| Escherichia coli diarrhea is one of the most common fatal diseases in veterinary clinic.Many researchers have created a test model of E.coli diarrhea such as rats and mice,rabbits and guinea pigs in order to study the mechanism of different drugs in the treatment of diarrhea caused by Escherichia coli to observe the animal detection model of clinical symptoms,repair of tissue and organ pathological changes of intestinal mucosa and related cytokines(such as EGF,TGF-a,TGF-?,alpha and beta)expression and intestinal mucosal repair cytokines can evaluate the drug for impaired intestinal function.In this study,polysaccharide can alleviate the symptoms of diarrhea Escherichia coli as the goal,based on the establishment of mouse Escherichia coli diarrhea model,with Jianpi Yiqi,curing diarrhea,protecting the intestinal function as the breakthrough point,through the statistics of Escherichia coli diarrhea mice diarrhea,diarrhea index,observed clinical symptoms,autopsy the symptoms,histological changes and ultrastructure changes and expression by immunohistochemistry and real-time fluorescence quantitative detection of relative intestinal mucosal repair by cytokines and genes,to explore the role of evaluation of Atractylodes macrocephala polysaccharide for Escherichia coli diarrhea,and to explore its mechanism of prevention of diarrhea.Experiment 1 Establishment of a mouse model of Escherichia coli diarrheaThis experiment through establishing the model of Escherichia coli infected mice,observe the diarrhea of mice,the clinical symptoms and autopsy symptoms,tissue slice,electron microscopy of mouse changes and ultrastructural changes were observed.Methods:ICR 20 mice were randomly divided into two groups.Divided into two groups,model group and blank group.The model group by intraperitoneal injection of Escherichia coli O111 5.4×107 each mice,control group injected with saline,rare level statistics of mice,loose stools rate calculation,dissected mice,H.E slice,slice,scanning electron microscope transmission electron microscope sample.Results:24h,model group mice diarrhea rate was 100%,the diarrhea index was 0.34,and the blank group had significant difference,the diarrhea model successfully modeled successfully;challenged 24h after model mice intestine were damaged;duodenal injury is more serious,the intestinal villi became short,villous structure damage,intestinal wall damage;observed model group under TEM,mitochondrial swelling,rough endoplasmic reticulum.Experiment 2 Effect of Atractylodes macrocephala polysaccharide on the prevention and treatment of Escherichia coli diarrhea in miceThis experiment was conducted to study the influence of Tremella Polysaccharide on mice diarrhea clinical symptoms,pathological changes,explore the prevention effect of Atractylodesmacrocephala polysaccharide on Escherichia coli caused diarrhea mice,providing the prerequisite for revealing atractylodesmacrocephala polysaccharide on mice diarrhea prevention mechanism.Methods:Atractylodes macrocephala powder was prepared by alcohol precipitation method.The crude polysaccharide was prepared and the content of polysaccharide was determined by phenol sulfuric acid method.Randomly selected 100 ICR mice were randomlydivided into 4 groups,namely blank group,treatment group,prevention group,positive group,each group of 25.The prevention group was given 3 days advanced of Atractylodes macrocephala polysaccharide 0.04mg/ml,0.2ml/(polysaccharide:0.0059mg/each mice).The treatment group,the prevention group,the positive group were injected with Escherichia coli 5.4×107/each mice(0.1ml/),the blank control group was injected with normal saline.Observation of diarrhea in mice.When 1h,8h,24h,48h,72h,96h,120h,respectively,killed 3 mice in each group and were dissected.Record.Results:the positive group at 6h began to appear symptoms of diarrhea,prevention group and treatment group at 8h began to appear symptoms of diarrhea,autopsy found prevention group of small intestine in mice with slight bleeding symptoms,treatment group mice duodenum red hyperaemia,the intestinal wall became thinner,the positive mice intestinal congestion and hemorrhage,twelve intestinal congestion is serious;24h positive group the diarrhea rate was 82.6%,significantly difference and prevention group 43.48%and treatment group 34.8%(P<0.05),diarrhea index positive group were significantly higher than those in the prevention group and treatment group(P<0.05);96h,prevention group and treatment group began to improve,male mice necropsy showed more serious symptoms,medication mice shorter recovery time,intestinal recovery is better.The results showed that the mice diarrhea caused by polysaccharide on Escherichia coli had certain control effects,mainly reflected in the initial slow diarrhea polysaccharide of atractylodesmacrocephala diarrhea,protect the intestinal mucosa,have certain effect for the subsequent treatment of diarrhea caused by Escherichia coli.Experiment 3 Effects of Polysaccharide from Atractylodes macrocephala on