Study On The Role And Mechanism Of Ferroptosis Induced By Erastin In Endometriosis

Part One Erastin induces ferroptosis in ectopic endometrial stromal cells ObjectiveTo clarify the role of ferroptosis induced by erastin in primary normal endometrial stromal cells(NESCs)and ectopic endometrial stromal cells(EESCs).MethodsFirstly,NESCs and EESCs were isolated and cultured to establish the cell models in vitro.Secondly,NESCs and EESCs were treated with erastin,the cell activity and markers of ferroptosis were measured before and after treatment.Specifically as follows: 1.NESCs or EESCs were treated with varying concentrations of erastin(0,0.5,0.8,1,1.5,2,2.5,5and 10 ?M)for 24 h,and the cell activity was measured by CCK8.2.NESCs or EESCs were treated with erastin(10 ?M)for 9-10 h,and the total reactive oxygen species(ROS)was assessed by H2 DCFDA fluorescent dye,the lipid ROS was assessed by C11-BODIPY fluorescent dye,and the content of malondialdehyde(MDA)was measured by colorimetric method.3.NESCs or EESCs were treated with erastin(10 ?M)in the absence or presence of inhibitors of regulated cell death for 24 h,and the cell activity was measured by CCK8.4.EESCs were treated with erastin(10 ?M)for 10 h,and the changes of cell microstructure mitochondria were observed by transmission electron scanning microscope.ResultsEESCs were more susceptible to erastin treatment,compared to NESCs.The cell viability was negatively correlated with increased erastin concentration,while the inhibition rate of cell growth was positively correlated.Treatment of cultured EESCs with erastin dramatically increased the total ROS level,lipid ROS level and the content of MDA.The ferroptosis inhibitors,ferrostatin-1 and liproxstatin-1,and iron chelator,deferoxamine(DFO),abolished erastin-induced death in EESCs,while apoptosis inhibitor ZVAD-FMK and necroptosis inhibitor necrostatin-1 failed to maintain the viability of EESCs.In EESCs with erastin treatment,shorter and condensed mitochondria were observed by electron microscopy.ConclusionEESCs were more sensitive to erastin than NESCs and erastin-induced cell death in EESCs displayed characteristic features of ferroptosis.Part Two Role of iron homeostasis in ferroptosis induced by erastin in ectopic endometrial stromal cellsObjective To study the role of iron homeostasis and iron-related proteins in ferroptosis induced by erastin in EESCs.Methods NESCs or EESCs were treated with erastin(10 ?M)for indicated time,the dynamic changes of iron content and iron metabolism related molecules were compared.Specifically as follows: 1.The levels of iron were assayed by Fe Rhonox-1 fluorescent dye.2.The mRNA expression levels of iron-related molecules were detected by reverse transcription and quantitative real-time polymerase chain reaction(RT-qPCR).3.The expression levels of ferroportin(FPN)protein were measured by Western blot(WB).The levels of iron in normal endometrium(n=11),eutopic endometrium(n=13)and ectopic endometrium(n=9)were detected by Prussian blue staining.The expression levels of FPN protein were detected by Immunohistochemistry.The expression levels of FPN protein in NESCs and EESCs were detected by WB.Results Treatment of erastin dramatically increased the iron levels in EESCs.However,erastin treatment did not alter iron levels in NESCs.Treatment of erastin markedly decreased FPN mRNA and protein expression levels in EESCs.Iron staining revealed remarkable accumulation of iron contents in the stromal cells of ectopic endometrium,whereas the normal and eutopic endometrium showed hardly any iron accumulation.Immunohistochemistry revealed that FPN was localized predominantly in the cell membrane.FPN protein levels in ectopic endometrium were lower than in nor-mal endometrium or endometrium of ovarian endometriosis.The protein level of FPN in EESCs showed a tendency to decrease as compared to NESCs.Conclusion Erastin induced ferroptosis in EESCs via iron accumulation.Part Three Ferroportin regulates iron accumulation to promote ferroptosis induced by erastin in ectopic endometrial stromal cellsObjective To study that whether FPN is involved in regulating erastin-induced ferroptosis in EESCs.Methods The expression levels of FPN gene in EESCs were up-regulated by adenovirus mediated