Study Of Effects And Molecular Mechanism Of Eutopic Endometrial Ischemia On The Formation Of Peritoneal Endometriotic Lesion

Part One Buiding endometriotic model in mice using normalendometriumObjectiveEndometriotic animal model in mice of C57BL/6was developed by an improvedmethod of intraperitoneal injection of normal endometrial fragments. The possibleinfluences on the development of endometriosis in mice model was studied, including thequantity of endometrial fragments for seeding and the times of injections.MethodsThe method of intrapelvic injecting of normal endometrial fragments was improvedby follow: The endometrium were chopped into1×1mm wide pieces, and put into20-gauge needle for intraperitoneal injection.Endometriosis model of mice was induced byintrapelvic injecting of endometrial fragments.（1）Effects of quantity of endometrialfragments on the development of endometriosis: According to the quantity of endometrialfragments for seeding, Sixty adult female C57BL/6were grouped into6groups（n=10）:1P group,2P group,4P group,8P group,16P group,32P-group.（2）Effects of injectiontimes on the development of endometriosis: Fifty adult female C57BL/6were divided into5groups according to the injection times with one piece of endometrial fragments（n=10）:1T group,1T group,2T group,3T group,4T group,5T group. Seven days after induction,implantation of endometriosis was checked to calculate the induction rate of endometriosisand the survival rate of endometrium. ResultsImproved method for development of endometriosis in mice was a good method forfollow advantage: Little endometrial fragments were remained in needle. The lesions werescattered and uniform in size. And induction rate of endometriosis and the survival rate ofendometrial fragments were stable.（1）Effects of quantity of endometrial fragments on thedevelopment of endometriosis: The induction rate of endometriosis was increased withquantity of endometrial fragments, which was20%in1P group and60%in8P group.When the quantity of endometrial fragments was more than16pieces, the induction ratewas100%. Quantity of endometrial fragments was positive correlated with the inductionrate of endometriosis assessed by the Pearson correlation coefficients, followed aQuadratic trend line of Y=1.095+0.863x-0.18x2（R2=0.944，F=33.45，p=0.003）;Quantityof endometrial fragments was positive correlated with the survival rate of endometrium,followed a Quadratic trend line of: Y=-1.167+3.72x-0.61x2（R2=0.972，F=69.127，p=0.001）（.2）Effects of injection times on the development of endometriosis: Chi--squaretest indicated that the induction rate of endometriosis of5Tgroup (100%,10/10）weresignificantly higher than1T group(20%,2/10）(p=0.001); the survival rate of endometrium.of5Tgroup (64%,32/50） were significantly higher than1T groups (20%,2/10）(p=0.027).Injection times was positive correlated with the induction rate of endometriosis,followed a Quadratic trend line of Y=1.095+0.863x-0.18x2（R2=0.944，F=33.45，p=0.003）; Injection times was positive correlated with the survival rate of endometrium,followed a Power trend line of: Y=1.99X0.949（R2=0.962，F=88.357，p=0.011.ConclusionImproved method for development of endometriosis in mice was a good method forfollow advantage: little endometrial fragments were remained in needle. The lesions werescattered and uniform in size. And induction rate of endometriosis and the survival rate ofendometrial fragments were stable. Quantity of endometrial fragments and injection timeswere positive correlated with the induction rate of endometriosis and the survival rate ofendometrium, suggesting that quantity of endometrial fragments and injection times are risk factors of e ndometriosis. Part Two Study of the implanting of ischemia endometrium on thesurface of peritoneumObjectiveContraction of spiral artery causes ischemia of endometrium in menstrual period formore than4-48hours. There are small endometrial fragments followed into pelvic cavity.But at present, the exact changes and ultimate destination of retrograde endometrialfragments are still not clear because of ethics reasons. The objective of the study was toobserve the dynamic changes and ultimate destination of retrograde endometrial fragmentsafter different time of ischemia.MethodsIschemia model of endometrium was made by ligature of uterine artery in mice.Donor mice and recipient mice were divided into6groups by the hour of ischemia:0h-group、0.5h-group、1h-group、2h-group、4h-group、8h-group. Endometriosis modelof mice was induced by intrapelvic injecting of endometrial fragments.Seven days afterinduction, implantation of endometriosis was checked to calculate the induction rate ofendometriosis and the survival rate of endometrium. Endometrial fragments were taken fordetecting apoptosis by TUNEL Assay before and24hours after induction.ResultsTime of ischemia was positive related to AI of endomerium before and afterinduction.