Experimental Study Of Integrated Traditional Chinese And Western Medicine On Intestinal Dysfunction Induced By Ileus Ⅱ

PARTⅠPhasic research of enterocyte,intestinal mucosal barrier andlumen flora of intestinal dysfunction induced by ileusExperimentⅠImproved methods and materials of reusable ileus model of rabbitsObjects:In order to make models easier to operate,more economical and morestable results.The improved the methods and materials were taken on the basis ofEstablished in the pre-experimental rabbit's mechanical ileus.Methods:taking the methods of"Intestinal extrinsic oppression",infusion set partswill be transformed into In vitro pull-type locking,made a hole in the Avascular zonemesenteric of the terminal ileum,trap tube,Leads to the incision of the abdominalwall next to the outside.1～2 days after operation was fixed by in vitro pull locking,resulting in the obstruction of the bowel.When the need to lift the obstruction onlyneed to cut the lock and withdrawing the device.At the same time,other methodssuch as in vitro-filled cystic obstruction device,plastic cable trap also have been tried.Results:This method is better than the original model in the safety,accuracy,etc.avoiding the existence of intra-abdominal infections,intestinal adhesion,intussusceptions,as well as intestinal perforation,and there is no Factors of instabilitysuch as Device off.Conclusion:on the basis of getting certainly more stable obstruction effect.theimproved methods has the advantage of trauma smaller,shorter-cycle model,simpleoperation and material economy,is a more satisfactory reusable mechanical model ofcomplete intestinal obstruction. ExperimentⅡInfluence of acute intestinal obstruction on damage and proliferativeand repair ability of intestinal epithelial cellsObjective:To observe the acute mechanical low small bowel obstruction for extentof intestinal epithelial damage,as well as the effects of their own re-proliferation,repair capacity.Methods:Healthy rabbits were randomly divided into normal control group(Norm),sham-operated group(Sham),obstruction lh group(H1),obstruction 6h group(H6),obstruction 12h group(H12),obstruction 24h group(H24),obstruction 48h group(H48),obstruction 72h group(H72),n = 6.Animals of each group were detected andanalyzed in serum citrulline levels,biochemical,intestinal tissue ornithinedecarboxylase activity,as well as in intestinal epithelial goblet cell morphology,changes in the number.Results:Early intestinal obstruction after the existence of the protection of intestinalepithelial cells to accelerate the process of proliferation,ODC activity showedincreased when 6h after the beginning of obstruction(p＜0.001),the damage factorsincreasing with the obstruction is not lifted,ODC activity decreased in obstructive24h,to 72h or even lower than normal level.Morphological observation of intestinalepithelial goblet cells found an increase in the trend,and patterns of phase changeoccured.Indicators over different parts of the small intestine changes in the degree ofphase and is not consistent,ileum more significant than changes in the jejunum.48h-72h to the obstructive phase,lower than the normal level of serum citrullinelevels(p＜0.05).In addition to biochemical indicators in detection of CK found in asignificant change in the earlier,more sensitive than other indicators(24h vs48h-72h).Conclusion:①Intestine epithelial repair has existed early after the obstructionoccurred,manifested as increased activity of intestinal tissue ODC;②Goblet cells may be involved in the repair of small intestinal mucosa,and may be the key cell toepithelial cell repair;③Late in the obstruction With the reduction of epithelialproliferative activity,serum citrulline levels which is a indirect indicators decreasedon their dysfunction,and it has a certain reference value on determining the severityof the small intestine;④After the intestinal obstruction,CK may be more sensitiveto the meaning of the diagnosis tips compared with other biochemical indicators ofmaterial injury to the intestine.ExperimentⅢPhase relationship among Intestinal Environment andmicro-ecological changes and bacteria and endotoxin translocationafter intestinal obstructionObjective:To observe the changes of Intestinal Environment and Microecologyafter the occurrence of the acute mechanical low small bowel obstruction in order tounderstand homeostasis after intestinal damage and intestinal barrier damage phase ofthe relationship.Methods:Healthy