triggrts,luminal microbial antigens and adjuvants,and so on,one of which becomes highlight recently:brain-intestinal peptide-hormones.Spexin is a highly conserved peptide which was identified through bioinformatics search strategy,based on a hidden Markov model and following biochemical characterization through application of another bioinformatics method based on an approach that models spatial structure along the genomic sequence simultaneously with the temporal evolutionary path structure across species,and has been named NPQ.And Spexin is widely distributed in the body communicate enteric nervous,endocrine and immune system,affecting the overall function of the body,plays an important role with binding protein receptors to work effects.UC researching is still in the initial stage,the correlation between Spexin and UC has not been known clearly yet.Neuropeptides in the intestinal mucosa and its corresponding antigen-antibody reaction between the receptor would be high-light with intestinal mucosal inflammation in future.Although the brain-gut peptide-hormones and UC also need to explore,previous studies have provided new useful insights through the brain-intestinal peptide-hormones.It may be deepen our understanding of the overall concept of UC,and it would be more clarify the nerve-endocrine-immune system relationship to explore the pathogenesis of UC in future.Methods and Results:1.To analyze the TCM Syndrome Distribution of UC patients in Guangzhou;to detect the expressions of Spexin.NPY and 5-HT in colorectal damp-heat type UC serum;the feather of UC patients in Guangzhou area was fit the TCM Syndrome Distribution.Methods:collecting the 94 patients from the First Affiliated Hospital of Guangzhou University of department of Gastroenterology wards and outpatient during January 2012-February 2016.Clinical samples data was collected,patients' symptoms were statistics,clinical data on the UC cluster were analysis,diagnose TCM syndromes were classified.The cluster of symptoms as a variable entry,establish syndromes name based clustering results,dialectical analysis.The protien expressions of Spexin,NPY and 5-HT in the intestine damp-heat type UC patients' serum were detectied by ELISA(double sandwich ELISA).Results:Cluster analysis results suggested that an eight-classified types were according to the entry information of all patients included in this study,the distribution feathures was fit the guideline of Chinese Medical Clinic Digestive Diseases Branch of consensus in 2003,where the majority of patients the intestine damp-heat type in TCM.Compared with the normal group,the protein expression of NPY,5-HT and Spexin were increased significantly in the intestine damp-heat in UC patients serum(P<0.05).2.To investigate the effect of spexin on the mucosal barrier factor MUC2 in TNBS rat model.Methods:50 SD rats were randomly divided into 5 groups,normal group(n=10),TNBS group(n=10),SASP group(n=10),SPX group(n=10),GALR2 group(n=10),which were observed 15 days after TNBS-inducing,the damage index,weight change,disease activity index(DAI),colon length and pathological changes of colonic mucosa in TNBS-induced rat were boserved;and the expressions of MUC2 in TNBS-induced colitis rats after Spexin and GALR2 intervention were detected by Fluorescent-immunohistochemistry and Western blot.In order to explore the relationship between Speixn and UC intestinal mucosal barrier.Results:? Compared with normal group,after inducing by TNBS,the trend of rats'body weight was persistent downward,and apathetic,dull coat,quiet,loss of appetite were appeared.After drugs intervention,the body weight of TNBS-induced rats still showed a continued downward trend,but compared to the previous intervention,the spirit were better than that before:shiny coat,increased food intake.? Compared with the normal group,the number of bowel movements was increased in TNBS-induced rats;stool pulpy stools,with mucus and blood was reduced after drug intervention.The DAI scores of SASP group,SPX group and GALR2 group were reduced than that in TNBS group.? Compared with normal group,colonic mucosa of TNBS group was showed severely damaged,glandular disorders,increased the number of goblet cells,inflammatory cell infiltration by light microscopy;SPX group and GALR2 colonic mucosa incomplete,glandular disorders,goblet cells increased,the optical microscope,inflammatory cell infiltration.? Compared with the normal group,mucosal MPO of TNBS group was significantly higher(P<0.05);Compared with TNBS group,SASP group,and SPX group,GALR2 group were decreased.? Compared with the normal group,IOD of TNBS group was significantly higher than the normal group(P<0.05);Compared with TNBS group,IOD value of SASP group and the SPX group was significantly increased(P<0.05).?Compared with the normal group,the MUC2 protein expression of TNBS group was significantly higher than that in normal group(P<0.05);Compared with TNBS group,the MUC2 protein expression of SASP group and the SPX group were significantly increased(P<0.05).3.EGFR,ZO-1 protein and ZO-1 mRNA.expression of Caco-2 cells by Spexin intervention.Methods:Caco-2 cell line was passaged 2-3 times,starting sheet for drug intervention and experimentation.cell density of 100,000/well was grown in12-well plate.After 24 hours adherent discarded and the original medium was intervention factor:TNF-?(100ng/ml)stimulated cells after 4h,were added Spexin(0.5?M),GALR2(1?M)intervention 3 h;added M871(1?g/ml)after stimulation pre 3 h,added Spexin(0.5?M)intervention 3 h;on the other hand,while observing the cells were added TNF-?(100ng/ml),Spexin(0.5?M),GALR2(1?M)intervention 3 h changes.EGFR and ZO-1 protein expression were detected by Western Blot;mRNA expression of EGFR and ZO-1 were detected by RT-PCR.Results:? Compared with normal group,cells after TNF-? intervention 3 hours,mRNA expression of EGFR was significantly increased(P<0.05),after SPX+GALR2 intervention for 3h,the EGFR protein expression was increased(P<0.05);after intervention by TNF-? for 3h,joined SPX,GALR2 respectively intervention for 3h,EGFR protein expression was no significant change;and adding blocker M871 intervention 3h,joined SPX intervention for 3h,EGFR mRNA expression was significantly reduced(P<0.05).?Compared with normal cells,after TNF-a intervention for 3h,ZO-1 protein expression was significantly reduced(P<0.05),after SPX+GALR2 intervention for 3h,the relative expression of ZO-1 protein seen no significant change;compared with TNF-? group,the intervention group after intervention by TNF-a 3h,joined SPX,GALR2 respectively intervention 3h,GALR2 group of ZO-1 expression was increased significantly(P<0.05);adding blocker M871 intervention 3h,joined SPX intervention 3h,EGFR mRNA expression was significantly increased(P<0.05).?after TNF-a intervention 3h,EGFR mRNA expression was significantly increased(P<0.05),SPX+GALR2 intervention 3h,EGFR mRNA expression of no significance;and after intervention by TNF-a 3h,joined SPX,GALR2 respectively intervention 3h,EGFR mRNA expression was significantly reduced(P<0.05);after adding blockers M871 intervention 3h,joined SPX intervention 3h,epithelial cells EGFR mRNA relative expression was significantly reduced(P<0.05).?compared to normal cells,cells after TNF-a intervention 3h,epithelial cells ZO-1 mRNA relative expression was increased significantly(P<0.05),SPX + GALR2 intervention 3h,ZO-1 mRNA expression was no significance;the intervention group after intervention by TNF-a 3h,joined SPX,GALR2 respectively intervention 3h,SPX,GALR2 group of epithelial cells relative ZO-1 mRNA expression was significantly lower(P<0.05).Conclusions? Guangzhou area by the rule of UC patients with TCM Distribution Distribution in line with guidelines,and in Guangzhou TCM clinical type might be mainly the large intestine damp-heat;? The expression of NPY,5-HT and Spexin the intestine damp-heat type of UC patients' serum were increased significantly.? Spexin may influence the mucosal barrier permeability of the mucosal barrier factor via inhibiting MUC2,EGFR,ZO-1. |
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