Study Of The Effects Of Cisplatin And Letrozole On Endometriosis In A Rat Model

BackgroundEndometriosis is a common,frequently-occurring and chronic disease in clinical gynecological practice.It presents diversiform clinical features and complicated pathogenesis,and is believed as genetic disease,inflammatory disease,immune disease,bleeding-induced disease,organ(uterus)-dependent disease and hormone(estrogen)-dependent disease.Currently,endometriosis is even regarded as endometrium disease,stem-cell disease and tumorous disease.Endometriosis is very invasive and easy to recur,which is the hot and hard point in the gynecologic research.For the restriction of clinic trial,animal model is very important and is widely applied in endometriosis research,and that is also the essential point of this study.In the development process of endometriosis,the reflux and implantation of menstrual blood should meets four "must" a.The menstrual blood refluxing through fallopian tube to the pelvic cavity must contain endometrial tissue;b.The glandular epithelium tissue and mesenchymal cells in the refluxing endometrium must be"alive";c.The "alive" tissue and cells must have the potential to implant on the pelvic tissues;d.The anatomic distribution of the pelvic endometriosis lesion must be in accordance with the disseminating pattern of fallopian tube.Further,the refluxing endometrium must break through "Three defensive lines",which include inflammatory factors in the peritoneal fluid,immune cells in the peritoneal cavity and extracellular matrix of peritoneum.With the decades of study,some scholars summarized a "The three stages" for the development of endometriosis,including attachment,aggression,and angiogenesis,which could be named as "3A procedure".Attachment is the first step of the ectopic endometrium's invasion on the pelvic peritoneum or the surface of other organ,then the endometrium aggress and break through the extracellular matrix,and angiogenesis is necessary for the further development,these steps are the so-called "the development of the roots,the development of the tissues,and the development of the disease",which is called The three development process.Based on the theory above,the evaluation of the endometriosis animal model should cover two aspects:(1):The macroscopic view and the histologic observation of the endometriotic tissues;(2):The detection of the biomarker expression in the endometriotic tissues.Many previous animal model studies have been conducted focusing on the mentioned pathogenic steps.However,study on the tumorous character of endometriosis has never been reported,and that is the initial idea and innovation point of this investigation.The three stages,attachment,aggression,and angiogenesis,for the development of endometriosis,and the recurrence and metastasis of endometriosis,have a high similarity with the character of tumor.Because of these reason,endometriosis is accepted as“benign disease,malignant behavior",which reflect more of endometriosis's tumorous character.In gynecologic clinical practice,endometriosis's malignant transformation is not a rare condition,the most common pathologic types of endometriosis 's malignant is endometrioid adenocarcinoma and clear cell carcinoma,which are belong to endometrial cancer and ovarian cancer.In the treatment of these two tumor,peritoneal perfusion chemotherapy plays an important role,cisplatin is the most widely applied and the most effective drug among the optional agents for the peritoneal perfusion chemotherapy.Cisplatin is a cell cycle nonspecific antitumor agent,it can enter tumor cell and inhibit DNA's replication and transcription by inducing the crosslink of DNA,resulting in DNA's breakage and error code,which can inhibit mitosis of the tumor cell and reach the antitumor effect.Aromatase is one complex enzyme of cytochrome P450,which catalyze androstendione and testosterone into estrone and estradiol.Animal studies showed that the expression of aromatase-related proteins elevated in the endometriotic lesion.Letrozole,a nonsteroidal aromatase inhibitor,was found having inhibiting effects on endometriotic tissue in rat model.In addition,through a persistent high-dose oral gavage,letrozole can induce a hyperandrogenism condition,polycystic change and ovulation failure of the ovary,and the loss of estrous cycle in the rats through its aromatase inhibiting effect.In the exploration of therapeutic methods for endometriosis using rat model,previous