rabbits were randomly divided into normal control group(Norm),sham-operated group(Sham),obstruction l h group(H1),obstruction 6h group(H6),obstruction 12h group(H12),obstruction 24h group(H24),obstruction 48h group(H48),obstruction 72h group(H72),n = 6.Observation of small bowel obstructionwithin hours of each PH,water content and understanding of the use of plate countand Enterobacteriaceae bacteria change in the number of Bifidobacteria.Also bloodendotoxin levels,the organs of the rate of bacterial translocation was observed atdifferent times.Results:The results showed that the PH value of terminal ileum was graduallydeclined while the water content in intestinal contents was gradually increases afterthe intestinal obstruction,both groups showed significant changes in the 12h-24hphase obstruction(p＜0.05,p＜0.01).Enterobacteriaceae in the site and found the cultivation of bifidobacteria,in the 12h-24h stage of obstruction there the ratio of thetwo inverted,Enterobacteriaceae become dominant bacteria.In addition,thecultivation of tissues and found that plasma endotoxin detection,also showedsignificant changes in the 12h-24h phase of the obstruction.Conclusion:Intestinal dysfunction after intestinal obstruction is the result of manyfactors,the destruction of intestinal homeostasis provides the basis for theenvironment for a large number of bacteria proliferation and of bacteria andendotoxin translocation.Obstruction compared to the earlier stages of 12h-24h maybe the intestinal environment,drastic changes microecological stage,but also may bestart of bacterial and endotoxin translocation.And gastro-intestinal mechanical barrierat this time often did not show extensive damage,it has also said that,mechanicalbarriers,biological barriers,chemical barriers intestinal damage are risk factors fordysfunction.ExperimentⅣThe changes of intestinal epithelial Claudin1 mRNA expression inthe occurrence after intestinal obstructionObjective:To understand the characteristics of phase of destruction of tightjunctions in intestinal epithelial cells after the occurrence of acute intestinalobstruction.Methods:Healthy rabbits were randomly divided into normal control group(Norm),sham-operated group(Sham),obstruction l h group(H1),obstruction 6h group(H6),obstruction 12h group(H12),obstruction 24h group(H24),obstruction 48h group(H48),obstruction 72h group(H72),n = 6.the relative quantitative analysis of theanimals of the last paragraph of the intestinal epithelial tight junction-associatedprotein Claudinl's mRNA's expression were made through the real-time fluorescencequantitative RT-PCR technology.Results:The experiments showed that after 6h obstruction Claudinl mRNA expression has been found to have significantly reduced levels to normal about half(p＜0.05),12h to 18%(p＜0.01),24h to 11%,48h-72h phase expression was very low.Conclusions:mechanical barriers have been destroyed at Early stage after theoccurrence of intestinal obstruction(6h),the manifestation was lowly expressedgenes for the tight junction protein upstream gene expression,with the obstruction ofprogressing,the low expression was more prominent at 48h-72h phase of obstruction.Indicated that this stage of intestinal epithelial integrity is completely lost,seriouslydestruction of intestinal mechanical barrier.ExperimentⅤChanges of the immune barrier function in small bowel obstructionin rabbitsObjective:To observe the impact of the immune system when occurrence of acutemechanical intestinal obstruction by making use of intestinal obstruction modelMethods:Healthy rabbits were randomly divided into normal control group(Norm),sham-operated group(Sham),obstruction lh group(H1),obstruction 6h group(H6),obstruction 12h group(H12),obstruction 24h group(H24),obstruction 48h group(H48),obstruction 72h group(H72),n = 6.By flow cytometry of low obstruction ofproximal ileum,two different parts of high jejunal Peyer intestinal lymph nodes(PP)of CD3+,CD4+,CD8+lymphocyte subsets in the analysis,using HE stainingsections in different parts of the two counts the percentage of IEL and LPL indifferent stages of obstructive changes.Another ELISA assay endocrine intestinalimmunoglobulin A(S-IgA)content.Results:The experiments showed that intestinal obstruction after 12h-24h stage,thesmall intestine of animals in the number of PP and the total area increased(p＜0.05),with the progress and gradually reduce obstruction to obstruction 72h significantlylower