| Objective:Endometriosis (EMs, referred to as endometriosis) is commonand popular gynecologic disease.Dysmenorrhea, chronic pelvic pain, andinfertility are the most frequent symptoms found in patients suffering fromendometriosis.life quality is affected seriously by endometriosis-associatedpain as well.Pain is associated closely with PG.Recently,Studies have showedthat never fibers proliferate abnormally involving in mechanism of pain.theaim of this experiment through animal experiment is to investigate theexpression of PGP9.5(Protein gene product9.5),NGF(never growth factor)and PGE2(Prostaglandin E2) in ectopic and eutopic endometrium,in order toreveal the part pathogenesis of endometriosis-associated pain, and the methodof using Bushenwenyanghuayu fang on mouse treated,we can discuss theeffect and function mechanism of the method.Method:80sanitary and healthy BALB/c mice，6~8weeks years old,20±2g in body weight.To administer0.5mg/kg Progynova to mouse two daysbefore operation to induce normal estrous cycle(Vaginal smears showed alarge number of keratin epithelial cells).65mice were used Wang Yan-Ping’smethod to duplicate the model of endometriosis.15are sham operated group.60mice were duplicated model triumphantly and divided into4group:modelgroup,Bushenwenyanghuayufang high-dose group,low-dose group,Gestrinonegroup.Those mice of low dose and high dose groups were administrated indose of15.41g/kg,61.76g/kg separately，Gestrinone group0.42mg/kg.The very next day began to establish a mouse model of primarydysmenorrhea model with Progynova (0.5mg/kg)and oxytocin(2U/mouse),totwelfth day.sham operated group was drenched with voluminal distilled water, 1ml/100g,twice a day;model group was drenched with the same voluminaldistilled water morning and Progynova afternoon;high-dose group andlow-dose group were drenched with Bushenweuyanghuayu fang morning andProgynova afternoon,high dose of crude drug6.17g/ml，low dose of crudedrug1.54g/ml.Gestrinone group was drenched with Gestrinone capsulemorning twice a week,other time drenched with voluminal distilled water，andProgynova afternoonThe twelfth day,one hours after the last dosage,peritoneally injectingoxytocinto induce writhing response,0.2ml/mouse or0.2U/mouse.sham-operated group were given the same voluminal distilled Water.Watched fastinghalf hours,decapitated,and asepticed out each ectopic and eutopicendometriosis caesarean organization, the sham operated group tookendometrial organization.Fixed endometrium in4%Polyoxymethylene,Carried them out immunohistochemical; At the same time, Checked anotherpart of the organization add liquid nitrogen to preserve cans quickly to-80℃refrigerator detected by Western-blotting and Real time PCR.andhomogenated to collect clear supernatant liquid for ELISA.Result:1Writhing responseMice of every group shows writhing response,except sham operatedgroup,the incidence was100%.Comparison of the writhing incubation,Compared with the model group,high-dose group and low-dose group,gestrinone group were increased significantly(P<0.01);Comparison of thewrithing frequency,compared with the model group,high-dose group andlow-dose group,gestrinone group were decreased significantly(P>0.05,P<0.01,P<0.01);there is no difference among three treatment groups.2The protein expression of PGP9.52.1PGP9.5protein by ImmunohistochemistryComparison of the ectopic endometrium,PGP9.5expressing positively intransplant ectopic endometrium interstitial cells in the cytoplasm.comparedwith the model group,expression of PGP9.5were decreased signficantly in three treatment groups(P<0.05);while there was no statistical significanceamong three treatment groups (P>0.05).Comparison of the eutopic endometrium,PGP9.5expressing positively ineutopic endometrium glandular epithelium cells in the cytoplasm.compared with the model group,expression of PGP9.5was decreasedsignficantly in sham opreated group(P<0.01);high-dose group was higher thansham opreated group(P<0.05);while there was no statistical significanceamong three treatment groups (P>0.05).2.2PGP9.5protein by Western-blottingComparison of the ectopic endometrium,compared with the modelgroup,expression of PGP9.5was decreased in three treatment groups(P<0.01);there was no statistical significance among three treatment groups (P>0.05).Comparison of the eutopic endometrium,compared with the modelgroup,expression of PGP9.5was decreased in sham opreated group(P<0.01);high-dose group was higher than sham opreated group(P<0.05);while therewas no statistical significance among three treatment groups (P>0.05).2.3PGP9.5mRNA by Real time PCRComparison of the ectopic endometrium,compared with the model group,expression of PGP9.5mRNA was decreased in three treatment groups(P<0.01);high-dose group was lower than gestrinone group(P<0.05);there was nostatistical significance among low-dose group and gestrinone group (P>0.05);high-dose group was lower than low-dose group(P<0.05).Comparison of the eutopic endometrium,compared with the model group,expression of PGP9.5mRNA was decreased in sham operated