| Objective: Inflammatory bowel disease(IBD),including Crohn's disease(CD)and ulcerative colitis(UC),is an unknown etiology,chronic,relapsing and non-specific inflammatory disease.In recent years,more and more researches evidenced that aberrant immune response and gut microbiota play an important role in the pathogenesis of IBD.Modern research has indicated that Amomi Fructus has better effect on the treatment of gastrointestinal diseases,However the further pharmacological activities and mechanisms of Amomi Fructus on IBD is still lacking.Therefore,this research is drafted from the balance of T-lymphocyte subpopulation and gut microbiota to observe the therapeutic effect of Amomi Fructus and make an inquiry into the mechanism on IBD in rat.The results will provide scientific basis for further study and exploitation of Amomi Fructus.Methods: 1.Induction of IBD and Treatment: A total of 90 Sprague-Dawley(SD)rats were randomly divided into control(CON)group;model(MOD)group;low-,medium-and high-dose volatile oil from Amomi Fructus(VOAV.L,VOAV.M,VOAV.H)groups;low-,medium-and high-dose water extracts from Amomi Fructus(WEAV.L,WEAV.M,WEAV.H)groups;mesalazin(MES)group,10 rats per group.Then 100 mg/kg of TNBS dissolved in 0.25 m L 50 % ethanol was injection into the colon,with 0.9 % saline treatment as control.CON and MOD groups,treated with drinking water.Water extract groups,treated orally with WEAV.L,WEAV.M and WEAV.H at doses of 0.2,0.4,0.8 g/kg.Volatile oil groups,treated orally with VOAV.L,VOAV.M and VOAV.H at doses of 0.007,0.014,0.028 g/kg.Positive control group,treated orally with MES at dose of 0.36 g/kg.All the treatments were started 6h after the modeling,once a day for 9 days.Food intake,body weight and disease activity index were measured daily.2.Assessment of therapeutic effect and Investigation of mechanism: Rats were sacrificed on d 9,colon length and weight,as well as spleen and thymus index were recorded.Macroscopic and histological damage were scored.The inflammatory response to IBD was assessed by measuring the expressions of MPO,TNF-?,IL-17,IL-10,IFN-? and TGF-? in serum and colonic tissue supernatants of rats by ELISA assays.The percentages of regulatory CD4+CD25+Foxp3+ Treg cells in rat spleen was measured by flow cytometry.Diversity of the colony in colon,the overall structural changes in gut microbiota were analyzed after sequenced on V4 region of 16 S r DNA.Clarifying the mechanism of Amomum villosum on Th cell subgroup balance,proportional changes of Treg cells and homeostasis of gut microbiota.Results: 1.Rats in model group compared with control group showed significant weight loss,poor appetite,pilo erection,diarrhoea and bloody mucus,the animals also displayed high DAI scores,the macroscopic evaluation of colonic mucosa appeared hyperemic,edematous,ulcerated,and the histological damage characterized by increased neutrophil numbers,mucosal glands disruption.Which prompt that the rat IBD models were successfully established.2.Treatment of animals with WEAV and VOAV significantly reduced the loss in body weight and stimulated with decreased diarrhea,the DAI scores in all treatments were significantly decreased compared with model group.Among them,treatments with VOAV.M could significantly inhibit weight and food loss(P<0.001),VOAV.L were effective to increase spleen index(P<0.05),while WEAV.M were effective to increase spleen index(P<0.05).Thus,these data suggest that Amomum villosum could improve the general physiological features of IBD rats.3.Administration of WEAV and VOAV could decreased the presence of adhesions to adjacent organs,reduce colonic wall thickening compared with model group,which was particularly obvious in VOAV.L group(P<0.001 or P<0.01).Ulceration with mucosal hyperaemia and edema were observed in model grooup.In contrast,such changes were markedly improved by VOAV.H,VOAV.M,WEAV.H treatment(P<0.01,P<0.05).Histological scoring revealed that all dose of VOAV,WEAV.M were effective to mitigated the severity of tissue lesions characterized by low level of inflammatory cell infiltration,intestinal villus arranged neatly and distinct of colon mucosa tissue(p<0.01 or p<0.05).Thus,these data suggest that Amomum villosum could diminished the severity of colonic injury.4.The activity of MPO and the level of TNF-? of colon in homogenate in model group were higher than that of the control group(P<0.001,P<0.01).WEAV.H could reduce the level of TNF-?(P<0.05),while VOAV at all dose have no significiant effects on MPO activity.Amomum villosum tested were effective in upregulate Th2 and Treg-related anti-inflammatory cytokine IL-10 and TGF-?,downregulate Th1 signature Pro-inflammatory cytokine IFN-?,treatment with VOAV.H could significantly decreased the content of IL-17,which is the key Pro-inflammatory cytokine of Th17.Based on the above results,Amomum villosum plays an important role in maintaining the homeostatic intestinal mucosal immune by balance of Th cell subpopulation signature inflammatory cytokine.5.Compared to model group,all dose of WEAV and VOAV.L treatment can marked increased the percentage of CD4+CD25+ Treg cells(P<0.001,P<0.05),the effect is better than control group.While,there was no significant difference between control group and model group.Foxp3+ is a key cellular transcriptional factor,which can differentiating naive T cells to Treg cells,Amomum villosum treatment could significantly increased the percentage of Foxp3+,the WEAV treatment is superior to VOAV treatment.Thus,the results indicated that the therapy of Amomum villosum on IBD was associated with an expansion of Treg cells for promoting the immunologic function.6.Determination of highly variable region V4 of 16 S r DNA showed that the gut microbiota diversities in model group were reduced significantly.Mainly dominated by decreased the abundance of Firmicutes and Bacteroidetes and increased the abundance of Proteobacteria and Spirochaetes.Pearson correlation analyzed that Firmicutes and Bacteroidetes were positive correlation to IL-10,TGF-? and Foxp3+and negative correlation to IL-17,IFN-?,MPO and LPS.Administration of Amomum villosum could significantly increased some bacteria belonging to the Firmicutes including Lactobacillus,Clostridium,Ruminococcaceae and Lachnospiraceae,as well as some bacteria belonging to the Bacteroidetes including Bacteroides and Prevotella.Meanwhile Amomum villosum could decreased the abundance of Proteobacteria such as Desulfovibrio,Sutterella and Helicobacter.Amomum villosum could also effectively increased short-chain fatty acids(SCFA)-producing bacteria and inhibit the release of enterogenic endotoxin and decrease the content of LPS,then modulate the imbalance of intestinal flora.Conclusion: The injection of 100mg/kg of TNBS in 0.25 m L 50 % ethanol can successfully establish the model of IBD.Here,Amomum villosum treatment marked decreased the DAI scores by reversed the lost body weight and decreased the extent of the diarrhoea and bleeding scores,the spleen and thymus index were increased.Consistently,Amomum villosum treatment also showed a marked reduction in the tissue disruption.Furthermore,pro-inflammatory cytokine expression level,such as TNF-? was also clearly decreased.Collectively,these results demonstrate that Amomum villosum is efective in protecting against acute TNBS-induced colitis.The immunomodulatory mechanisms of Amomum villosum are related to regulating Th1/Th2 and Th17/Treg-Related cytokine production,promoting the CD4+CD25+Foxp3+ Treg levels in spleen of rats.On the other hand,Amomum villosum may maintenance the internal environment by balance the ratio of Firmicutes,Bacteroidetes,Proteobacteria and Spirochaetes,increase SCFA-producing bacteria and decrease the content of LPS. |
PDF Full Text Request