than the normal stage level(p＜0.01).PP lymphocytes by flow cytometry onCD3+,CD4+,CD8+sub-group analysis showed that after the lifting of PP within intestinal obstruction CD3+subpopulations for the increase in the trend of low ilealSham group was 29.44±2.54%,12h increased to 35.92±2.08%(p＜0.05),72h to49.48±6.37%(p＜0.01);CD4+subsets in the 12h stage of the same wassignificantly higher than Sham group(84.68±2.80% vs 80.51±2.22%,p＜0.05),Been steadily declining since then to a rapid decline in 72h was significantly lowerthan Sham group(61.62±6.96%,p＜0.01);CD8+subsets and CD4+Trends Incontrast,12h-24h phase of decline than the Sham group(8.47±1.41% vs 11.52±1.04%,p＜0.05),after 72h was gradually increased to the highest(22.97±4.83%,p＜0.05);CD4+/ CD8+ratio of the change in the trend of first increase for the earlystages,24h stage was significantly higher than Sham group(12.10±2.17 vs 7.97±1.40,p＜0.01),the late phase of gradual decline to 72h was significantly lower thanSham group(3.35±1.03,p＜0.01).Campylobacter high above in the results observedsignificantly later than the low ileum,and the changes are not dramatic.Through theHE staining of the percentage of IEL and LPL counting results showed that thepercentage of IEL in different parts of the time with the obstruction found nosignificant difference,and LPL in the percentage of obstruction increased 6h stage,inwhich parts of the jejunum than the Sham group,significant difference(p＜0.05).With prolonged obstruction,the percentage of the two sites decreased LPL.Mesocaval s-IgA in the ELISA results showed that after the occurrence of theintestinal obstruction content increased,24h stage higher than the Sham group(p＜0.05),then dropped to the lowest level in 72h,and 24h are different levels,but withthe Sham group found no significant difference between.Conclusions:①when obstruction occurred,the number of the small intestine andthe total area of PP increased,which may be related to obstruction by inflammatoryfactors stimulate more relevant,while its lymphocyte subsets is also a correspondingchange,CD3+showed increasing trend,the proportion of CD4+subsets,CD4+/CD8+were increased in the early days,and then gradually decreased with theprogress obstruction.In contrast,CD8+subsets to reduce the proportion of earlystage,the latter increased,which indicates that intestinal immunity and transientenhanced after the decline;②By intraepithelial IEL,lamina propria LPL percentage of the total cell number of statistics,can be seen,IEL changes in a certain change,butthis experiment has not yet found the difference statistically.LPL is characterized bythe percentage of early obstruction increased,the latter decline,this may belymphocyte homing to the lamina part of the effect of the increase,and the latter withthe increase in lymphocyte apoptosis and other inflammatory cell infiltration anddecreased to its;③s-IgA changes in the amount of further verify theabove-mentioned phenomenon,with an increase in the early mesocaval to graduallylower than the level of the latter;④intestinal immune system activation occursearlier,and intestinal obstruction caused by changes in the immune barrier there maybe differences between sites,ileum earlier,the emergence of a stronger immune tochange.The effect of position on the lamina propria lymphocytes Such differencesmay not be apparent,to be confirmed. PARTⅡPhasic research of enterocyte,intestinal mucosal barrier andlumen flora of intestinal dysfunction induced by ileusExperiment VIRepair of intestinal epithelial role of Sijunzi tang After theobstruction was removedObjective:To use reusable mechanical model of complete intestinal obstruction,tocompare and observe the natural recovery and influence of traditional Chinesemedicine Sijunzitang on the impact of intestinal epithelial cell proliferation andrepair.Methods:Healthy rabbits were randomly divided into sham operation group(Sham),obstruction of 48 model group(M48),natural recovery 48h group(S48),Chinese medicine treatment of 48h group(T48),natural recovery 96h group(S96),Chinese medicine treatment of 96h group(T96),each group 6.The lifting of ananimal model of obstruction after 48h,96h.Making intestinal goblet cell morphology,organization of small intestinal ornithine decarboxylase activity and serum citrullinelevels of tight junction-associated protein expression Claudinl mRNA's detection andobservation,respectively.Results:The experiments showed that the number of goblet