The Molecular Mechanism Of Endometrial Abnormal Bleeding Caused By Copper Intrauterine Device

Part Ⅰ: Effect of copper-containing intrauterine device on endometrium in macaca mulatta [Purpose] In order to understand the mechanism of endometrial abnormal hemorrhage caused by copper-IUD,we constructed a copper-IUD implantation model in macaca mulatta,and analyzed the concentration of Cu²⁺ in uterine cavity fluid,serum estrogen and progesterone levels,the expression of endometrial hemorrhage-related factors and histopathological changes in different menstrual cycles.[Methods] Twelve adult female macaca mulatta were selected,including 9 in the copper-IUD group and 3 in the control group.Surgical implantation of copper-IUD in the copper-IUD group（according to the equivalent dose conversion method between animal and human body,the size of materials needed for the experiment was calculated on the basis of 220 mm2 copper-containing surface area of IUD implanted in normal human body）,the control group was only treated with uterine incision.After a complete menstrual cycle was observed,the uterine lavage fluid,arterial blood and endometrial tissue were collected during menstruation（the 2nd day of menstrual cycle）,proliferative period（the 10 th day of menstrual cycle）and secretory period（the 20 th day of menstrual cycle）in the next menstrual cycle.Among them,three macaca mulattas were taken from the copper-IUD group in different menstrual cycles,and one from the control group in different menstrual cycles.The Cu²⁺ concentration in uterine lavage fluid of two groups of rmacaca mulattas in different menstrual cycles was detected Flame Atomic Absorption Spectrometry（FAAS）;the endometrial morphology was stained by hematoxylin-eosin staining（HE）;enzyme-linked immunosorbent assay（ELISA）was used to detect the level of estrogen and progesterone;furthermore the expressions of cyclooxygenase-2（COX-2）,vascular endothelial growth factor（vascular endothelial growth factor）,nitric oxide synthase（NOs）were detected by RT-q PCR and Western blot,respectively.[Results] FAAS results showed that the concentration of Cu²⁺ in different menstrual cycles of the copper-IUD group was significantly higher than that of the control group（P < 0.05）,and the greatest difference was in the menstrual period（P < 0.05）;HE results showed that the damage of endometrium during menstruation in the copper-IUD group was more obvious than that in the control group,and there was no significant difference between proliferative and secretory phases;ELISA results showed that there was no significant difference in estrogen and progesterone levels in different menstrual cycles between the copper-IUD group and the control group;The m RNA and protein expression levels of COX-2,VEGF,NOs in the endometrium of the menstrual period in the copper-IUD group were significantly higher than those in the control group（P < 0.05）,and there was no significant difference between proliferative and secretory stages（P > 0.05）.[Conclusions] The release of Cu²⁺ in the uterine fluid of macaca mulatta increased significantly after implantation of copper-IUD,which led to endometrial injury and the increase of the expression of hemorrhage-related factors during menstruation,but did not affect the levels of serum estrogen and progesterone.Part Ⅱ: Preliminary exploration of signaling pathways related to damage repair of endometrium induced by copper-IUD in macaca mulatta [Purpose] In order to investigate the changes of gene expression profiles in endometrial tissues of macaca mulatta after implantation of copper-IUD,and preliminarily explore the signaling pathways involved in the damage repair of endometrium.[Methods] RNA was extracted from endometrial tissues of different menstrual cycles of macaca mulatta in the copper-IUD group and the control group,and high-throughput sequencing was carried out for the second generation.The differentially expressed genes in the two groups during menstruation were screened and bioinformatics were used to analyse the biological processes and molecular functions of these differentially expressed genes.RT-q PCR was used to validate some of the differentially expressed genes and screen the target genes.Western Blot was used to further validate the key target genes.Furthermore,Western Blot and immunohistochemistry were used to detect the expression levels of the related upstream and downstream genes,and to explore the possible pathways involved in endometrial abnormal bleeding induced by copper-IUD.