(R=0.865，p=0.0029; R=0.904，p=0.0048). Chi--square test indicated that AI ofendometrium after induction were significantly lower than before induction (p<0.05). Theinduction rate of endometriosis and the survival rate of endometrium were decreased withthe time of ischemia. There was no endometrium implanting on the surface of peritoneumof4h-group. Time of ischemia was negatively related to the induction rate ofendometriosis(R=-0.757，p=0.041) and the survival rate of endometrium（R=-0.986，p=0.004） by Spearman’s Rank Correlation Analysis.ConclusionThe damage of endomerium increases with the time of ischemia, expressed by necrosis or apoptosis of cells. The damage of endomerium increases after injected intopelvic cavity. After more than4hours of ischemia, the endomerium can not implant on thesurface of peritoneum because of necrosis and apoptosis. Part Three Study of Effects and molecular mechanism of Ischemicpreconditioning(IPC) on the implanting of ischemia endometrium on thesurface of peritoneumObjectiveIschemic-preconditioning (IPC) is a phenomenon that slight ischemia and reperfusionincrease tissue tolerance to the subsequent lethal ischemia. Retrograde endometrialfragments don’t implant on the surface of peritoneum because of ischemia in womenwithout endometriosis, while on the other hand, in women with endometriosis retrogradeendometrial fragments do. Mild ischemia of endometrium in women with endometriosismight play important role in endometriosis, which may protect endometrium fromischemia damage.IPC model of endometrium was made in mice to study the effects andmolecular mechanism of IPC on the implanting of ischemia endometrium on the surface ofperitoneum.Methods(1)Building IPC model of endometrium in mice: Model of IPC of endometrium wasmade by muscle injection of.Hemabate which causes the contraction of the uterus. Donormice and recipient mice were divided into5groups by the dose of Hemabate: A group(0.125mg/kg weight)， B group (0.25mg/kg weight)， C group(0.5mg/kgweight)，Dgroup (1.0mg/kg weight)。E group(control group)(0.1ml water for injection instead ofHemabate). The oxygen concentration of tissue of uterus was checked.Seven days afterinduction, implantation of endometriosis was checked to calculate the induction rate ofendometriosis and the survival rate of endometrium.(2) Role of IPC on the implanting of ischemia endometrium on the surface ofperitoneum: Mice were divided into6groups: IPC group, ISCH2h group (ischemia for2hours)，ISCH4h group，IPC+ISCH2h group， IPC+ISCH4group and CON group。Endometriosis model of mice was induced by intrapelvic injecting of endometrialfragments. Seven days after induction, implantation of endometriosis was checked to calculate the induction rate of endometriosis and the survival rate of endometrium.24hoursafter induction, Endometrium was taken for detecting apoptosis by TUNEL Assay beforeand after induction.(3) Molecular mechanism of IPC: Mice were divided into4groups: CON group,ISCH group (ischemia for2hours),IPC+ISCH group, LY+IPC+ISCH group （LY:LY294002）。 Endometrium was taken for detecting apoptosis byTUNEL Assay,histological examinations using HE staining, and protein molecules were detected usingWEST test after ischemia of endometrium for2hours.Results(1) The dose of Hemabate was negative correlated with the lowest oxygenconcentration of tissue of uterus (p=0.049，R=-0.88) and positive correlated with the timeof hypoxia(p=0.000，R=1.0).(2) Role of IPC on the implanting of ischemia endometrium on the surface ofperitoneum: AI of every group were higher after induction than before (p<0.05). Beforeinduction, AI of IPC+ISCH2h group was lower than ISCH2h group (p<0.05), AI ofIPC+ISCH4h group was lower than ISCH4h group (p<0.05). After induction, AI ofIPC+ISCH2h group was lower than ISCH2h group (p<0.05), AI of IPC+ISCH4h groupwas lower than ISCH4h group (p<0.05). Both induction rate of endometriosis andsurvival rate of endometrium of IPC+ISCH2h group were higher than ISCH2hgroup(p=0.028，p=0.000).Both induction rate of endometriosis and survival rate ofendometrium of IPC+ISCH4h group were higher than ISCH4h group(p=0.035，p=0.024).(3) Molecular mechanism of IPC: HS of IPC+ISCH group was higher than ISCH andIPC+ISCH+LY group(p<0.05).AI of IPC+ISCH group was lower than ISCH group andIPC+ISCH+LYgroup (p<0.05). There was no different in AI between IPC+ISCH+LYgroup and ISCH group (p>0.05). Protein molecules of p-Akt、p-Bad、p-GSK-3β、p-mTOR、p-4EBP1、 p-S6in IPC+ISCH group were higher than CON group and ISCHgroup(p<0.05), but there was no difference in p-Akt,p-GSK-3β,p-mTOR,p-4EBP1,p-S6between IPC+ISCH+LY group and ISCH group(p>0.05). p-Bad of LY+IPC+ISCH group was lower than ISCH group (p<0.05). Cleaved caspase-3of IPC+ISCH group was lowerthan ISCH group and LY+IPC+ISCH group(p>0.05), there was no difference betweenIPC+ISCH+LY group and ISCH group(p>0.05).ConclusionIPC improved implantation of endometrium on the surface of peritoneum. IPC protectthe endometrium against apoptosis by acted the phosphatidylinositol3(PI3) kinase/Aktpathway. Akt phosphorylates various downstream elements, including Bad, GSK-3βandmTOR. Phosphorylation of Bad and GSK-3β by Akt suppress caspase-3activity and blockapoptosis. Phosphorylation of mTOR promote cell growth and proliferation.