Role Of UPR-mediated Autophagy In Genital Injury Induced By Flutamide To Female Mice

Objective:(1)To investigate the expression of HSPA5 in human ovarian tissue and its potential as a biomarker for the development of ovarian cancer;(2)to study the role of unfolded protein response(UPR)and its induced autophagy in flutamide induced female rat reproductive system injury model adds some theoretical basis for the study of environmental endocrine disruptors on female sexual genital injurey.Methds:(1)Extract the HSPA5/GRP78 gene expression data in various tissues of normal human body,HSPA5/GRP78 gene expression data of ovarian tissue and ovarian tissue of ovarian cancer patients and protein interaction data with HSPA5 from from BioGPS,Oncomine and String databases.Methods of bioinformatics and biostatistics were used for statistical analysis.Systemic review was used to analyze the relationship between HSPA5/GRP78 gene expression and ovarian cancer,and protein interaction network model was used to analyze proteins interacting with HSPA5/GRP78.(2)ICR mice were intragastrically administered with 300 mg/kg·bw flutamide for 12-18 days of pregnancy,the control group was given an equal volume of soybean oil.The shape and body mass of the female rats were observed after 7 weeks of birth.The uterus and ovaries were taken and weighed,and the organ index was calculated.The uterus and ovarian tissues of the mice were sliced,and pathological changes were observed and the follicles at various levels were counted.Measuring the superoxide dismutase(SOD),glutathioneperoxidase(GSH-Px)and malonaldehyde(MDA)of the uterus and ovary in each group,and then detected the mRNA abundance of UPR,autophagy,apoptosis,angiogenesis,oxidative stress and cell cycle related genes in uterus and ovarian tissues by RT-PCR.Finally,the expression of UPR key proteins in uterus and ovarian tissues was detected by Western blot.To investigate the role of UPR and autophagy in uterine and ovarian tissue injury.Results:(1)BioGPS and Oncomine database results showed that HSPA5/GRP78 was expressed in various normal tissues of human body.The expression level of HSPA5/GRP78 in ovarian cancer tissues was higher than that in normal ovarian tissues(P<0.05);String database results showed HSP90B1,ERN1,DNAJC10 DNAJB1,CANX,ATF6,PDIA3,DNAJB6,DNAJB11,DNAJC7 have an interaction relationship with HSPA5.(2)The female offspring mice in the experimental group had lower body mass than the control group(P<0.05).(3)Histopathological examination revealed that the uterus and ovary were retarded.The results of follicle count showed that the number of primordial follicles and mature follicles in the experimental group were less than those in the control group(P<0.05).(4)The content of GSH-Px and SOD in the uterus of the female mice of the flutamide group were lower than those of the control group,but the MDA content in the uterus tissue was higher than that of the control group(P<0.05).The SOD activity and GSH-Px content in the ovary of the experimental group were lower than those in the control group(P<0.05).(5)The expression in uterus of the experimental group were up-regulated: UPR key genes Grp78,Perk,Ire1,eIF2?,Atf4,Xbp1;autophagy-related genes Atg3,Lc3 b,Akt1,Bcl-xl,Nf-?b;apoptosis-related genes Ask1,Jnk1,Jnk2,Jnk3,Caspase3,Caspase9,Caspase12,Chop,Bax;oxidative stress related gene Noxo1;cell cycle related gene Ccnd1(P<0.05).The expression of down-regulated in uterus was Sod1;angiogenesis-related genes Vegfa and Hif-1?(P<0.05).The genes up-regulated in ovary of the experimental group were: UPR key genes Grp78,Perk,Ire1,Atf6,Atf4,Xbp1;autophagy-related genes Atg3,Aktl,Bcl-xl,Nf-?b;apoptosis-related genes Ask1,Jnk3,Caspase3,Caspase9,Caspase12,Chop,Bax;oxidative stress related gene Noxo1(P<0.05);down-regulated: angiogenesis-related genes Vegfa,Hif-1?;autophagy-related genes Lc3 b,Ulk1;oxidative stress-related gene Gpx1(P<0.05).The protein expression of Grp78,Perk and Ire1 in uterus and ovary tissues of the experimental group were higher than those in the control group(P<0.05).(6)Use the String database to predict protein interactions: Significant gene interactions in uterine tissue are Hspa5,Ddit3/Chop,Xbp1,Atf4;differential gene interactions in ovarian tissue are significant: Hspa5,Ddit3/Chop,Xbp1,Atf4,Ern1/Ire1.Conclusions:(1)The expression level of HSPA5 mRNA in ovarian tissue of ovarian cancer patients is higher than that of normal human ovarian tissue.(2)Exposure to flutamide in pregnancy can induce uterus and ovarian injurey in offspring female mice.(3)Flutamide induced females mice uterine ovarian tissue injurey,follicular reserve reduction may be related to UPR and autophagy.