studies focused on the macroscopic changes of the endometriotic lesion after the letrozole medication,while the investigation about the rat's estrous cycle and hormonal change of the ovary was rarely reported.For this deficiency,in the present study,we study the endometriotic tissue's changes after the letrozole medication,at the same time,the changes of the rat's estrous cycle,hormones and ovulation were also studied.We intend to find the exact mechanism of letrozole on endometriosis,to find that whether the direct inhibition of the aromatase in the endometriotic lesion is the primary function,or the systemic low estrogen condition induced by the letrozole in the rat is the dominant factor.The molecular biology,proteomics,and animal model studies showed that expressions of MMP-2?TIMP-2 and TGFb2 mRNA elevated in the endometriotic tissues of animal model and human patients.Researches also found that IL-1b?TNF-a?VEGF?MCP-1 and PEDF play important roles in the development of endometriosis in rat model.In studies of endometriosis rat model,through peritoneal perfusion of the therapeutic drugs,and study of the change of the endometriotic lesion and the expression of the related proteins and factors,to evaluate the effect of the therapeutic drugs and to explore the potential therapeutic methods.Based on the theory above,the idea of the present study was to explore the possible therapeutic method of endometriosis using the animal model.The biomarkers detected in the study include P450arom,MMP-2,VEGF,and TGF-?.The effect of P450arom was described as above;VEGF is the essential growth factor in the development and metastasis of endometriotic tissue and tumor tissue,its expression decide directly the activity of the endometriotic lesion,MMP-2 is an important member in the MMP family,MMP/TIMP is the main enzyme system dominating the degradation of ECM,and is the critical factor in the invasion and metastasis of endometriotic tissue and tumor tissue.TGF-? is also an important growth factor,and plays a vital role in the growth of endometriotic lesion and tumor.In this study,the endometriosis model was induced by the autologous transplantation of the rat's endometrium,after the evaluation of the success of the animal model,the model rats were divided into groups randomly.In the research process,macroscopic view and volume of the endometriotic lesion,histopathologic observation,biomarker expressions in the ectopic endometrium,the change of the rat's estrous cycle,morphologic,histologic and immunologic changes of the rat's ovary,sex hormone detection of the rats,were all studied.Cisplatin's effect was compared with letrozole.The aim of the study was to explore the effect of the peritoneal perfusion of cisplatin and more mechanism of letrozole's effect on endometriosis,to study more of the pathogenesis and therapeutics of endometriosis,to provide more experimental proof for the clinical diagnosis and treatment of endometriosis.This study contains four parts as described bellow.ObjectivePart 1:To explore the surgical procedure,technical points,and evaluation of the endometriosis rat model,and to provide theoretical support for the medication research of endometriosis using rat model.Part 2:To investigate the effects of cisplatin(CDDP)on surgically induced endometriosis in a rat model.Part 3:To investigate the ovarian function.changes induced by letrozole in an endometriosis rat model.Part 4:This study was to investigate the effects of cisplatin(CDDP)and letrozole on surgically induced endometriosis and comparison of the two drugs in a rat model.Methods and ResultsPart 1.Forty-five SPF(specific-pathogen free)level female SD(Spraue Dawley)rats,were randomly divided into blank group,control group,and treatment group,15 rats each.Endometriosis model was surgical induced by the autologous transplantation of endometrium.In the treatment group,endometrium was separated from serosa layer.In the control group,endometrium was not separated from serosa layer.In the blank group,no endometrium transplantation was conducted.Peritoneal perfusion of 1ml normal saline were performed in the blank and treatment groups every four days.No perfusion was performed in the control group.The growth of the implants,the mean size of the endometriotic lesion,and histopathologic observation were applied to evaluate the success of the model.No died or infected case through the medication process.Endometriotic lesions were successfully formed in the thirty