[Results] The results of high throughput sequencing showed that there were 953 differentially expressed genes in the endometrium during menstruation in the copper-IUD group compared with the control group,of these,425 were up-regulated and 528 were down-regulated.Bioinformatics analysis showed that some of the above genes were involved in bleeding injury,Cu²⁺ metabolism,tissue repair and other related processes.The target genes identified by RT-q PCR were MMP1,MMP2,MMP9,MMP14,MT1 A,MT1L,TGF-beta 1,TGF-beta 3.Western Blot results showed that the expression levels of MMP1,MMP2,MMP9 and MMP14 in endometrial tissues during menstruation in the copper-IUD group were significantly higher than those in the control group（P < 0.05）,the protein expression level of MT was significantly lower than that of control group（P < 0.05）,and the protein expression level of TGF-beta 1 was significantly lower than that of control group（P < 0.05）,and the above results were consistent with RT-q PCR.In additionly,Western Blot and immunohistochemical results showed that the protein expression level of TSP1（upstream gene of TGF-beta）was significantly lower in the menstrual endometrium of the copper-IUD group than that of the control group（P<0.05）,and the protein expression level of P-SMAD2/3（downstream gene of TGF-beta）was significantly lower than that of the control group（P<0.05）;The protein expression level of P-P65（phosphorylated NF-κB,upstream gene of MMPs）in endometrial tissues of the Cu-IUD group was significantly higher than that of the control group（P < 0.05）.[Conclusions] Cu-IUD can significantly affect the expression of genes and proteins related to endometrial hemorrhage,Cu²⁺ metabolism and tissue repair in macaca mulatta,and which may participate in the signaling pathways damage repair of endometrium: NF-κB/MMPs（damage pathway）and TSP1/TGF-beta s/SMAD2/3（repair pathway）Part Ⅲ: NF-κB and TGF-β signaling pathways regulates behaveiors in the damage/repair of human endometrial epithelial cells（HEEC）induced by copper-IUD [Purpose] To explore the mechanism of NF-κB and TGF-beta signaling pathways involved in endometrial damage/repair induced by copper-IUD in vitro.[Methods] HEEC was selected as the research object,Cu²⁺ was added to the culture medium for treatment.In order to determine the appropriate concentration of Cu²⁺ for subsequent experiments,Cell Counting Kit-8（CCK-8）was used to detect the cell viability of Cu²⁺ treatment group and untreated group,and scratch test was used to determine whether Cu²⁺ had any effect on the damage repair of HEEC.Western Blot and cellular immunofluorescence were used to detect the damage repair related protein expression levels in Cu²⁺ treatment group and untreated group,including MMP1,MMP2,MMP9,TGF-beta 1,P-P65,P65,P-SMAD2,SMAD2,P-SMAD3,SMAD3 and TPS-1.In the presence of different expression levels of the above proteins,the related pathway of damage repair of HEEC induced by copper-IUD was explored by interfering with P-P65 or TGF-beta 1.Damage pathways: In the case of Cu²⁺ treatment,different concentrations of NF-κB inhibitors（QNZ）were added to the HEEC culture medium of the experimental group.Western Blot was used to detect the protein expression levels of P-P65 and MMP9,scratch test was used to detect the damage repair of HEEC at the optimum concentration.Repair pathway: Under the condition of Cu²⁺ treatment,different concentrations of cytokine TGF-beta 1 were added to HEEC culture medium of experimental group.Western Blot was used to detect the protein expression levels of P-SMAD2,SMAD2,P-SMAD3 and SMAD3.Scratch test was used to detect the expression of HEEC at the optimum concentration.[Results] After 48 hours of treatment with 5 μg/ml Cu²⁺ for HEEC,the cell proliferation activity was about 50% of that of the control group,and scratch test showed that the repair rate of 5μg/ml Cu²⁺ treatment was significantly lower than that of the control group（P < 0.05）;the protein expression levels of MMP1,MMP2,MMP9,P-P65 were significantly higher than those of the control group（P < 0.05）,and the expression levels of TGF-beta,P-SMAD2,P-SMAD3 and TSP1 were significantly lower than those of the control group（P < 0.05）.In the case of Cu²⁺ treatment,the protein expression levels of P-P65 and MMP9 decreased significantly after adding QNZ（100 n M）,and the damage repair rate of HHEC significantly increased.After adding TGF-beta 1（10 ng/ml）,the protein expression levels of P-SMAD2 and P-SMAD3 increased significantly,and the damage repair rate of HHEC significantly increased.[Conclusions] Cu²⁺ can induce the up-regulation of MMPs and P-P65 protein expression and the down-regulation of TGF-beta protein expression in HHEC,and may inhibit the proliferation and repair of HEEC by activating the NF-kappa B/MMP9 signaling pathway（damage pathway）and inhibiting the TSP1/TGF-beta/SMAD2/3 signaling pathway（repair pathway）.