| 1.Background and objectiveUlcerative colitis(UC)is a chronic inflammatory disease of the colon char acterized by continuous mucosal ulceration of the colon starting in the rectum.The disease is characterized by periods of quiescence and flares.Most commo nly presents with blood in the stool and diarrhoea,mucus discharge,abdominal discomfort,fever,weight loss,etc.Long course of disease and repeated attack s seriously affecting the quality of patients life,finally lead to colorectal cance r.Urgent need for new therapy because of Ulcerative colitis corticosteroids are often needed to induce remission in moderate-severe UC but should never be a maintenance therapy.Traditional Chinese medicine consider that,the same di sease often presents different pathogenesis and syndromes depending on the sta ge of disease development,individual constitution or environment.After dialecti cal treatment of its external manifestations,the dialectical prescriptions should be treated according to different principles and methods.Ulcerative colitis belo ngs to the category of 'diarrhea' in traditional Chinese medicine treatment theo ry.It shows different syndromes at different stages of disease,such as damp-h eat type of large intestine,liver-depression and spleen-deficiency type,spleen-ki dney-yang deficiency type.So.different prescriptions are often used for treatme nt in clinic.Huangqin Tang(HQT)is a compound from Shanghan Lun,it's cli nically used to treat damp-heat diarrhea of large intestine;Sishen Wan(SSW)from Zhengzhi Zhunsheng,the major function is treat spleen kidney yang defic iency;Tongxie Yaofang(TXYF)is contained in Danxi Xinfa,it's have signific ant effect on diarrhea due to liver depression and spleen deficiency,this three prescriptions are commonly used in clinical treatment of UC.This paper is bas ed on the theory of TCM corresponding to prescriptions and syndromes,establi shed an animal model of UC,to compare and identify the therapeutic effect a nd mechanism of Huangqin Tang,Sishen Pill and Tongxie Yaofang.2.Methods and results 2.1 A comparative study on the effects of three kinds of compound prescriptio ns on inflammatory response in UC model rats.2.1.1 Behavioral signs of rats:The models of UC were made by TNBS compound method,rats were ran domly divided into normal group,model group,SASP group,HQT group,SS W group and TXYF group.Rats were modeled beside normal group,then give n rats intragastric administration three days later,the praxiology and pathematol ogy of UC rats were observed and recorded during administration.According t o praxiology evaluation,UC rats models were successfully established,HQT an d SSW can improve the behavior of rats,manifested as increased food intake,q uickened activity and improved mental state.The effect of HQT is the most si gnificant.There is no significant in fecal epilepsy of rats in TXYF group,and have no significant difference in weight change of rats in each group.2.1.2 Pathematology examination:After five days intragastric administration of UC rats,taken colon tissues t hen executed,Microscopic observation of colon after HE stain showed that HQ T and SSW could alleviate the pathological changes of colon tissues in rats,th e manifestations were decreased inflammatory infiltration of colonic mucosa an d improved glandular ucler.The improvement of HQT was most significant,bu t there was no significant improvement in TXYF group.2.1.3 The changes of serum inflammatory cytokine in UC rats were detected b y Elisa method:After five days intragastric administration of UC rats,collected blood from orbit,separated upper serum,the levels of IL-6,IL-8,PGE2 and TNF-a in rat s were determined by sandwich enzyme-linked immunosorbent assay.The result s showed that the levels of IL-6,IL-8,PGE2 and TNF-a in serum of model c ontrol were significantly higher than those in normal group(P<0.05).Compare d with the model group,the contents of IL-6,PGE2 and INF-a in SASP grou p were decreased(P<0.05),the level of IL-8 decreased most significantly(P<0.01);The contents of IL-6,IL-8 and TNF-a in HQT group were decreased in common(P<0.05),and the contents of PGE2 were significantly decreased(P<0.01);The levels of four inflammatory cytokine in SSW group were all decrea sed,but there was no statistical difference:The contents of IL-6,TNF-a in TX YF group were decreased,the levels of IL-8 and PGE2 were decreased signific antly(P<0.05).The results showed that HQT had significant inhibitory effects o n IL-6,IL-8,PGE2 and TNF-a,TXYF had inhibitory effects on IL-8 and PGE 2,while SSW had no significant inhibitory effects.2.1.4 The expression of MAPK pathway in UC rats was detected by PCR seq uencing technology:After five days intragastric administration of UC rats,taken colon tissues t hen executed,after extracted the protein,extracted RNA for PCR detection.Th e results showed that the expression of Erk1?p38 in colon tissues of SASP,H QT,SSW and TXYF group was significantly lower than those of model group(P<0.01),especially in HQT group.2.2 The changes of brain-gut peptide in colon tissues of UC rats were detected by Elisa and immunohistochemistry.After five days intragastric administration of UC rats,collected blood from orbit,taken colon tissues then executed,detected the content