Protective Effects Of SCUBE1 On Oxidative Stress-induced Apoptosis In Human Ovarian Granulosa Cells

Premature ovarian insufficiency(POI)is one of the common gynecological diseases that endanger female reproductive endocrine health.The essence of POI is the acceleration of follicular atresia to premature exhaustion caused by various factors,and the apoptosis of ovarian granulosa cells(GCs)is the initiating factor of follicular atresia.Studies have shown that oxidative stress(OS)is closely related to POI and the apoptosis of GCs.OS refers to the imbalance of the oxidation and antioxidant systems in the body,resulting in an abnormal increase in reactive oxygen species(ROS).High ROS levels induce changes in mitochondrial DNA and lead to mitochondrial dysfunction,impaired oxidative phosphorylation,and thus damages oogenesis,promotes GCs to accelerate apoptosis,affecst the quality of embryos,and ultimately lead to a decline in female fertility.In the early stage,our research group used exon sequencing technology to screen out 12 genes in 10 sporadic POI patients,including 22 mutation sites,as candidate pathogenic mutations for POI.Sanger sequencing verification was then performed on 20 expanded POI samples.As a result,the gene mutation SCUBE1(c.1169C>G,p.P390R)was confirmed to have a mutation frequency of 0.15 in 20 POI samples.Studies have shown that SCUBE1 is involved in various pathophysiological processes such as inflammation,cell proliferation and repair,and OS plays an important role in the above processes.However,it is not clear whether SCUBE1 can protect human GCs from OS damage.The use of hydrogen peroxide(H2O2)to induce oxidative damage of ovarian GCs in vitro to induce cell apoptosis is a common and reasonable and effective method to explore the factors and mechanisms that affect follicular development and ovarian function,and to analyze SCUBE1 in human GCs.The role of apoptosis provides new ideas for the prevention and treatment of POI,and provides scientific theoretical and practical basis for further research on POI.ObjectiveExplore the expression pattern of SCUBE1 protein on human and mouse ovaries.To explore the role and mechanism of SCUBE1 in protecting human GCs from apoptosis induced by H2O2.MethodsFirstly,the expression of SCUBE1 on the ovaries of humans and mice of different ages was analyzed by qRT-PCR,western blot and immunohistochemistry.Subsequently,the H2O2 treated GCs were pretreated with SCUBE1 recombinant protein.The CCK-8 method was used to detect cell viability and proliferation.Flow cytometry combined with DCFH-DA and rhodamine 123 were used to measure ROS and mitochondrial membrane potential levels,respectively.The Annexin V-FITC/PI apoptosis detection kit was used to determine the cell apoptosis ratio.The expression levels of pathway related proteins were detected by western blot.Finally,the bioinformatics changes before and after the point mutation of SCUBE1 gene were analyzed based on the software.ResultsSCUBE1 protein is mainly distributed in the medulla area in the center of the ovary,and it is strongly expressed in the vascular cavity and surrounding areas with abundant blood vessels.In vitro cell experiments show that SCUBE1 pretreatment can reduce H2O2-induced cell apoptosis and improve cell viability.SCUBE1 can also block the production of ROS in cells and improve the mitochondrial membrane potential.After SCUBE1 pretreatment,the expression of anti-apoptotic protein Bcl-2 was up-regulated while down-regulating the expression of pro-apoptotic proteins Bax,Bax/Bcl-2,Caspase-3 and p53.In addition,it can also weaken the expression of p38 MAPK and JNK protein,and enhance the expression of ERK1/2.Finally,analyze the effect of SCUBE1(c.1169C>G,p.P390R)mutation from the aspects of mutation pathogenicity,protein stability,gene haplotype insufficiency,etc.A large number of data show that the p.P390R mutation is significantly pathogenic.ConclusionSCUBE1 protects GCs from H2O2-induced damage by reducing the generation of ROS,increasing the mitochondrial membrane potential,up-regulating the expression of anti-apoptotic proteins and down-regulating the expression of pro-apoptotic proteins.