Study On The Mechanism And Effective Fractions Of Xilei San To Promote Mucosal Ulcers Healing

Xilei-San,a traditional Chinese medicine,is composed of seven crude drugs:indigo naturalis,elephant-tusk scraps,margarita,uroctea compactilis,calculus Bovis,human nail and borneolum syntheticum.It was firstly recorded in Jin Kui Yi written by Zai-Jing You in Qing Dynasty. Its action is clearing away heat;removing toxic materials and eliminating putridity.In traditional usage,it is efficacious for ulcer of oral mucosa,stomatitis,acute pharyngitis and laryngitis,abscess of retropharynx and acute epiglotti- ditis,etc.Modern clinical application showed that recurrent oral ulcer and ulcerative colitis were its major indications,but the therapeutic mechanism of Xilei-San has never been fully understood.In this paper,we cultured mice oral primary fibroblasts and used it as an in vitro model to investigate the effects of Xilei-San on the stimulation of fibroblasts growth,collagen synthesis,MMP-2,9 activities and TIMP-1 production;and to investigate the major effective fractions of these seven drugs which formulate Xilei San.The major content of this study is as following:1.Antimicrobial,analgesic and anti-inflammatory test of Xilei San.â‘ The antimicrobial activity of Xilei San was investigated by slip method and tube double dilution method,the result showed that the minimal bactericide concentration(MBC) of Xilei-San for Escherichia coli, Candida albicans,Pseudomonas aeruginos and Staphyliccus aureus were 25,12.5,12.5,12.5mg/mL respectively.â‘¡The analgesic activity of Xilei San was investigated by acetic acid-induced writhing and hotplate tests,the result showed that the latency time in hotplate test was significantly(P＜0.01) increased, acetic acid-induced writhing was significantly(P＜0.05) inhibited.â‘¢The anti-inflammatory activity of Xilei San was investigated by xylene-induced ear edema,carrageenan induced paw edema and cotton pellet-induced granuloma tests,the result showed that Xylene - induced ear edema,carrageenan-induced paw edema and the weight of the cotton pellet-induced granuloma were significantly(P＜0.05 and 0.01) inhibited.2.Primary mouse oral fibroblasts were cultured in vitro,and the result showed:â‘ After 5 days of incubation,fibroblasts began to grow out of the incubated mucosal fragments.After 20 days,80%area of the culture flask was occupied by fibroblasts,then the cells could be subcultrued.â‘¡The cell proliferation activity of different generation was tested by MTT method,and the result showed that the cell proliferation activity had no significant difference within 6 generations.â‘¢The best quantity of inoculation for 96-well plate(×200μL) had been determined as 4×10⁴ cell/well.3.The effects of Xilei San on fibroblast proliferation,collagen accumulation,matrix metalloproteinases-2,9(MMP-2,9) activities and tissue inhibitor of metalloproteinase 1(TNP-1) production were investigated by MTT,chloramine T method,gelatin zymography and enzyme-linked immunosorbent assays(ELISA) respectively,and the result showed:â‘ The aqueous extract of Xilei san could significantly(P＜0.05) promote fibroblasts proliferation and collagen production,but the alcoholic extract of Xilei san could not.â‘¡Both of the aqueous(125μg/mL) and alcoholic(125μg/mL) extract of Xilei san could significantly(P＜0.05) increased the MMP-2 activity, but had no effect on MMP-9 activity.â‘¢Both of the aqueous(125μg/mL) and alcoholic(125μg/mL) extract of Xilei san could significantly(P＜0.01) increased TIMP-1 production.4.The reducing power was investigated by Oyaizu method,the effect of Xilei San on SOD,MDA and NO of fibroblasts injured by H₂O₂ were also measured,and the result showed:â‘ Both of the aqueous and alcoholic extract of Xilei san per se have high reducing power,the reducing power of 1000μg/mL aqueous extract of Xilei san was equivalent to 40μg/mL Vc.â‘¡The concentration and duration of H₂O₂ which was used to injure the fibroblasts were determined as 100μmol/mL and 16 hours.â‘¢Both of the aqueous and alcoholic extract of Xilei san could significantly(P＜0.O1 or 0.05 ) increase the SOD activity;very significantly(P＜0.01) decrease the MDA releasing amount;and also very significantly(P＜0.01) increase the NO releasing amount.5.The effects of the drugs and their different fractions,which formulates Xilei San,on fibroblast proliferation,collagen accumulation, matrix metalloproteinases-2,9(MMP-2,9) activities and tissue inhibitor of metalloproteinase 1(TIMP-1) production were investigated by MTT, chloramine T method,gelatin zymography and enzyme-linked immunosorbent assays(ELISA) respectively,and the result showed:â‘ The major fractions which could promote fibroblasts proliferation were the aqueous extract of indigo naturalis,the fraction(MR＞14KDa) of margarita,the fraction(MR＜14KDa) of elephant -tusk scraps and the aqueous and alcoholic extract of human nail.â‘¡The major fractions which could inhibit fibroblasts proliferation were the alcoholic extract of indigo naturalis,Calculus Bovis,Borneolum Syntheticum and the alcoholic extract of Uroctea compactilis.6.The reducing powers of the drugs and their different fractions, which formulate Xilei San,were investigated by Oyaizu method,the effects on SOD,MDA and NO of fibroblasts injured by H₂O₂ were also measured,and the result showed that the aqueous extract of indigo naturalis,the aqueous and alcoholic extract of human nail had high antioxidant activity.In conclusion,this study has shown that the mechanism of Xilei San to promote ulcers healing is related with its ability of promoting fibroblasts proliferation,collagen production,increasing MMP-2 and TIMP-1 activities.Among these seven drugs which formulates Xlei San,the major effective fractions,which promote fibroblasts proliferation, collagen production,increassMMP-2 and TIMP-1 activities,are the aqueous extract of indigo naturalis,aqueous and alcoholic extract of human nail, aqueous extract of elephant-tusk scraps(MR＜14kD) and aqueous extract of margarita(MR＞14kD).The alcoholic extract of indigo naturalis,calculus Boris and borneolum syntheticum have inhibiting effects on fibroblasts proliferation and collagen production,but they all have anti-inflammatory activity,which are also important for ulcers healing.This study also showed that uroctea compactilis has inhibiting effects on fibroblasts proliferation and collagen production,and little literatures about uroctea compactilis can be looked up,so its function in ulcers healing is not known.