| Inflammatory bowel disease(IBD)is a chronic,non-specific,recurrent intestinal diseases and its incidence shows a sharp increase.However,up to now,the therapeutic effect of existing drugs is not satisfactory.IBD is prone to relapse and the repeated attack of IBD has been proven to be a main cause of colitis-associated cancer(CAC).Therefore,for the prevention and treatment of IBD and CAC,it is extremely urgent to develop more desirable drugs.Many traditional Chinese medicines have been proven to have positive therapeutic effects on a variety of incurable diseases.Based on the clinical practice of traditional Chinese medicine,for some Chinese medicine with special efficicy,using modern research methods to develop and verify often have some surprises.Dendrobium Officinale,known as‘the first of the nine major herbs’,is a valuable traditional Chinese herbal medicine in our country,which can‘nourish the intestines and stomach’and used to treat‘Chang Pi’.Based on this,in the present study,we will further explore whether Dendrobium Officinale could prevent IBD and inhibit the development of IBD into CAC,as well as provide experimental basis for further development and application of Dendrobium Officinale.Objective:Dendrobium Officinale can‘nourish the intestines and stomach’,and there are medicinal records for the treatment of‘Chang Pi’.Polysaccharides as the major active ingredient in Dendrobium Officinale,which have been reported to possess extensive pharmacological effects such as improvement of intestinal barrier function,anti-tumor,anti-inflammatory and immune regulation.Thus,in this study,we set out to highlight the therapeutic effect of DOPS on treat IBD,and further investigate whether it can control the formation and development of CAC and its mechanism of action.Methods:1.The preparation,quality analysis and pharmacodynamic screening of Dendrobium Officinale polysaccharide1.1 The preparation and quality analysis of Dendrobium Officinale polysaccharidePolysaccharides from Dendrobium Officinale was extracted by juicing extraction.Then DOPS was fractionated and purified by DEAE-52 cellulose column chromatography with alkaline borate buffer solution.After these,total sugar,protein,uronic acid and sulfate content of purified fraction was detected by using phenol sulfuric acid method,coomassie brilliant blue method,meta hydroxydiphenyl method,barium chloride gelatin colorimetry.Molecular weight distribution,functional group characteristics and monosaccharide composition characteristics of DOPS fractions were determined by HPGEC,FTIR,and nitronitrile acetate derivative gas chromatography respectively to evaluate its quality.1.2 The pharmacodynamic screening of DOPS1,DOPS2 and DOPS3 in vitroRAW264.7 cells as the research object,the optimal concentrations of drugs in vitro were determined by MTT assay.The inflammatory model was established by using LPS in RAW264.7 cells,which was used to detected the pharmacodynamic differences of DOPS1,DOPS2 and DOPS3 were assayed by the level of TNF-α.2.Experimental study on the prevention and treatment of acute IBD by DOPS12.1 The therapeutic effect of DOPS1 on IBD animal model induced by DSSAccording to the yield of each Dendrobium Officinale polysaccharide,the highest yield of DOPS1 was firstly studied its pharmacodynamic effects.IBD animal model was induced by administering 4%dextran sodium sulfate(DSS,dissolved in drinking water),meanwhile,DOPS1 was administration to study the treatment effect.DOPS1 was divided into low-,medium-,and high-dose groups.The gavage doses of three groups were 50,100,and 200 mg/kg·day,respectively,and they were administered once daily.Normal group and DSS model group were given distilled water with equal volumes.Overall condition of animals,the percentage of weight loss percentage,disease activity index(DAI),colonic macroscopic evaluation,histopathological examination,levels of inflammatory cytokine and immune organs were observed.Intestinal permeability was detected by FITC-dextran.All of these indicators were used to investigate the treatment effect of DOPS1 on IBD induced by DSS.2.2 The effects of DOPS1 on inhibiting inflammation induced by LPS in NCM460 cellsNCM460 cells were selected as the research object,the concentrations of drugs in vitro were confirmed by MTT assay.In vitro cell model,inflammatory injury was induced by LPS in NCM460 cells,the levels of TNF-αand IL-1βwere detected to investigate the effect of DOPS1 on inhibiting inflammatory injury in NCM460 cells.3.Experimental study on the prevention and treatment of CAC by DOPS13.1 The therapeutic effect of DOPS1 on AOM and DSS-induced CAC animal modelAOM(10 mg/kg)was used for a single intraperitoneal injection,then 2%DSS was administered.The CAC animal model was induced by three 2%DSS treatment cycle(To simulate the recurrent episodes of IBD in clinic),each of which consisted of 2%DSS continuous free drinking for 7 days and 14 days of free drinking distilled water.After the first cycle,DOPS1 was given.The oral doses of DOPS1 low-,middle-and high-dose groups were 50,100,and 200 mg/kg·day,respectively and given once daily.The normal and the AOM/DSS model group were given distilled water with equal volumes,individually.The therapeutical action