intestinal morphology in mice with Escherichia coli diarrheaTo further explore the effect of Atractylodes macrocephala polysaccharide on intestinal mucosal damage in mice,the experiment using Atractylodes macrocephala polysaccharide effect on Escherichia coli diarrhea model of mice was observed,the morphology and ultrastructure morphological changes,to further explore the mechanism of prevention and treatment of Atractylodes polysaccharide on mice diarrhea.Methods:polysaccharide preparation and packet processing method with two experimental mice,when 1h,8h,24h,48h,72h,96h,120h in each group of 3 mice were necropsied,duodenum making H.E slice,scanning electron microscope and transmission electron microscope slice slice,the results show that the anti bacteria 1h,prevention group obvious lesions;treatment group of intestinal gland hemorrhage,mild edema,scanning electron microscopy of intestinal villus is neat;positive group of intestinal gland bleeding,intestinal villi began to shrink,SEM villus shedding damage,partial villous atrophy.24h,the positive group of intestinal villi a large number of damage,symptoms than the prevention group and treatment group.96h prevention group and treatment group began to improve,positive group symptoms.Observed under TEM,mid 24h mice diarrhea,intestinal epithelial cell microvilli medication group mice were arranged in order,mitochondrial swelling,nuclear deformation,dilatation of endoplasmic reticulum;positive group mouse intestinal epithelial microvilli,lodging,nucleus is highly irregular,a large number of mitochondriadegeneration myelin like structures,endoplasmic reticulum expanded containing granular inclusions.The results show that the Atractylodes macrocephala polysaccharide treatment group for intestinal epithelial cell microvilli better protection,Atractylodes polysaccharide has protective role of intestinal mucosal lesions,rough endoplasmic reticulum,mitochondria and other organelles involved in cell metabolism is that Atractylodes macrocephala polysaccharide on intestinal factor may be beneficial regulating effect.Experiment 4 Study on the mechanism of Polysaccharide from Atractylodes macrocephala on intestinal repair in Escherichia coli diarrhea miceThe experimental study of intestinal injury by immunohistochemical method and real time PCR,with the change of time,the skin in the intestine of polysaccharide of Atractylodes macrocephala Koidz on growth factor receptor EGFR and TGF-beta 1(transforming growth factor beta 1)effect.Methods:preparation and test of two mice with packet processing drugs,respectively in 1h,8h,24h,48h,72h,96h,120h group of 3 mice were culled at necropsy,making immunohistochemistry and real time PCR method to detect the expression of EGFR and TGF-beta 1 in the gut.Results:early diarrhea,blank group and other groups of mice EGFR positive staining occurred mostly in crypt epithelial cell cytoplasm;TGF-beta 1 positive staining in intestinal lymphocytes,macrophages and intestinal epithelial cells in the cytoplasm;the groups EGFR and TGF-beta 1 expression and control group had no significant difference;the middle of diarrhea the most serious injury,intestinal mucosa,while EGFR and TGF-beta 1 a large number of positive staining,the EGFR and TGF-beta 1 expression reached peak,each expression was significantly higher than those of control group(P<0.05);late diarrhea prevention group and treatment group,the expression of EGFR and 24h decreased but the difference was not obviously,the positive expression of EGFR was increasing significantly(P<0.01),indicating the Atractylodes macrocephala polysaccharide can affect the expression of EGFR,which maintained at a higher expression level In promoting the rapid proliferation of epithelial cells;TGF-beta 1 expression was decreased,but the prevention group and the treatment group TGF-beta 1 expression level is high,that the Atractylodes macrocephala polysaccharide TGF-beta 1 regulate by regulating TGF-beta I to promote intestinal mucosal repair.Conclusion:Atractylodes macrocephala polysaccharide has a certain effect on the prevention and treatment of diarrhea induced by Escherichia coli in mice.The results showed that there was a significant protective effect on the intestinal tract of diarrhea mice,and the protective effect was the best.It was found that the polysaccharide had protective effects on the microvilli,mitochondria and endoplasmic reticulum of intestinal epithelial cells,and accelerated the recovery of normal metabolism.The main reason is that the Atractylodes macrocephala polysaccharide to modulate intestinal mucosal repair factor EGFR and TGF-beta 1,promote intestinal mucosal repair,resulting in diarrhea of early intestinal mucosal protection polysaccharide can effectively,diarrhea in the late continuous use of Atractylodes macrocephala polysaccharide can make the cell earlier to restore normal metabolism so that the body can quickly recover from the disease,enter the vesting period. |
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