model rats.Before the medication,there was no significant difference between the control group and the treatment group in the mean size of the lesion.When the medication ended,significant increases of the mean size were observed in the two groups,but the mean size of the two groups were similar after the medication.The histopathologic score of the two model groups were similar.The histopathologic score of the two model groups were all similar with the score of the eutopic endometrium of the blank group.Part 2.Animalstudy.Specific-pathogen free(SPF)level animal research facility.Thirty-six Spraue Dawley(SD)rats.Endometriosis(EM)was surgically induced by autologous transplantation.The rats in group 1(control group,n=12)received no medication.The rats in group 2(n=12)were given 3;mg/m2 CDDP every four days.The rats in group 3(n=12)were given 70mg/m2 CDDP every four days.All the rats were administered for a total of 24 days.The growth and histologic scores of the implants were measured.The expression of protein markers,including vascular endothelial growth factor(VEGF),aromatase P450(P450arom),transforming growth factor beta(TGF-?),and matrix metalloprotein(MMP)-2,were assessed using immunohistochemistry,enzyme-linked immuno sorbent assay,and western blot analyses.After the CDDP treatment,the mean implant sizes decreased more in the 2 and 3 groups than in the control group.Mean histologic scores were significantly lower in the 2 and 3 groups than in the control group.When compared with the control group,the protein expressions of VEGF,P450arom,TGF-?,and MMP-2 were significantly lower in group 2 or in group 3.Loss of hair of the rats were observed only in the group 3.A dose-dependent effect was observed between the two CDDP-treated groups.Part 3.Design:A prospective,animal model study.Specific-pathogen free(SPF)level animal research facility.Spraue Dawley(SD)rats were used.Endometriosis(EM)was surgically induced in 36 rats.Twelve rats served as controls(group ?)and received ImL normal saline per day orally.Twelve rats in low-dose letrozole group(group ?)received 0.5 mg/kg/day of oral letrozole.Another twelve rats in high-dose letrozole group(group ?)were given 1 mg/kg/day of oral letrozole.Daily vaginal smears were obtained to observe the rats' estrous cycles.The serum concentrations of FSH,LH,E2,T and P were assayed using chemi-luminescence method.Histological examination of endometriotic tissues and ovaries using macroscopic measurement and HE staining.Compared with the control group,the rats in letrozole groups lost regular estrous cycles after administering letrozole for 14 days and had significantly higher levels of FSH,LH and T,while levels of E2 and P were significantly lower in the letrozole groups.Ovaries in letrozole groups showed many cystic dilating follicles.The ovulation signs were not found in letrozole groups.The mean areas and the mean histologic scores of the implants at the end of the medication in letrozole groups were lower.Part 4.A prospective,animal model study.Specific-pathogen free(SPF)level animal research facility.Spraue Dawley(SD)rats were used.Endometriosis was surgically induced by autologous transplantation of endometrial pieces.Thirty model rats were divided into three groups,randomly.Group 1(n=10)served as control and received no medication.Group 2(n=10)received 0.2 mg/kg/day of oral letrozole.Group 3(n=10)received 35 mg/m2 CDDP via peritoneal perfusion every four days.All the rats were treated for 24 days.The growth and histologic score of the implants were evaluated.The proliferation-and angiogenesis-associated proteins were assessed using immunohistochemistry and western blotting.The serum sex hormones were assayed using ELISA.After the medication,the growth and histologic score of the implants were significantly lower in the 2 and 3 groups than in the control group.The protein expressions of vascular endothelial growth factor(VEGF),aromatase P450(P450arom),transforming growth factor-beta(TGF-?),and matrix metalloproteinase(MMP)-2,were significantly lower in groups 2 and 3 than in the control group.Further,the P450arom level was lower in the letrozole group than in the CDDP group.The TGF-? and MMP-2 levels were lower in the CDDP group than in the letrozole group.Serum T level was significantly higher in the letrozole group,and serum E2 level was lower in the letrozole group.Conclusions:1.The endometriosis rat model is feasible,reliable,and easy to establish,some technical points