of 5-HT in seru m by Elisa method.The results showed that compared with the normal control,the model group were significantly lower(P<0.01).Compared with model co ntrol,the contents of 5-HT in SASP group and TXYF group were increased si gnificantly(P<0.05),while the contents of HQT and SSW group were no sign ificant difference increased.The contents of VIP and SP in colon tissues were detected by immunohistochemistry.The results of VIP showed that the conten ts of VIP in model group were significantly lower than those in normal group(P<0.01).Comeared with model control,the contents of VIP in SASP group,HQT group.TXYF group were significantly increased in common(P<0.05),S SW group also increased but no significantly.The results of SP showed that c ompared with the normal control,the contents of SP in the other five groups were decreased,but there was no significant difference.Compared with the mo del group,the contents of SP in SASP group,HQT group and TXYF group w ere increased significantly(P<0.05),but the contents of SP in SSW group incr eased without any significant difference.All above that proved HQT could sig nificantly increase the contents of VIP and SP in colon tissues,but no 5-HT;SSW could promote the content of three brain-gut peptides,but there was no s atistical difference;TXYF could significantly promote the content of three brai n-gut peptides.2.3 The changes of aquaporins in colon tissues of UC rats were detected by Western-B ot method.After five days intragastric administration of UC rats,taken colon tissues t hen executed,after extracted the protein,detected the contents of AQP3,AQP4 in colon tissues.The results of AQP3 showed that the contents of AQP3 in model control was higher than those in normal group.Compared with the mod el control,the contents of AQP3 in HQT group were significantly decreased(P<0.01);The contents of AQP3 in SASP,SSW,TXYF group decreased.The results of AQP4 showed that the contents of AQP4 in model control was sig nificantly lower than that in normal group(P<0.01).Compared with the model group,the contents of SASP and HQT groups increased significantly(P<0.05);The contents of AQP4 in SSW and TXYF group didn't increased and there was no statistical significance.This experiment proved that HQT could signific antly promote to decrease the contents of AQP3 and increase the content of A QP4 in colon tissues,also could significantly decrease the content of AQP4;S SW could significantly decreased the contents of AQP3 in colon tisssues,but had no effects on AQP4;TXYF ould significantly decreased the contents of A QP3 in colon tisssues,but had on effects on AQP4.2.4 The changes of intestinal bacteria in UC rats were detected by high-throug hput sequencing.After five days intragastric administration of UC rats,collected fresh rats f eces,Sequencing and analysis were carried out at 16sMiseqPE300V3-4 segment.The results showed that the sequencing quantity was sufficient to reflect the majority microbial information in samples.Compared with the model group,th e similarity between SSW group and model group was higher.HQT or TXYF control were obvious different with model group:The species richness of SS W group was highest,followed by was HQT group,there was no sigificant dif ference between TXYF and model group.TXYF group had the highest similari ty among samples,then was SSW group,HQT group had significant difference s among samples;The content of beneficial bacteria in intestine in HQT group was higher than that in SSW group and higher than that in TXYF group.Lact obacillales and Streptococcae were significant different flora in SSW group;F-Prevotellaceae,f-Rikenellaceae and g-clostridium-innocuum-group were significan tly different flora in TXYF group;there were significant differences in the HQ T group among the three groups:f-Lachnospiraceae,f-Ruminococcaceae,f-Gam ma proteobacteria,g-Escherichia-Shigella and f-PorphyromonaceaeUC is an uncertain etiology,resulting from inflammatory,brain-gut axis,water transport,intestinal bacteria and so on.These three compounds are widel y used to treat UC in clinic,but the therapeutic mechanism and effect of each compounds are different.According to combination of prescription and syndro me in TCM,Huangqin Tang is suitable for diarrhea due to wetness-heat of lar ge intestine,have an efficacy in the treatment of inflammation and water trans port;Sishen Wan is suitable for diarrhea due to spleen and kidney yang defici ency type,there may be a benefit of intestinal bacteria balance and intestinal f unction restore;Tongxie Yaofang is suitable for diarrhea due to liver depressio n and spleen deficiency,adjust the mood and restore the balance of brain-gut axis to achieve the goal of treatment.This paper used the model of diarrhea d ue to wetness-heat of large intestine,the UC model corresponding to modern c linical practice.Compared effects of three compounds act on same model by measuring inflammatory cytokine and pathways,brain-gut peptide,aquaporin an d intestinal bacteria.The incidence and prevalence of ulcerative colitis have be en increasing over time worldwide,our goal is to explore the pathogenesis of UC and do better guide for clinical treatment medication. |
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