of DOPS1 on AOM combined with DSS-induced CAC mice were investigated by overall condition of animals,weight loss percentage,DAI score,colon macroscopic score,tumor number and size,levels of inflammatory cytokines and colon histopathological examination.All of these indicators were used to investigate the therapeutic effect of DOPS1 on CAC model.4.The functional mechanism of DOPS1 to control CAC4.1 Effect of DOPS1 on intestinal mucosal barrier and the number of CD8~+T cells in CAC miceIntestinal permeability detected by FITC-dextran and used to study the therapeutic effect of DOPS1 on AOM combined with DSS-induced CAC animals.The number of CD8~+T cells and the protein expressions of ZO-1and Occludin in colonic tumor marginal area and tumor area were detected by immunofluorescence.These results were used to analyze the effect of DOPS1 on intestinal barrier mechanical regulation proteins and immune-inflammatory cells in colonic tissues.4.2 Effect of DOPS1 on the cell viability of CD8~+T in tumor microenvironment of CAC animal modelThe content of lymphocyte ATP in tumor microenvironment was detected to investigate the effect of DOPS1 on the energy metabolism of T cells.The level of IFN-γin tumor tissue was detected by ELISA kit.The mitochondrial number of CD8~+T cells and the expression of CD8~+T cells PD-1 in tumor microenvironment were detected by flow cytometry.These indicators were used to evaluate the effect of DOPS1 on the cell viability of CD8~+T in tumor microenvironment of CAC animal model.Results:1.The preparation,quality analysis and pharmacodynamic screening of Dendrobium Officinale polysaccharide1.1 The quality of Dendrobium Officinale polysaccharideThe yield of crude polysaccharide was 14.75%.After separation,three components of DOPS-1,DOPS-2 and DOPS-3 were obtained.The average total sugar content of DOPS-1,DOPS-2 and DOPS-3 were 92.47%,94.64%and 90.85%,respectively.They do not contain proteins,aluronic acids or sulphuric acid groups.The molecular weights of the 3purified components were 393.8 k Da,408.7 k Da and 364.2 k Da,respectively.DOPS-1,DOPS-2 and DOPS-3 were composed of mannose and glucose,mainly mannose.The mole ratio of mannose to glucose was 3.33:1,3.12:1 and 3.20:1,respectively.1.2 The pharmacodynamic screening of DOPS1,DOPS2 and DOPS3 in vitroThe result of MTT showed that the optimal concentration of DOPS1,DOPS2 and DOPS3 is 200,200 and 400μg/m L,respectively.The anti-inflammatory effect of DOPS1is the best,followed by DOPS2,and DOPS3 has slight anti-inflammatory effect.2.DOPS1 can control acute IBD on animal models2.1 The therapeutical action of DOPS1 on IBD animal models induced by DSSAfter model establishment,the body weight,food and water intake of the animals in the model group were decreased significantly(P<0.01)when compared to normal group.There was a significant increase in the number of animals loose stools,blood stools and DAI score,the length of the colon decreased significantly(P<0.05 or P<0.01).Colon macroscopic score was markedly increased(P<0.01).The infiltration of inflammatory cells in colon histopathology was obvious and spleen coefficient increased significantly(P<0.01).The levels of pro-inflammatory cytokines IL-1β,IL-6,IL-18,TNF-αand IFN-γwere significantly increased(P<0.05 or P<0.01).The expression level of anti-inflammatory factor IL-10 was also increased,but with no significant difference(P>0.05).The ratios of IL-1β/IL-10,IL-18/IL-10 and TNF-α/IL-10 were increased visibly(P<0.01).After DOPS1was administrated,the body weight of animals,food and water intake improved obviously(P<0.05 or P<0.01),the number of animals loose stools,blood stools and DAI score were visibly reduced(P<0.05 or P<0.01).The condition of colon shortening and inflammatory infiltration in colon were significantly improved(P<0.05 or P<0.01).Spleen coefficient significantly decreased(P<0.05 or P<0.01).The expression levels of pro-inflammatory cytokines IL-1β,IL-6,IL-18,TNF-αand IFN-γwere decreased in a dose-dependent manner(P<0.05 or P<0.01).The expression level of anti-inflammatory factor IL-10 was increased but without significant difference(P>0.05).The ratios of IL-1β/IL-10,IL-18/IL-10 and TNF-α/IL-10 were decreased visibly(P<0.01).In addition,the intestinal permeability was significantly increased in DSS group(P<0.05).However,after giving DOPS1,the intestinal permeability was decreased dose-dependently(P<0.05).2.2 The effects of DOPS1 on inhibitiong inflammation induced by LPS in NCM460 cellsAccording to the results of MTT assay,the concentrations of DOPS1 in subsequent cell experiments were:50,100 and 200μg/m L,respectively.After using LPS to stimulate NCM460 cells,compared with those in the blank control group,the levels of TNF-αand IL-1βwere significantly increased(P<0.05 or P<0.01).Compared with the LPS group,DOPS1 was able to reduce the expression of TNF-αand IL-1βin a dose-dependent manner.The middle-dose group of DOPS1 could significantly down-regulate the levels of IL-1β(P<0.05).The high-dose group of DOPS1 was the most effective group in inhibiting the secretion of TNF-αand IL-1βwith significant differences(P<0.05 or P<0.01).3.DOPS1 could control CAC on animal modelsCompare to normal group,the body weight,food intake and water intake