Role Of UPR-mediated Autophagy In Genital Injur Induced By Flutamide To Malemice

Objective:To study the role of unfolded protein response(UPR)and its induced autophagy in the testis and penile malformation in duced by flutamide,and provide a theoretical basis for the study of genital malformation and tumor mechanism.Methods:(1)Use bioinformatics and biostatistics methods,thro ugh BioGPS,Oncomine,and String databases.The expression of Pe rk,Ulk1 and Bax genes in testicular tumors was studied;(2)Then,wi th the use of flutamide,the ICR mice and the control group were given an equal volume of soybean oil at 300 mg/kg·bw for 12-18 days of pregnancy,and the shape and body mass of the male rats were observed after 7 weeks of birth.(3)Then the penis and testis were taken and their length,size and volume were calculated.The mouse penis and testicular tissue were sliced and the pathological c hanges were observed;(4)Real-time PCR was performed by randoml y measuring the content of superoxide dismutase(SOD),glutathione peroxidase(GSH-Px)and malondialdehyde(MDA)in the penis an d testis of 12 rats.The method was used to detect the expression a bundance of UPR,autophagy,apoptosis,angiogenesis,oxidative stre ss and cell cycle related genes in penis and testis.(5)The expressio n of UPR key protein in penis and testis was detected by Western blot to investigate the role of UPR and autophagy in penile and tes ticular tissue damage.Results:(1)The results of BioGPS and Oncomine database sho wed that the expression of UPR gene Perk,autophagy gene Ulk1 a nd apoptosis gene Bax were lower in normal tissues,and higher in normal testis tumors than in normal tissues(P<0.05).The String database shows that the most obvious interaction with PERK is HSPA5/GRP78.The most obvious interaction with ULK1 are ATG17,MT OR,PIK3R4,PIK3C3,ATG10,RPTOR,ATG13.The most obvious interaction with BAX are BCL2L1,MCL1,TP53 and MAPK8;(2)The incidence of hypospadias in the experimental group was 100%,and the weight of the mice in the experimental group was lower t han that in the control group(P<0.05).The AGD of the experiment al group was shorter than that of the control group(P<0.05);the a verage length of the penis was about 40% shorter than the control group(P<0.05).Histopathological sections showed that the urethra of the experimental group did not form a closed structure,and the la yers of the layers were disordered.The testicular volume was small er than that of the control group,showing a phenomenon of "small testicles".The testicular sections showed that the lumen of the exp erimental group was mostly irregularly enlarged,the sperm cells we re less than the control group,and the spermatogenic cells in the l uminal side were dissociated and autolyzed;(3)The changes of oxid ative stress related indexes in the mouse model of hypospadias: the content of MDA and SOD in the testis was higher than that in th e control group,and the content of GSH-Px was lower than that in the control group(P<0.05).The abundance of Noxo1 was higher t han that of the control group.Sod1 and Gpx1 were lower than the control group(P<0.05);(4)Up-regulated expression in the penis: U PR-related genes Atf6,Elf2?,Grp78,Ire1,Perk,Xbp1;autophagy-re lated genes: Atg7,Lc3 b,Ulk1;apoptosis-related genes: Ask1,Aktl,B cl,Bcl-xl,Caspase3,Caspase9,Caspase12,Chop,Jnk3;The expressi on of downregulation in the penis is: cell cycle related genes: Ccna,Vegf(P<0.05).(5)Up-regulated expression in the testis: UPR-related gene expression: Atf6,Grp78,Ire1,Perk,Xbp1;autophagy-related genes: Atg7;apoptosis-related genes: Ask1,Aktl,Bax,Bcl-xl,Chop,Jnk1,Jnk3.The expression of testis was down-regulated: Ccne,Cd44,Vegf(P<0.05);(6)The expression of UPR-related proteins: the ex pression of Grp78,Ire1 and Perk in the unfolded protein in the pe nis increased.The expression of Grp78 and Perk in the testis was higher than that in the control group(P<0.05).(7)The gene expressi on results were predicted by the String database to predict the prot ein interaction.The most significant differences in the protein intera ctions in the penis were Hspa5,Ern1,Ulk1,Map1lc3 b,Atf6,Eif2 a k3,Map3k5 and Atg7;Casp3,Aktl,Casp9,Bax,Mapk10,Bcl2l1,Map3k5 and Pmaip1.The most significant correlations between the differential proteins in the testis are Map3k5,Hspa5,Atg7,Aktl1,Mapk10,Mapk8,Bcl2l1,Bax,Hspa5,Ern1 and Eif2ak3.Conclusions:(1)The expression of Perk,Ulk1 and Bax in norma l tissues and tumor tissues is different,and their increased expressio n can inhibit the progression of testicular tumors and promote the a poptosis of tumor cells.(2)Female rats exposed to flutamide during pregnancy can induce hypospadias and "small testicles" in male rats.(3)Flutamide can cause oxidative stress and UPR in mouse penis and testis,and then lead to autophagy,apoptosis and cycle arrest,which lead to hypospadias and "small testicles" in male rats..