group(P<0.01);sham operated group was lower than high-dose group(P<0.05);high-dosegroup was lower than gestrinone group(P<0.05);there was no statisticalsignificance between low-dose group and gestrinone group(P>0.05);high-dosegroup was lower than low-dose group(P<0.01).3The protein expression of NGF3.1NGF protein by ImmunohistochemistryComparison of the ectopic endometrium,NGF expressing positively in transplant ectopic endometrium interstitial cells in the cytoplasm.comparedwith the model group,expression of NGF were decreased signficantly in threetreatment groups(P<0.01);while there was no statistical significance amongthree treatment groups (P>0.05).Comparison of the eutopic endometrium,NGF expressing positively ineutopic endometrium interstitial cells in the cytoplasm.compared with themodel group,expression of NGF was decreased signficantly in sham opreatedgroup(P<0.01);high-dose group was higher than sham opreated group(P<0.05);while there was no statistical significance among three treatment groups(P>0.05).3.2NGF protein by Western-blottingComparison of the ectopic endometrium,compared with the modelgroup,expression of NGF was decreased in three treatment groups(P<0.01);there was no statistical significance among three treatment groups (P>0.05).Comparison of the eutopic endometrium,compared with the modelgroup,expression of NGF was decreased in sham opreated group(P<0.01);there was no statistical significance among high-dose group,low-dose groupand sham opreated group(P>0.05);while there was no statistical significanceamong three treatment groups (P>0.05).3.3NGF mRNA by Real time PCRComparison of the ectopic endometrium,compared with the model group,expression of NGF mRNA was decreased in three treatment groups(P<0.01);high-dose group was lower than gestrinone group(P<0.01);low-dose groupwas lower than gestrinone group(P<0.05);high-dose group was lower thanlow-dose group(P<0.05).Comparison of the eutopic endometrium,compared with the model group,expression of NGF mRNA was decreased in sham operated group(P<0.01);there was no statistical significance between high-dose group and shamoperated group(P>0.05);high-dose group was lower than gestrinonegroup(P<0.01);there was no statistical significance between low-dose groupand gestrinone group(P>0.05);high-dose group was lower than low-dose group(P<0.01).4Expression of PGE2by ELISAComparison of the ectopic endometrium,compared with the modelgroup,expression of PGE2was decreased in three treatment groups(P<0.01);high-dose group was lower than gestrinone group (P<0.05);there was noStatistical significance between low-dose group and gestrinone group(P>0.05);high-dose group was lower than low-dose group(P<0.01).Comparison of the eutopic endometrium,compared with the modelgroup,expression of PGE2was decreased in sham operated group (P<0.01);high-dose group was higher than sham operated group(P<0.05);expression ofPGE2in high-dose group was lower than gestrinone group(P<0.05);there wasno statistical significance between low-dose group and gestrinone group(P>0.05);high-dose group was lower than low-dose group(P<0.01).5Correlation analysis among PGE2,PGP9.5and NGFComparison of theectopic endometrium,there was positive correlationbetween expression of PGE2and PGP9.5in peritoneal endometriumtissue(r=0.755,P=0.0005);the expression of PGE2and NGF had a positivecorrelation in peritoneal endometrium tissue(r=0.747,P=0.0025).theexpression of PGP9.5and NGF had a positive correlation in peritonealendometrium tissue(r=0.906，P<0.01).Comparison of the eutopic endometrium,there was positive correlationbetween expression of PGE2and PGP9.5in peritoneal endometriumtissue(r=0.456,P=0.044);the expression of PGE2and NGF had a positivecorrelation in peritoneal endometrium tissue(r=0.336,P=0.11).the expressionof PGP9.5and NGF had a positive correlation in peritoneal endometriumtissue(r=0.964,P<0.01).Conclusions:1the model of endometriosis-dysmenorrhea is successful confirmed bybehavior, gross specimen and pathology, can be used in the study of clinicaldisease. 2NGF,PGP9.5are overexpression in ectopic and eutopic endometriumof mouse endometriosis model,there is a positive correlation between NGFand PGP9.5.It shows that never fibers proliferate abnormally inendometriosis lesion,and NGF can promote neurogenesis in peritonealendometriosis,thus involve in the endometriosis-associated pain pathogenesis.3Expression of PGE2is higher in ectopic and eutopic homogenatedendometrium of mouse endometriosis model,and having a positive correlationwith NGF and PGP9.5,shows that PGE2can promote expression of NGF in ec-topic endometrium to enhance nerve fibers hyperplasia.4Bushenwenyanghuayu chinese medicine can reduce endometrial tissue,transplantation and be of analgesia effect, At the same time,it can attenuat thehigh level of NGF,PGP9.5,PGE2.suggest that the method can ajust level ofNGF,PGP9.5,PGE2in both ectopic and eutopic endometrium to releaseendometriosis-associated pain,the effect is as well as Gestrinone. |
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