should be applied in the surgical procedures.The traditional method and modified method all can meet the requirement of the research.The rat model is valuable to the therapeutic drug research of endometriosis.2.The expression of proliferation-and angiogenesis-associated proteins were significantly lower after the cisplatin treatment.Cisplatin caused a significant regression on the size of the endometriotic implants and induced atrophy of these lesions in rats.Cisplatin might has an effect on the patients in the clinical treatment of endometriosis.3.A Polycystic ovary syndrome like state was produced after letrozole administration in the endometriosis rat model.Decreased sizes and lower histologic scores of endometriotic implants were also observed in the letrozole-treated rats.Letrozole might cause the effects on endometriosis in the rats through a joint action directly in the endometriotic lesions and indirectly in the ovaries.4.Cisplatin and letrozole caused similar regression of the implants in the endometriosis model rats.But their effects on the proliferation-and angiogenesis-associated protein expressions and the serum sex hormone levels were different.Cisplatin and letrozole might cause the effects in the endometriotic foci through different mechanism.Background Salpingectomy is a common surgery in clinical gynecological practice,which is widely applied in the treatment of gynecological diseases.In recent decades,great efforts have been devoted to the improvement of salpingectomy,various methods,including abdominal salpingectomy,vaginal salpingectomy,and laparoscopic salpingectomy etc.,have been developed.With the development of the minimally invasive medicine concept,especially the patient's requirement for the quality of life and the childbearing demand after the surgery,the adverse effects on the ovarian blood supply and ovarian function of salpingectomyshouldn't be neglected.Then,to develop a more minimally invasive method,which can minimize the unwanted surgical impacts on ovarian blood supply and ovarian function to a lower level,and can treat the tubal disease at the same time,is of an urgent problem to be solved for gynecologists worldwide.ObjectiveThe present study aimed to evaluate the feasibility,safety,and efficacy of laparoscopic "core-pulling" salpingectomy for tubal pregnancy.And to investigate the potential application value of the novel and minimally invasive salpingectomy variant.Methods and Materialsln this retrospective study,one hundred and fifty-four patients with tubal pregnancy underwent laparoscopic salpingectomy.In total,76 and 78 patients underwent laparoscopic "core-pulling" salpingectomy(LCPS)and conventional multi-port laparoscopic salpingectomy(MPLS),respectively.Then,clinical characteristics,intraoperative findings,and operative outcomes were compared between the two groups.Results The 154 patients underwent laparoscopic surgery(76 LCPS and 78 MPLS)successfully without switching over to abdominal surgery.The patients were 29.9±4.4 and 31.0±4.1 years old in the MPLS and LCPS groups,respectively.Body mass index(BMI).values for the patients were 27.4±4.0 kg/m2(MPLS)and 27.1±3.3 kg/m2(LCPS).There were no statistically significant differences between the two groups(P>0.05).In addition,both groups had similar pathological statuses with regard to the medical history of abdominal surgery,pelvic diseases(including endometriosis and chronic inflammation),and other pathological states(P>0.05).Nosignificant difference was observed with regard to the state of the fallopian tube and location of tubal pregnancy(P>0.05).Grades of tubal adhesion were also similar between the two groups.There were no significant differences between the LCPS and MPLS groups in terms of operative time,estimated blood loss,incidence of postoperative fever,and duration of hospitalization after surgery(P>0.05).No intraoperative complications,including bowel injury,wound infection,heavy bleeding,severe shock,and blood transfusion were observed in either group(P>0.05).Finally,no wound-related problems were observed during postoperative follow-up.Conclusion Laparoscopiccore-pulling salpingectomyis safe,feasible,and effective;It constitutes a practical alternative to conventional salpingectomy and a promising future,and may be applied to prevent unwanted adverse effects on ovarian blood flow and ovarian function in salpingectomy.More studies are needed to reveal its exact effects on ovarian function and pragnancy.