Role Of Autophagy And Apoptosis Mediated By Oxidative Stress In The Development Of Hypospadias

Background: Hypospadias is one of the most common congenital malformations in children and it ranks second in the children’s genitourinary system malformations.Epidemiological surveys at home and abroad show that the incidence rate is 1/200 to 1/300,which is increasing year by year.trend.The main clinical manifestations of hypospadias are penile curvature and abnormal position of urethral opening,which have a great influence on children’s mental health and adult fertility.The incidence of hypospadias is high,which has a significant impact on the quality of life of patients and significantly increases the medical burden.With the progress of Chinese society,the incidence of hypospadias in China,like that of developed countries,has shown an upward trend year by year.At present,hypospadias is considered to be a disease caused by multiple factors such as genes,environment and endocrinology.The specific mechanism is not yet clear.Di(2-ethylhexyl)phthalate(DEHP)is a member of phthalates(PEs)and is the most widely used general-purpose plasticizer in China.It acts as a typical environmental endocrine.Contaminants can cause abnormal development of the male reproductive system.DEHP can enter the body through various pathways and cause intracellular mitochondria to produce large amounts of reactive oxygen species(ROS).Autophagy and reasonable apoptosis are essential for mammalian embryonic development and organogenesis.Recent studies have found that oxidative stress,autophagy,and apoptosis are closely related to each other,and that they occur in hypospadias.The role played in the process is not yet clear.Objective: This experiment aims to explore the effects of DEHP exposure during pregnancy on the development of male offspring,as well as oxidative stress,autophagy,and withering during the development of genital tubercles in male offspring of SD rats.The change of death further explored the possible molecular regulatory mechanisms during the development of the urethra.Methods: 20 SPF pregnant SD rats were randomly divided into normal control group(corn oil,1000 mg/kg·d,n=10)and DEHP exposure group(DEHP,750 mg/kg·d,n = 10),gavage treatment in GD8.5 ~ 18.5.At GD19.5,10% chloral hydrate solution was intraperitoneally injected into pregnant mice(4 ml/kg)and the fetuses were removed by cesarean section.Female rats of the fetuses were judged with or without testes,weighed,counted,and dissected microscope.Observed under the occurrence of hypospadias,vernier caliper measurement of anal reproductive distance(AGD).The Genital Tubercles(GTs)in each group were collected and divided into normal control group,normal group after DEHP exposure,and hypospadias group according to the occurrence of hypospadias.H&E staining and scanning electron microscopy(SEM)were used to observe the structural changes of GTs.After ultrathin sections of GTs were made in each group,the level of autophagy was observed by transmission electron microscopy(TEM);the GTs of each group were extracted.Total proteins and Western blotting(WB)were used to detect oxidative stress-related proteins(Nrf2,HO-1,SOD1,Gpx),autophagic markers(p62,LC3,Beclin1),and autophagy signaling pathways.Protein(PI3K,Akt,m TOR,p-AktSer473,p-m TORSer2448)and apoptosis-related protein(Cleved-Caspase3,Bax,Bcl-2)expression levels;TUNEL assay detected apoptosis in the GTs in each group.Results: Compared with the normal control group,hypospadias occurred in the male offspring of the DEHP-exposed group and the incidence was 27.58%(P<0.05),accompanied by a decrease in AGD and AGI(P<0.05).In the GTs of the offspring hypospadias,the expression of Nrf2-HO1 signal pathway was abnormal,and the expression of endogenous antioxidative substances SOD1 and Gpx was decreased(P<0.05),and Vit A with antioxidation in vivo was also found.Vit E was also significantly decreased(P<0.05);at the same time,the level of autophagy was abnormally increased and the phosphorylation of Akt/m TOR was inhibited(P<0.05);the proapoptotic protein Bax and the apoptotic protein Cleaved-Caspase were abnormally increased(P<0.05).Abnormally increased apoptotic cells in the reproductive group were mainly concentrated around the urethral suture.Conclusion: DEHP enters the SD rats and is rapidly hydrolyzed to MEHP.MEHP can cause mitochondrial respiratory depression in the cells,eventually resulting in excessive ROS production,leaving the embryonic tissue cells in oxidative stress.A large number of antioxidant substances,such as SOD1,Gpx,Vit A,Vit E,etc.are consumed,the balance between active oxygen and antioxidant systems is broken,and the PI3K/Akt/m TOR pathway is inhibited,which in turn causes reproductive nodules to self Defects.Thus,we speculated that under the combined effects of oxidative stress imbalance and autophagy defects,abnormal apoptosis of the GTs of the offspring of male SD rats resulted in the failure of urethral fold fusion,resulting in the occurrence of hypospadias.