Induction Of Apoptosis By Metformin In Experimentally Induced Endometriosis In A Rat Model

Objective: Endometriosis (Endometriosis, EMs) is a common diseasesamomg child-bearing aged women. EMs treatment methods mainly includedrug therapy and surgery. Because surgical treatment can lead to menopausesymptoms and the recurrence rate is higher, it is not easy to accept forchild-bearing aged women. At present, Chinese herbal medicine and medicinetreatment of EMs has become the hot spot. But its pathogenesis is not yet fullyclear,so the lack of effective treatment methods. In recent years, Sampsontheory of EMs pathogenesis leading theory has been supplemented and revised,countercurrent endometrium needs the adhesion, invasion and the formation ofblood vessels (the "three A" model),which can be the pathogenesis. Todetermine whether EMs occurrence of the key is the endometrium itself, It isnamely "The eutopic endometrium determinism". Research founds thatendometrial apoptosis is the key premise of endometrial organizations tomaintain the normal structure and the function. EMs endometrial cells are ofincreased antiapoptotic activity and reduced promote apoptosis activity, so itcan evade immune clearance, which allow the ectopic endometrial cellsplanting, survival and growth in the uterine cavity, endometrial cells apoptosiscan promote the occurrence and development of EMs.Metformin (metformin, Met) has been first-line treatment of type2diabetes (Diabetes mellitus, DM) more than50years. Recent studies haveshown that metformin have the role in significantly anti-proliferation andpromoting apoptosis wither on the different types of glandular epitheliumcarcinoma by activation of AMPK signaling pathway, for example epithelialovarian carcinoma and endometrial cancer. Metformin can inhibit endometrialstromal cell proliferation and foreign studies have shown the effective ofmetformin on experimentally induced endometriosis in a rat model. This study to investigate the effects of metformin on experimentally inducedendometriosis in a rat model and to identify the pathways involved in theeffects.Studies have shown that the antiapoptotic Bcl-2gene protein expressionin patients ectopic and eutopic endometrial tissues with EMs weresignificantly higher than the expression in normal endometrium. Apoptosisregulatory protein Bcl-2/Bax and P53play a crucial role in EMs development.Apoptosis regulatory genes Bcl-2/Bax and P53may be therapeutic targets forEMs; Based on the above research and apoptosis playing an important role inendometrial metastasis, invasion and angiogenesis, further study to verifymetformin can induce apoptosis by down-regulating apoptosis regulatoryprotein Bcl-2and up-regulating Bax and P53expression and to explore themolecular mechanism, which provide new strategies and targets for theclinical treatment of Ems.Methods: Experimentally induced endometriosis in a rat model byautologous endometrium transplanting during non-estrus.55rats were used,5of which died to intestinal obstruction.40rats met the inclusioncriteria(height>2mm), ectopic endometrial tissue of two model rat are forhistopathological observation. The remaining38mouse randomly divided intoA, B, C, D group by digital table method, Group A (n=10) was given50mg kg~-1d~-1of oral metformin. Group B (n=10) was given100mg kg~-1d~-1oforal metformin. Group C (n=10) was given200mg kg~-1d-1of oralmetformin. GroupD (n=8) was given saline as placebo. respectively, for28days. Collect samples for index determination.1Measurement the volume of ectopic endometrium before and after drug;Statistical analysis the volume of ectopic endometrium after treatment.2Eutopic endometrium and ectopic endometrium for pathologicalmorphology observation by HE.3TUNEL was used to assay in the eutopic endometrium and ectopicendometrium, apoptosis were expressed by apoptotic index.4Bcl-2, Bax and P53protein expression was determined by SABC immunohistochemistry in ectopic endometrial tissue.5Bcl-2, Bax and P53mRNA level of gene transcription was determinedectopic endometrial tissue by reverse transcriptase