The Effect Of Topical Application Of Mitomycin C On The Benign Bile Duct Stricture In Vitro

Benign biliary stricture is one of the severe common complications from biliary tract surgery.lt is difficult to be treated and obstructive jaundice consequently impairs liver function,leading to biliary cirrhosis.However,surgical treatment is still the mainstay approach for the management of bile duct stricture secondary to fibrous tissue proliferation and scar formation. Some studies reported the using of adrenal cortex hormones in prevention of restenosis of bile duct through inhibiting scar hyperplasia. But its efficacy is uncurtained.Thus,it is very important to investigate the further mechanisms that related to biliary tract stricture and find a better way for the prevention and treatment.Many anti-fibrosis drugs are shown to be effective to inhibit cell proliferation. Mitomycin C (MMC) is an anti-tumor antibiotics isolated from Streptomyces.It can inhibit the proliferation of fibroblasts.This effect is 100 times than fluorouracil and in a time-dependent way.The effect of MMC is closely related to dosage and large dose can cause irreversible inhibition.Topical application of MMC has been shown to play a role in the prevention and treatment of scars,inhibiting the proliferation of an indefinite period of time.It is used in the ophthalmology,orthopedics,ear-nose-throat,urology,orthopedics and upper digestive tract to prevent and eliminate scar formation with very good effects.ObjectiveThe aim of present study is to establish a new model of benigh biliary stricture in rats by electrocoagulation and to evaluate the effect of MMC on prevention of benign biliary stricture.Methods48 Sprague Dawley rats were used in this study.40 rats were randomly divided into four groups:sham-operation group,electrocoagulation group,electrocoagulation+drug 1 (treatment I group,MMC dosage:0.4mg/ml),electrocoagulation+drug 2 (treatmentⅡgroup,MMC dosage:0.6mg/ml).The rest 8 rats were subjected to control group.After anesthetics,the common bile duct (CBD) was dissected in all the rats.Tissues 1 cm around the hilar were separated.For the electrocoagulation group,control group and treatment groups,0.5 cm area of the CBD at the hilar were electrocoagulated using the energy of 8J. Gelatin sponge (1 X 1 X 0.5cm3) with MMC(lml) were covered aroud the injury part of the CBD for 5 minutes for the treatment groups.The control group subjected to water for injection of lml treatment.After the operation,all the rats were fed under the same conditions for 15 days before sacrifice.Body weight,diet intaking,mental status and jaundice were recorded.On sacrificing,blood were extracted from portal vein for the analysis of blood routine, serum total and unconjugated bilirubin,and glutamic-pyruvic transaminase (ALT).Bile ducts were removed and fixed with 10%formalin,embedded with paraffin and sectioned for pathological examination(except control group).The samples were stained for HE staining,Masson trichrome staining (MTS) and examined for the thickness of bile duct wall.Scanning electron microscope was performed.The expression of transforming growth factorβ(TGF-β),a-smooth muscle actin (a-SMA),proliferating cell nuclear antigen (PCNA) and vascular epithelial growth factor (VEGF) were analysed by immunohistochemistry (IHC) and quantified under the microscopy.Micro vessel density (MVD) was also counted.All the variables were compared using ANOVA.ResultsThe serum bilirubin of electrocoagulation group and control group was significantly higher than sham-operated group and the treatment group (P<0.05).Treatment I group had higher serum bilirubin than treatment II group (P<0.05).Serum ALT was significantly higher in the electrocoagulation group than the sham-operated and treatment groups (P<0.05).It was higher in the treatment I group than the treatment II group.WBC was higher in the electrocoagulation group than sham-operation group (P<0.05) and RBC was lower in the sham-operated group than the other groups (P<0.05).In electrocoagulation group and control group,the proximal part of the bile duct over the lesion was dilated and the diameter was 5 times larger than normal bile duct.Three of the 8 rats had ascites.The diameter of proximal part of the bile duct over the lesion is about 1-3 times larger than normal bile duct in treatment I and II groups.No ascites was present in these groups.Pathological findings showed that:in the electrocoagulation group,there was benign stricture of bile duct presenting as fibroblasts proliferated actively with significant fibrosis.The bile duct wall thickened and inflammatory cells extensively infiltrated.MST showed that large quantity of collagen fibers present in the submusoca of bile duct,condensely arranged withour order.The thickness of bile duct wall was significantly higher in the electrocoagulation group than the sham-operated group (5.88±0.67 vs 2.76±0.22 cm,P< 0.01),while no significant difference existed between the treatment I group, treatment group I with the sham-operation group.IHC staining showed that:(1) TGF-β,a-SMA, PCNA-positive cells were significantly differed between the groups (P<0.01);(2) The VEGF-positive points and MVD of electrocoagulation group and treatment group were significantly higher than sham-opeation group (P<0.01). It was lower in the two treatment goup than the electrocoagulation group with no difference between the treatment I group and the treatment II group;(3)The MVD was lower in the electrocoagulation group than in the treatment group (P＜0.01).No difference was found between the two treatment groups.ConclusionsIn this study we established a new model of benign biliary stricture in rats by use of electrocoagulation method.In this model,the main pathological changes of benign bile duct stricture are the prolifereation of fibroblasts of the bile duct wall and the hyperplasia of the collagen fibers.In addition,the expression of TGFβ,α-SMA,PCNA and VEGF were significantly elevated as shown by IHC.Our study also showed that topical application of MMC may play a role in the prevention of bile duct stricture through inhibition of the excessive fibroblast proliferation and collagen fiber crosslinking without affecting the blood routine and liver function.Increase the dosage of MMC from 0.4 mg/ml to 0.6mg/ml may provide a more potent suppression of bile duct stricture.