The Effect Of TLRs/MyD88 Signaling Pathway And Inflammatory Factors In Model Rats Of Ulcerative Colitis With Different Combinations Of Compound Lizard Powders

Objectives By observing different combinations of compound lizard powders(CLP)intervention of the general conditions,ulcerative colitis model rats colon tissue pathology,and TLRs/MyD88 signaling pathways downstream of the signal factor myeloid differentiation factor 88(MyD88,tumor necrosis factor receptor related factor 6(TRAF6)protein expression and serum tumor necrosis factor-ɑ(TNF-ɑ),interleukin 10(IL-10)content,the influence of explore the CLP in the treatment of ulcerative colitis(UC)the mechanism of action,provide experimental basis for clinical use of CLP for UC and theoretical basis.Methods 84 SD male rats,aged from 4 to 6weeks,weight 200±20 g,since the purchases in barrier system raised after 7 days,randomly divided into 6 groups,each group of 14,that is,the blank control group,model group,SASP group,CLP80 group,group CLP100,CLP80+100 group,followed by numbered A~F group.All rats fasting water after 24 hours,in addition to the rats using amount of saline enema treatment group A,the other groups rats by trinitrobenzene sulfonic acid(TNBS)enema with ethanol mixed solution preparation of UC model.Groups of the enema were randomly assigned to take a rat test for his dyeing on the third day.The treatment groups were treated therapeutic respectively after the diagnosis criteria of ulcerative colitis under the microscope,group A and group B were given normal saline gavage,continuous 14 days,once a day.After 14 days administration,the rats were fasted for 24 h,the rats were dissected under anesthesia,the abdominal aorta was taken out of the blood and the colon tissue was taken to be observed by naked eyes.HEstaining was used to observe the pathological morphology of colonic mucosa in rats and to evaluate the histological grading.Immunohistochemistry was used to detect the expression of MyD88 and TRAF6 protein in adjacent sections.Determination of TNF-ɑ and IL-10 by ELISA.Analysis of experimental data using statistical methods.Result1.Observation on general situation: No death occurred in the process of modeling,there were 2 deaths in group B to E and there were only 1 deaths in group F during the treatment.Blank control group rats eyes,responsive,feeding water is good,body fat,warm surface,white coat,thick,smooth,glossy,stools granule,defecation is normal,no crissum pollution situation.Most model group rats,glassy-eyed sleep,unresponsive,feeding water is poor,thin shape,surface cooling,the coat is damp,the colour yellow light,or curled up lazy move,abdominal bulge,loose stool with mucous purulent blood,defecate time more,perianal contamination is larger.The SASP group,CLP80 group,CLP100group of rats were shown to be better in the model group by the degree of thickness and the hair color,the abdomen,the volume,and the volume of the mucous abscess.SASP group,CLP80 group,CLP100 group generally the difference between: The number and amount of defecation,purulent stools symptoms of SASP group rats were rapidly improved,but the spirit is still weak,lumpy,eating less water,slow weight gain;The number and amount of defecation,purulent stools symptoms of rats in CLP80 group and CLP100 group medication early abdomen of rats were slowler improved then SASP group in the early,but in the whole treatment process large water rat spirit,activity,diet,weight of rats in CLP80 group and CLP100 group were improved more obviously then the SASP group,and the treatment of late CLP80,CLP100 group rats,quantity,quality and time returns to normal.CLP80+100 group of rats had good mental state,the look,rapid response,activity increased,eating into the water to increase,body,warm coat tightly without moist,the colour white of a less luster,defecate is normal.2.Pathological and histological observation:See to the naked eye: The blank control group rats colon normal form,without the shortening of the circuity,good stretch,in neat rows and dense,intestinal wall edema,erosion,haemorrhage;The colonic of SASP group,CLP80 group,CLP100group had different degrees of tortuous shrinkage,poor stretchability,brittleness change,intestinal stenosis,mucosal uplift and so on.Rats colon shape of CLP80+100 group is improved.Under a microscope to see: In the blank control group,the colon mucosa was clear and the glands were normal;The rats in the model group were blurred,the glandular structure was disturbed and the crypt abscess was seen.The inflammatory cells were infiltrated in the mucosal layer and submucosal layer;Compared with the model group,the degree of inflammation improved.SASP group colon mucosa and submucosal layer see a small amount of inflammatory cell infiltration,mild inflammation;CLP80 group of rats colonic mucosa widely seen scattered in inflammatory cell infiltration,submucosal see a small amount of inflammatory cell infiltration,mild inflammation;CLP100group mucosa showed a small amount of inflammatory cells,submucosal see scattered in the inflammatory cells and granulation tissue proliferation,was between mild and moderate inflammation between the performance;CLP80+100group only seen in the mucosa Inflammatory cells,glands arranged neatly.3.Colon histological damage score:Compared with group A,colon histological injury score increased significantly,there was significant difference(P < 0.05).The treatment groups compared with group B,rats colon tissue injury score decreased significantly,there was significant difference(P<0.05).Comparison among groups CLP80,CLP100,SASP,there was no significant difference(P>0.05).CLP80+100 group compared with CLP80 group,CLP100 group,SASP group,colon histological injury score decreased,there was significant difference(P<0.05),the results showed the effect of CLP80+100 on inflammatory injury and repair is the best.4.ELISA results: Compared with group A,the content of serum TNF-ɑ was increased,the content of serum IL-10 was decreased,there was significant difference(P<0.05).The treatment groups compared with group B,the content of serum TNF-ɑ was decreased,the content of serum IL-10 was increased,there was significant difference(P < 0.05).Comparison among groups of CLP80,CLP100,SASP,there were no significant differences(P>0.05).CLP80+100 group compared with CLP80 group,CLP100 group,SASP group,the content of serum TNF-ɑ was decreased,the content of serum IL-10 was increased,there were significant differences(P < 0.05),the results showed the effect of CLP80+100 group reduce proinflammatory factor TNF-ɑ and promote anti-inflammatory factor IL-10 is the best.5.Immunohistochemical results: Compared with group A,the expression level of colon MyD88 and TRAF6 protein was significantly increased,there was significant difference(P<0.05).The treatment groups compared with group B,the expression level of colon MyD88 and TRAF6 protein was significantly decreased,there was significant difference(P<0.05).Comparison among groups CLP80,CLP100,SASP,there was no significant difference(P>0.05).CLP80+100 group compared with CLP80 group,CLP100 group,SASP group,the expression level of colon MyD88 andTRAF6 protein was significantly decreased,there was significant difference(P < 0.05),the results showed that MyD88 and TRAF6 protein in the CLP80+100 group was the lowest.Conclusion1.Different combinations of compound lizard powders significantly improved the general situation and the pathological and histological observation in the ulcerative colitis model rats.2.Different combinations of compound lizard powders significantly decreased the secretion level of TNF-ɑ,and reduce the score of IL-10 increased colonic tissue injury,which plays an important role in regulating the immune balance,to resist inflammatory factor attack further intestinal mucosa.3.Different combinations of compound lizard powders significantly decreased the expression of TLRs/MyD88 signaling pathway downstream signal molecule MyD88,TRAF6 protein,which control intestinal inflammation,maintain intestinal homeostasis,to promote mucosal repair function.4.Application of 80 mesh and 100 mesh mixed of the different combinations of compound lizards powder plays a better role on improving the colon inflammation elimination and ulcer width,depth on rats with ulcerative colitis.