Ubiquitin-like Modifier 1 Ligating Enzyme 1 Relieves Cisplatin-induced Premature Ovarian Failure By Reducing Endoplasmic Reticulum Stress In Granulosa Cells

Objective:The aim of this study was to investigate the effect of UFM1 specific ligase 1(UFL1)on chemotherapy-induced premature ovarian failure(POF)by regulating the endoplasmic reticulum stress(ER stress)state in granulosa cells(GC).The study aims to elucidate the mechanism of ovarian function protection by UFL1,and to provide new ideas and theoretical basis for the clinical treatment of premature ovarian failure,infertility and remodeling of ovarian function.Methods:In this paper,a POF model was constructed using cisplatin(Cis)intraperitoneally injected into 4-6 weeks KM female rats,as well as Cis treatment of primary extracted granulosa cells(Ovarian granulosa cell,GC)to observe the effect of Cis on UFL1 and ER stress at the animal level and cellular level;further,UFL1 lentivirus was constructed sh RNA plasmid/overexpression plasmid,packaging viral infection/Cis co-treatment of in vitro cultured ovaries and primary GC,using HE,IHC,CCK-8,q PCR,WB,IF,ELISA and other techniques and methods to investigate the protective role of UFL1 in Cis-injured GC leading to POF at the levels of ovarian histological morphology and function,GC characterization and related marker molecules and specific molecular mechanisms.The specific methods are as follows.1.The ovaries of 1D,7D,2W,2M,and 10 M mice were extracted to detect the expression levels of UFL1 in the ovaries at each age;4-6 weeks female mice were injected intraperitoneally with saline,2.5 mg/kg and 5.0 mg/kg of Cis,respectively,to detect changes in UFL1 and FSHR levels,serum FSH and E2 concentrations and follicle counts at all levels;4-6 W female mice ovaries were taken for in vitro culture which was treated with different concentrations of Cis to detect the changes of UFL1 level in vitro.2.Compared with the control group,the ovaries in the KD-1 and KD-2 groups showed a decrease in the total number of normal follicles,a decrease in the relative ratio of primordial follicles,an increase in the proportion of atretic follicles,and a decrease in the levels of FSHR,AMH,and E2 in ovarian tissues.Compared with the Cis group,the expression of ovarian function indexes FSHR and AMH were lower,and the ratio of apoptosis-related molecular proteins BAX/BCL-2 and Cleaved caspase-3/caspase-3was increased in the KD-1 + Cis and KD-2 + Cis groups,whereas the expression of FSHR and AMH was increased in the OE-UFL1 + Cis group,and the expression of BAX/BCL-2,and Cleaved caspase-3/caspase-3 ratios were decreased.3.Primary cultured GCs were treated with 5 μM,10 μM,15 μM,20 μM Cis,and at 3h,6 h,12 h,and 24 h time points(20 μM Cis)to detect changes in GC viability,UFL1 expression and levels of ER stress-related marker molecules GRP78,XBP1 s,CHOP,and ATF4.The ER stress inhibitor 4-PBA was used to co-treat GC with Cis to observe whether alleviating ER stress could reduce the damage of Cis on GCs.4.Compared with the control group,the GCs in the KD-1 and KD-2 groups showed decreased cell proliferation capacity,lower levels of E2,and significantly increased expression of ER stress-specific indicators GRP78,XBP1 s,and CHOP.Compared with the Cis group,the GRP78,XBP1 s,and CHOP levels were further increased in the GCs of the KD-1 + Cis and KD-2 + Cis groups,and the ratios of apoptosis-related proteins BAX/BCL-2 and Cleaved caspase3/caspase3 were significantly increased.5.GCs were co-treated with OE-UFL1 lentiviral particle suspension and Cis to detect GC proliferation and E2 secretion ability,expression levels of UFL1,GRP78,XBP1 s,and CHOP,expression levels of apoptosis-related molecules BAX,BCL-2,Cleaved caspase-3,and caspase-3 and mitochondrial membrane potential alteration.6.The ovaries were treated with UFL1-sh RNA and overexpressed UFL1 lentivirus alone or in combination with Cis,and the expression levels of UFL1 and ER stress marker molecules GRP78,XBP1 s,and CHOP were detected.Result:1.UFL1 was differentially enriched in the ovaries of mice at all ages,and UFL1 expression gradually increased during mouse development,with the highest expression in the ovaries of 2M mice and decreased in the ovaries of 10 M mice.Compared with the control group,the expression of UFL1 and GC-specific marker FSHR in ovaries of the 5.0 mg/kg Cis group was significantly reduced.Different concentrations of Cis treatment induced a stressful increased expression of UFL1 in ovaries cultured in vitro,with a significant increase in UFL1 expression at 24 h of 20 μM Cis treatment and a decrease in expression at 48 h.2.After in vitro cultured ovaries were infected with UFL1-sh RNA lentivirus,the total number of normal ovarian follicles decreased,the relative ratio of primordial follicles decreased,the relative ratio of atretic follicles increased,and the levels of FSHR,AMH,and E2 in ovarian tissues decreased in the KD-1 and KD-2 groups alone compared with the control group.Compared with the Cis group alone,the expression of ovarian function indicators FSHR and AMH were lower in the UFL1-sh RNA lentivirus infection combined with the Cis treatment group,and the expression levels of ER stress-specific markers GRP78,XBP1 s and CHOP were further increased.The ratio of apoptosis-related molecular proteins BAX/BCL-2,Cleaved caspase-3/caspase-3 was increased.3.Cis treatment decreased GC cell viability,causing a concentration effect-related and time-effect-related increase in UFL1 expression and ER stress.Compared with the control,20 μM Cis treatment for 12 h led to an increase in the expression of UFL1 and ER stress indicators GRP78,XBP1 s,and CHOP in GC.However,at 24 h of treatment,the expression of UFL1,GRP78,and XBP1 s decreased,but CHOP expression further increased.4.Cis treatment led to a decrease in GC cell viability compared to the control group.Cis treatment of GC caused a concentration effect-related and time-effect-related increase in ER stress and UFL1 expression.Compared with the control,20 μM Cis treatment for 12 h resulted in increased UFL1 expression and was accompanied by elevated expression of ER stress indicators GRP78,XBP1 s,and CHOP,and decreased UFL1 expression at 24 h of Cis treatment and was accompanied by decreased expression of GRP78 and XBP1 s,but further increased CHOP expression.5.Compared with the Cis group,the OE-UFL1 + Cis group had enhanced GC cell viability,increased secretory E2 levels,relatively lower expression of ER stressspecific indicators GRP78,XBP1 s,and CHOP,and significantly lower ratios of apoptosis-related proteins BAX/BCL-2 and Cleaved caspase3/caspase3.6.In cultured ovaries in vitro,compared with the Cis group,the expression levels of ER stress-specific markers GRP78,XBP1 s,and CHOP were further increased in the KD-1 + Cis and KD-2 + Cis groups,whereas were decreased in the OE-UFL1 + Cis group.In conclusion:Our study shows that UFL1 regulates Cis-induced endoplasmic reticulum stress and granulosa cell apoptosis and is involved in protecting against Cis-induced ovarian dysfunction,providing a potential therapeutic target for clinical prevention of chemotherapeutic drug-induced POF.