Mitochondrial Superoxide Dismutase Extends Antioxidant Functions In Ovarian Endometriosis

BackgroundsEndometriosis,as an ectopic location for endometrial tissues outside of the uterine cavity,mostly located in ovarian,is a considerable threat to the physical,psychological,and social integrity of women,causing pelvic pain and infertility in 10-15%women.Despite intensive research,its mechanistic rationale still remains elusive.Although the precise etiology of endometriosis is unclear,it is generally considered to be multifactorial.According to Sampson's theory,one of the main theory about endometriosis,retrograde menstruation carry highly pro-oxidant factors like iron,heme and apoptotic endometrial tissues into the peritoneal cavity.This mechanism can induce increased activated macrophages and production of pro-inflamatory cytokines factors,which promote proliferation and survival of ectopic lesion.During these events,cells produce many reactive oxidative species(ROS),which are involved in endometriosis etiology.Mitochondria are major sources for ROS generation.It is also an essential organelle crucial in calcium homeostasis,cellular proliferation regulation and apoptosis-programmed cell death.Studies have shown that mitochondrial DNA mutations are associated with endometriosis.However,there are few further studies on mitochondria and the development of endometriosis.Mitochondrial superoxide dismutase(SOD2),localized in the mitochondria,is an antioxidant enzyme,playing a crucial role in maintaining cellular ROS balance.It has been reported that SOD2 highly expressed in the ectopic endometrium.Despite its antioxidant role,to date,little is known about its other functions in endometriosis.To study the oxidative stress and anti-oxidation system of ectopic cysts in the ovary,and provide new ideas for further study of endometriosis.Objectives:To study the function of mitochondria and mitochondrial superoxide dismutase in the development of endometriosis.To investigate the oxidative stress and anti-oxidation system of ectopic cysts in the ovary,and provide new ideas for further study of endometriosis.Method:151 endometrial specimens were collected in this study,of which 78 were ectopic endometrium(EC)from patients with endometriosis,38 were eutopic endometrium(EU)from patients with endometriosis,35 were controlled endometrium(CE)from patients without endometriosis.The ROS of the three groups were detected by flow cytometry.The morphology of mitochondria was observed by transmission electron microscopy.The metabolic function of mitochondrial was detected by Seahorse.The localization and expression of mitochondrial superoxide dismutase(SOD2)were detected by immunohistochemistry,real-time PCR and Western blot.The expression of SOD2 in ectopic endometrial stromal cells was knocked down by small interfering RNA.Then the EC were grouped into si-Ctrl group(control group)and si-SOD2(transfection group).The changes in mitochondrial oxidative metabolism were observed by Seahorse,the effects of decreased SOD2 on endometriotic stromal cell migration and proliferation were assessed by cell motility and proliferation assays,and the cell membrane potential and apoptosis were detected by flow cytometry.Results:1.The production of mitochondrial superoxide in ectopic ESCs was increased by 90%and 98%compared with eutopic ESCs and controlled ESCs respectively(P<0.01,P<0.01).No significant difference was found in mtDNA copy numbers and transcription among CE,EU and EC groups.2.The numbers of mitochondria was incremental in ectopic ESCs compared to eutopic ESCs and controlled ESCs.The mitochondria tended to gather in the central part,with varying sizes and shapes without swelling in ectopic ESCs.The mitochondrial mean length of ectopic ESCs was 720.5 nm,which was longer than that of eutopic ESCs and controlled ESCs(P<0.01,P<0.0001).3.The basal OCR in ectopic ESCs was 95%higher than eutopic ESCs(P<0.0001)and 51%higher than controlled ESCs(P<0.01).ATP-linked OCR in ectopic ESCs was 79%higher than eutopic ESCs(P<0.001).Reserve capacity in ectopic ESCs was 77%lower than eutopic ESCs(P0.0001)and 81%lower than controlled ESCs(P<0.001).As for proton leak and non-mitochondrial,ectopic ESCs was significantly increased than eutopic ESCs and controlled ESCs.Glycolysis in ectopic ESCs was 14%higher than eutopic ESCs(P<0.001)and 132%higher than contr4olled ESCs(P0.001),glycolytic capacity in ectopic ESCs was 81%higher than controlled ESCs(P<0.0001).No significant difference of glycolytic capacity was found between ectopic ESCs and eutopic ESCs.The basal OCR/ECAR ratio in ectopic and eutopic ESCs were obviously decreased compared to controlled ESCs(P<0.01)4.SOD2 immune expression was distributed strongly in the cytoplasm of the surface epithelial and stromal cells,especially stromal cells in the ectopic endometrium,and also in glandular epithelium cells and stromal cells of eutopic endometrium,while controlled endometrium showed low expression of SOD2.The SOD2 mRNA expression was significantly elevated in ectopic ESCs compared to eutopic ESCs(P<0.01)and controlled ESCs(P<0.01).The SOD2 protein also expressed higher in ectopic ESCs than eutopic ESCs(P<0.05)and controlled ESCs(P<0.0001).The ratio SOD2/GPxl in mRNA levels was significantly elevated in ectopic ESCs compared to eutopic ESCs(P<0.01)and controlled ESCs(P<0.001).5.Significant reduction of basal OCR,maximal OCR,respiratory reserve capacity,ATP-linked OCR and non-mitochondrial OCR were observed in si-SOD2 treated cells compared with si-Ctrl treated cells(P<0.0001,P<0.0001,P<0.001,P<0.0001,P<0.05,respectively).No significant decreases in ECAR were observed between si-Ctrl and si-SOD2 transfected cells.The basal OCR/ECAR ratio,which was higher in si-SOD2 treated cells than that in si-Ctrl treated cells(P<0.0001).6.OD was lower in si-SOD2 treated cells than si-Ctrl treated cells(P<0.001).The number of migrated cells in the si-SOD2 treated groups was significantly decreased compared with that in the si-Ctrl group(P<0.001).The MMP was significantly decreased in si-SOD2 treated cells than control group(P<0.001).Conclusion:Endometriosis is associated with oxidative stress.The number of mitochondria and energy metabolism in ectopic endometrial stromal cells were higher than those in the other two groups.The mitochondrial antioxidant enzyme SOD2 is highly expressed in the ectopic endometrium and is associated with endometriosis disease.