of animals in AOM/DSS model group were significantly decreased(P<0.01).The condition of animal loose stools,bloody stools and DAI scores were markedly increased(P<0.01),and colon length was significantly decreased(P<0.01).Inflammatory cell infiltration was evident in colonic histopathology(P<0.05).The number of tumors in the colon and the number of tumors with large diameters increased significantly(P<0.01).The spleen was significantly enlarged(P<0.01);The levels of pro-inflammatory cytokines IL-1βand TNF-αwere significantly increased(P<0.05 or P<0.01)in the non-tumor and tumor areas of the AOM/DSS model group.The expression of anti-inflammatory cytokine IL-10 was also increased but without significant difference(P>0.05),the ratios of pro-/anti-inflammatory factor were significantly increased(P<0.05 or P<0.01).After giving DOPS1,animal body weight,food intake,and water intake were significantly improved(P<0.05 or P<0.01).There was a significant reduction in animal loose stools,bloody stool and DAI scores(P<0.05 or P<0.01).The condition of colon shortening and inflammatory infiltration in colon were significantly improved(P<0.05 or P<0.01).The number of colonic tumors and the number of tumors with large diameters were significantly decreased(P<0.05 or P<0.01).In the non-tumor area,the expressions of pro-inflammatory cytokines IL-1βand TNF-αdecreased in a dose-dependent manner(P<0.05 or P<0.01),and the levels of anti-inflammatory cytokines IL-10 increased without significant difference(P>0.05),the ratios of pro-/anti-inflammatory factor were decreased without significant difference(P>0.05),as compare to normal group.In the tumor area,the ratio of pro-inflammatory/anti-inflammatory cytokines dose-dependently decreased after DOPS1administration.Compare to normal group,it was the most obvious in DOPS1 high-dose group about the recovery of intestinal inflammation balance.The results of intestinal permeability showed that the intestinal permeability in the model group was significantly higher than that in the normal group.However,after DOPS1 was administered,DOPS1was able to restore intestinal permeability which means that it has a protective effect on the mucosal barrier of the colon.4.The functional mechanism of DOPS1 to control CAC4.1 The regulating effect of DOPS1 on intestinal mucosal barrier and CD8~+T in CAC animal modelsThe results of intestinal permeability showed that the intestinal permeability of the animals in model group was significantly higher than that in normal group(P<0.01).After DOPS1 administration,DOPS1 could significantly restore the intestinal permeability(P<0.01)and protecte the colonic mucosal barrier.According to the result of immunofluorescence,in non-tumor area,the expression of CD8~+T was significantly increased in the colon of the AOM/DSS model group(P<0.01),and the expressions of ZO-1 and Occludin were decreased significantly(P<0.05 or P<0.01).After administration of DOPS1,the expression of CD8~+T in the colonic tissue of the animals in AOM/DSS model group was significantly decreased(P<0.05 or P<0.01),and ZO-1 and Occludin’s expressions were significantly increased(P<0.05 or P<0.01).In the tumor area,the expression of CD8~+T was significantly increased in the AOM/DSS model group(P<0.01),and the expressions of ZO-1 and Occludin decreased significantly(P<0.05 or P<0.01).After DOPS1 was administrated,the expression of CD8~+T in animal colon tissues increased,but without significant difference(P>0.01),when compare to model group.The expressions of ZO-1 and Occludin were obviously increased(P<0.01).4.2 The effect of DOPS1 on the function of CD8~+T cells in tumor microenvironment of CAC animal modelCompare to noamal group,the content of lymphocyte ATP in the tumor microenvironment of AOM/DSS model group was decreased significantly(P<0.01).However,the expression of IFN-γwas not significantly increased in AOM/DSS model group(P>0.05).The content of mitochondria in CD8~+T cells decreased significantly(P<0.01)and the expression of PD-1 in CD8~+T cells was significantly increased in the tumor microenvironment of the AOM/DSS model group.After giving DOPS1,the content of lymphocyte ATP in tumor microenvironment and the number of mitochondria in CD8~+T cells were significantly increased(P<0.05),and the expression of IFN-γin tumor tissues was also significantly increased(P<0.05).In addition,we also found that the expression of PD-1 in CD8~+T cells was significantly decreased(P<0.05 or P<0.01).Conclusion:1.DOPS1 is the most abundant polysaccharide in Dendrobium Officinale,the yield of DOPS1 is the highest,as well as the anti-inflammatory effect of DOPS1 is the best.2.DOPS1 could improve the related symptoms of DSS induced IBD model enteritis and alleviate the inflammatory damage of NCM460 caused by LPS.3.DOPS1 could improve the related symptoms of of CAC animal model induced by AOM and DSS,inhibit the formation and development of colon cancer,and have therapeutic effect on CAC disease.The effect of DOPS1 in preventing and treating CAC animals model is related to improve the expression of intestinal mechanical protein and improve the metabolic immune function of CD8~+T Cells in tumor microenvironment. |
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