ferroportin overexpression(FPN OE)vector.RT-qPCR and WB were used to detect the transcription and protein levels of FPN.After the EESCs,which overexpressed FPN,were successfully constructed,and then the makers of ferroptosis were measured before and after treatment of erastin.Specifically as follows: 1.Adenovirus upregulated the expression of FPN in EESCs,and then treated with different concentrations of erastin(0,0.5,1.5,2.5,5 and 10 ?M)for 24 h.The cell viability was detected by CCK8.2.Adenovirus upregulated the expression of FPN in EESCs,and then treated with erastin(10 ?M)for 9 h.The total ROS was assessed by H2 DCFDA fluorescent dye and the lipid ROS was assessed by C11-BODIPY fluorescent dye.The expression levels of FPN gene in EESCs were down-regulated by siRNA-mediated transfection(FPN siRNA-1,siRNA-2,siRNA-3).RT-qPCR and WB were used to detect the transcription and protein levels of FPN.After the EESCs,in which the gene of FPN was knocked down,were successfully constructed,and then the makers of ferroptosis were measured before and after treatment of erastin.Specifically as follows: 1.siRNA downregulated the expression of FPN in EESCs,and then treated with different concentrations of erastin(0,0.5,1.0,1.5 ? 2.5 ?M)for 24 h.The cell viability was detected by CCK8.2.siRNA downregulated the expression of FPN in EESCs,and then treated with erastin(10 ?M)for 9 h.The total ROS was assessed by H2 DCFDA fluorescent dye and the lipid ROS was assessed by H2 DCFDA fluorescent dye.Results The mRNA and protein levels of FPN considerably elevated after adenovirus infection.Ferroportin overexpression ablated the sensitivity to erastin.Compared with Vector+erastin group,the levels of total ROS and lipid ROS were decreased in FPN OE+erastin group.FPN mRNA and protein levels significantly decreased after siRNA transfection.Knockdown of FPN increased sensitivity to erastin compared to negative control group.Compared with control+erastin group,the levels of total ROS and lipid ROS were elevated in FPN siRNA-2+erastin and FPN siRNA-3+erastin group,respectively.Conclusion FPN regulates iron accumulation to promote ferroptosis induced by erastin in EESCs.Part Four Erastin could regress endometriotic implants in the mouse endometriosis modelObjective To study whether erastin could reduce endometriotic implants in the mouse endometriosis model.Methods Endometriotic lesions were surgically induced by auto-transplantation of uterine horns onto the peritoneal wall,and endometriotic-like lesions were allowed to become established for 2 weeks.Then they were randomly divided into two groups: erastin group and control group.In the erastin group,each mouse received erastin by intraperitoneal injection over a 14-day period.In the control group,in place of erastin,soybean oil was used.At the day of 28,the ectopic lesions in abdominal wall of mice were obtained,and the following experiments were performed: 1.Hematoxylin-eosin staining(HE)was performed on the ectopic lesions in mice.The models were evaluated by observing the appearance of lesions with naked eyes and microscopic structure under microscope.2.The long and short diameters of ectopic lesions in mice were measured,and the volume of lesions was calculated.Compare the volume of ectopic lesions between erastin group and control group.3.To observe whether there is any difference in mental state,coat color,reaction and diet between erastin group and control group.The weights of mice were measured and the difference between the two groups was compared.Results Observed by naked eyes,the plants are completely fused with the peritoneum of the planting site,which is spherical and contains transparent liquid,and obvious filamentous vascular crawling can be seen on the surface of some plants.Under the microscope,it was observed that the glandular structure was similar to that of endometrium,which contained epithelial and stromal cells.It is suggested that the abdominal wall endometriosis model was successfully established.Ectopic lesions size was reduced after treatment with erastin.In addition,erastin had little or no impact on body weights of mice and hair of mice displayed neat and glossy.Conclusion Erastin could regress endometriotic lesions in the mouse endometriosis model.