PCR (RT-PCR).6Phosphorylation AMPK (P-AMPK) and phosphorylation mTOR (P-mTOR)protein expression was determined in ectopic endometrium by the Westernimmunoblotting.7Cell cycle analysis was performed in ectopic endometrium by flowcytometry.8P-AMPK activity correlation with G1cell ratio was determined by Pearsoncorrelation analysis.9Experimental data are showed with X±s, and are analyzed by the statisticalsoftware SPSS13.0. Materials meet normality and Homoscedasticity analizedby One-Way ANOVA and SNK-q. Others compared with the Kruskal-Wills Hand Mann-Whitney U. All data P≤0.05was deemed to have statisticalsignificance.Results:1Experimentally induced endometriosis in a rat model by autologousendometrium transplanting during non-estrus, rate for80%.2The volumes of ectopic endometrium in group A,B,C,D before treatmentwere not significantly different (P>0.05); The average volumes of ectopicendometrium in group A,B,C are smaller than that in control groupD (P<0.05),which has statistical difference; The average volumes of ectopicendometrium in group A,B,C are not different,which has not statisticaldifference (P>0.05); But the volume of ectopic endometrium using metformin100mg kg-1d-1group is reduced more obviously.3The ectopic endometrium and eutopic endometrium were observed after HEstaining. Compared with the control group D, glands relatively small numberof small glandular and glandular epithelium was flat or low cuboidalpart,mesenchymal cells become smaller in ectopic endometrium of the group A, B,C; The eutopic endometrial glands was more significantly reduced in group A,B, C than that in the control group D,The majority of the glandular epithelium was columnar or cubic, stromal cytoplasmic dense in the group A, B, C.4The ectopic endometrium apoptosis and eutopic endometrium apoptosis aredetected by TUNEL: the apoptotic index of the ectopic endometrium in groupA, B, C are respectively (15.29±0.035)%,(29.65±0.259)%,(25.96±0.178)%, were significantly higher compared with the control group D (7.14±0.052)%(P<0.05). The increased apoptotic index in metformin100mg kg~-1d~-1group is the most obvious (P<0.05); The apoptotic index of the eutopicendometrium in group A, B, C are respectively (26.07±0.155)%,(29.65±0.259)%,(28.37±0.105)%, are higher thanthat in contrast group D (7.14±0.052)%(P<0.05).The increased apoptotic index in metformin100mg kg~-1d~-1group the most significant (P <0.05).5Bcl-2, Bax and P53protein expression was determined by SABCimmunohistochemistry in ectopic endometrial tissue. It is established thatmetformin can induce apoptosis in ectopic endometrium by down-regulatingBcl-2protein expression and mutation P53protein expression, andup-regulating Bax protein expression (P<0.05).6RT-PCR demonstrated the inhibition of Bcl-2mRNA levels and adose-dependent induction of Bax mRNA and P53mRNA levels in alltreatment groups (P<0.01).But also metformin reduced the ratio ofBcl-2mRNA and Bax mRNA(P<0.01).7Western immunoblotting showed an increase of phospho-AMPK activatedform, and an decrease of phospho-mTOR activated form in group A, B, C(P<0.05). The P-AMPK protein expression are increased and the P-mTORprotein expression are reduced more significantly in metformin100mg kg-1d-1group (P <0.05).8Metformin provoked a cell cycle arrest in the G0/G1phase (in group A, B,C, D respectively:71.66±0.526,79.48±0.465,71.01±0.387,58.66±0.358,P<0.05) in an AMPK-independent manner by Flow cytometry. The ectopicendometrium cells G0/G1phase ratio is the highest in metformin100mg kg~-1d~-1group (P <0.05).9Moreover, protein expression of phospho-AMPK was positively correlated with the number of cells in the G0/G1phase (r=0.95, P=0.05).Conclusions：Metformin caused a statistically significant regression of endometrioticimplants in rats. Metformin induced apoptosis in ectopic endometrium in anAMPK-independent manner and provoked a cell cycle arrest in theG0/G1phase. Moreover, we established that metformin can induce apoptosisby down-regulating Bcl-2and up-regulating Bax and P53expression, thusinhibiting the progress of the of ectopic endometrim.