Effects Of Pulsatilla Alcohol Extract On Neutrophil Elastase Of TNBS Induced Colitis In Rats

Objective: To explore the effects of Pulsatilla alcohol extract onneutrophil elastase of Trinitrobenzene sulfonic acid induced colitis in rats.Methods: Acute colitis of rats was induced by giving TNBS.Twenty-eight rats were randomized into TNBS group (n=10),Pulsatillaalcohol extract treated group(n=10),normal controlled group(n=8).Toestablish the model of TNBS-induced colitis in rats:all rats were fasting for24hours but drinking water freely,2%pentobarbital sodium injection (2.3ml/kg)after intraperitoneal injection of anesthetic rats;With a diameter of about2mmand length about12cm silicone tube gently insert a depth of about8cm intoanus of TNBS group and Pulsatilla alcohol extract treated group,withcontaining0.85ml solution with80mg/kg TNBS equal volume of50%ethanol solution slowly into the colon,and let rats keep anus raise at45degreefor2-3minutes to avoid that enema reflows,Promote absorption,and then keeprats lie low,to its natural awake and back feeding in a cage.The normalcontrolled group enema with an equal volume of saline into the colon underthe same anesthesia as control,the rest of the process are the same as before.After the success of the building complete colitis,normal controlledgroup,TNBS model group were each daily enema once with1ml of distilledwater for7days.Pulsatilla alcohol extract treated group rats were perfusedwith1ml Pulsatilla alcohol extract at the dosage of2g/kg/d for7days.Ratsin each group with normal feeding,Given free access to water and food in thesame particles.activity,stools,body weights were observed.The8thday,aftersodium pentobarbital anesthesia and take the inner canthus blood to determination plasma D-lactic acid,neutrophil elastase,TNF-alpha and serumIL-8.All the rats were cut the neck off on the8thday,to remove the entirelength of the colon and the longitudinal cut open, and use saline wash dirt andobserve the entire colon observe whether of colon ulcer,congestion edemaand of active bleeding;take the same part of2cm colon tissue of each rat,formalinfixed for HE staining,Simultaneously disease activity index (DAI) andhistopathological injury score were calculated.The remaining specimens werepre-fixed with3%glutaraldehyde for observation the ultrastructural changesof colon tissue with transmission electron microscopy (TEM).Results: General condition:Rat animals24hours after the modelappeared apathetic,Activity and food intake was significantly lower thanbefore,Peripheral stimulation weakened,Decreased body weight,Some ratshemorrhagic diarrhea with blood in the stool or fecal occult blood positive forthe naked eye.TNBS model group,four days before the severe diarrhea,hairfluffy,crisp,After three days of diarrhea gradually ease gradually,A slightincrease in body weight and food intake,Pulsatilla alcohol extract treatedgroup compared with the model group,diet, activity gradually increased,slowly increasing body mass,the spirit of improvement,gradually reducediarrhea,symptoms improved significantly over the forming stool, bloodystool showed more positive occult blood, and weight loss is not obvious.Histologically model group colonic epithelial necrosis, mucosa, submucosaand grassroots lymphocytes, plasma cells, glands rupture, structuraldisorder,loss of goblet cells,crypt abscess formation,shows multipleulcers,Ulcers deep muscularis propria. Pulsatilla group of inflammatory cellinfiltration less light microscope under visible mucous membranes,ulcersinvolving the submucosa, glandular structure improved ulcers was hyperplasia of epithelial cells covering the edge of granulation tissuefibrosis.(2)Disease activity index (DAI), The DAI of TNBS group、Pulsatillaalcohol extract treated group and normal controlled group respectively was8.80±3.04、5.10±1.85and1.00±0.74,histologic lesion scores were4.00±2.16、2.70±1.15and0.37±0.51,all have significant difference compared with normalgroup,(P <0.05).(3)Serum TNF alpha,IL-8and NE,D-lactic acid content inplasma: TNBS induced colitis in rats after elevated levels of serum TNF alpha,IL-8,NE,D-lactic acid levels in plasma,but after Pulsatilla alcohol extracttreatment,plasma NE level,serum TNF alpha level,and the level of serum IL-8、D-lactic acid levels were lower (P <0.05).(4)TNBS model group bytransmission electron microscopy (TEM) in sparse, irregular, swollenmitochondria, rough endoplasmic reticulum,ribosomes are lost and freebubble formation.Occasional nuclear chromatin edge set. Pulsatilla alcoholextract treated group compared with TNBS model group improvedsomewhat,there are still differences compared with normal controlled group.Conclusion: Pulsatilla alcohol extract has significant anti-inflammatoryeffect in IBD,and the possible mechanism of action is: Pulsatilla alcoholextract inhibition of the NE release,further inhibiting inflammatory mediatercascade reaction,achieve the anti-inflammatory effect.