cells of small intestineepithelium was gradually decline after the lifting of the obstruction,the significantdifferences have emerged 48h later,96h returned to normal level,traditional Chinesemedicine treatment group was more effectively.Serum citrulline levels graduallyreturned to normal levels,the Chinese Medicines Board was earlier than the natural recovery group(48h,p＜0.05),omithine decarboxylase activity was significantlyincreased,especially in Chinese medicine the treatment group was significantlyhigher than the natural recovery group(P＜0.05).Also found in traditional Chinesemedicine Sijunzitang Claudinl mRNA can increase the expression level higher thanthe natural recovery group.Conclusions:①Sijunzi tang medicine can promote intestinal obstruction intestinalepithelial cells after the lifting of the re-proliferation and epithelial integrity of therepair process,can accelerate the performance of their intestinal intraepithelial gobletcells of the intestinal epithelium of the completion of the reconstruction progress;②itcan improve ODC activity in intestinal tissue,suggesting Sijunzitang the ability toincrease epithelial regeneration;③epithelial regeneration and the results reflected inserum citrulline levels,④Sijunzitang can increase intestinal epithelial tightjunction-associated protein gene expression in the upper reaches of the Claudinl,andpromote the integrity of intestinal epithelial repair⑤The results showed throughafter the lifting of the intestinal obstruction dysfunction exist in the differentiation ofthe spleen and stomach empty,the evil is already Qu,the essence of righteousnessmay be damaged,including damage to intestinal epithelium,and the role ofSijunzitang one target may be the promotion of intestinal crypt stem cell proliferationso as to achieve the integrity of epithelial repair.ExperimentⅦIntestinal immune opsonization role of Sijunzi tang After theobstruction was removedObjective:To use reusable mechanical model of complete intestinal obstruction,tocompare and observe the natural recovery and influence of traditional Chinesemedicine Sijunzitang on the impact of intestinal immune barrier.Methods:Healthy rabbits were randomly divided into sham operation group(Sham),obstruction of 48 model group(M48),natural recovery 48h group(S48), Chinese medicine treatment of 48h group(T48),natural recovery 96h group(S96),Chinese medicine treatment of 96h group(T96),each group 6.after the lifting of ananimal model of obstructive 48h,96h,getting PP-lymphocyte subsets(CD3+,CD4+,CD8+,CD4+/ CD8+)for analysis Through flow cytometry,s-IgA in intestinalcontent were detected by using ELISA method as well as through the biopsy of IEL,LPL percentage count.Results:The experiments showed that after the Chinese intervention in intestinalobstruction to lift the T48 group,T96 group than in the natural restoration of the S48,S96 Group PP lymphocytes CD3+,CD4+,CD8+subsets and CD4+/ CD8+ratiohas a regulatory role,so that faster restoration of or close to the former level ofobstruction;in iIEL,LPL relative number of observation,Sijunzitang LPL earlierintervention group the proportion of the near normal;the treatment group the level ofs-IgA did not find significant difference among them.Conclusion:the barrier function of the immune disorder was not restored withoutdelay,after 48h natural CD4+,CD4+/CD8+ratio and other indicators have notrecovered,and has declined more than the M48 group,which may indicate theimmune status of intestinal function has not been affected by intestinal obstructionand an immediate resumption of disarmament,it may be gastro-intestinal disorderwithin the environment,imbalance of intestinal flora,as well as the integrity ofintestinal epithelial damage can not be immediately improved.Sijunzitang andapplications can be in the promotion of small bowel obstruction after the lifting of theexistence of dysfunction of the immune barrier.Reflected in the intervention groupSijunzitang more natural restoration of the PP group Vallarta leaching faster return tonormal group,and may be part of the intestinal effects of the distribution or thenumber of lymphocytes with a regulatory role.Obstruction after the lifting of thesubstance of the spleen and stomach function may be covered by the intestinalepithelial dysfunction,as well as the immune system,intestinal disorders,and the roleof Sijunzitang strengthening spleen's target is likely to include the intestinal immunesystem opsonization.