Zinc Ion Protects Against Premature Ovarian Failure Through PI3K/AKT/GSK3β Signaling Pathway

ObjectiveThis study aims to explore the protective effect of zinc ion（Zn2+）on cisplatin-induced Premature ovarian insufficiency（POI）and its mechanism and to provide a current idea for treating POF.MethodsPart I.In vivo experiments of Zn2+ treatment for cisplatin-induced ovarian damage in rats1.SD rats with regular motility cycles were randomly divided into three equal groups: normal control group（Ctrl group）,model group（Cis group）,and treatment group（Cis+ZnSO₄ group）.2.The effect of Zn2+ on ovarian endocrine function was assessed by vaginal smear to compare the motility cycle;ELISA kits were used to detect the levels of E2 and FSH.3.Comparison of serum Zn2+ levels by zinc colorimetric test kit4.The effect of Zn2+ on ovarian tissue structure in rats was assessed by ovarian index and HE staining.5.The rate of apoptosis of granulosa cells（GCs）in the ovary was observed by TUNEL staining.6.The expression of apoptosis-related proteins Bax,Bcl2,cleaved Caspase3,and key proteins of PI3K/AKT/GSK3β signaling pathway in rat ovarian tissues was detected by Western Blot.Part II In vitro experiments of Zn2+ treatment of cisplatin-induced GCs injury in rat ovaries1.Primary GCs were extracted from SD rat ovaries and cultured.The GCs were divided into three groups: blank control group（Ctrl group）,model group（Cis group）,and treatment group（Cis+ZnSO₄ group）.2.GCs viability was detected by the CCK8 method.3.Morphological changes in GCs were observed by crystalline violet staining.4.Detection of Zn2+ level in GCs by zinc ion fluorescence probe.5.Detection of GCs apoptosis rate by Annexin V-FITC/7ADD Apoptosis Detection Kit6.Intracellular levels of reactive oxygen species（ROS）were measured by DCFH-DA fluorescent probe combined with flow cytometry.7.Intracellular mitochondrial membrane potential（MMP）levels were measured by Rhodamine 123 and Mito-Tracker Red CMXRos fluorescent probes.8.The expression of apoptosis-related proteins Bax,Bcl2,cleaved Caspase3 and key proteins of PI3K/AKT/GSK3β signalling pathway in GCs was measured by Western Blot.ResultsPart I.In vivo experiments of cisplatin-induced ovarian damage in rats treated with Zn2+1.Compared with the Ctrl group,rats in the Cis group had a disturbed motile cycle,increased FSH（P<0.01）,and decreased E2（P<0.01）in serum;compared with the Cis group,rats in the Cis+ZnSO₄ group had a regular motile cycle,decreased FSH（P<0.01）and increased E2（P<0.01）.2.Compared with the Ctrl group,the ovarian index in the Cis group was significantly lower（P<0.01）,with fewer follicles at all levels,more atretic follicles,thinner vascular walls,and different degrees of interstitial fibrosis;compared with the Cis group,the ovarian index in the Cis+ZnSO₄ group was significantly higher（P<0.01）,with dense ovarian tissue,thickened vascular walls and normal follicle development at all levels.3.The serum level of Zn2+ was significantly lower in the Cis group compared with the Ctrl group（P<0.01）;the level of Zn2+ was increased in the Cis+ZnSO₄ group compared with the Cis group（P<0.01）.4.The results of TUNEL and the Western Blot method showed that the serum level of Zn2+ was significantly lower in the Cis group compared to the Ctrl group;the level of Zn2+ was increased in the Cis+ZnSO₄ group compared to the Cis group.Compared with the Ctrl group,the Cis group showed increased protein expression of Bax,cleaved Caspase3（P<0.05）and decreased protein expression of Bcl2（P<0.01）,and increased apoptosis of ovarian cells（P<0.01）;compared with the Cis group,the Cis+ZnSO₄ group showed decreased protein expression of Bax,cleaved Caspase3（P<0.05）,while Bcl2 protein expression increased（P<0.05）.The rate of ovarian cell apoptosis decreased（P<0.01）.5.Compared with the Ctrl group,the expression of p-PI3K/PI3 K,p-AKT/AKT,and p-GSK3β/GSK3β was significantly lower in the Cis group（P<0.05）;compared with the Cis group,the expression of p-PI3K/PI3 K,p-AKT/AKT,and p-GSK3β/GSK3β was significantly higher in the Cis+ZnSO₄ group（P<0.05）.Part II In vitro experiments of Zn2+ treatment of cisplatin-induced GCs injury in rat ovaries1.Compared with the Ctrl group,the cell viability of the Cis group was significantly decreased（P<0.01）;compared with the Cis group,the cell viability of the Cis+ZnSO₄ group was significantly increased（P<0.01）.2.Compared with the Ctrl group,the number of GCs decreased,and apoptotic features appeared in the Cis group;compared with the Cis group,the number and cell morphology were significantly improved in the Cis+ZnSO₄ group.3.The Zn2+ level was significantly reduced in the Cis group compared to the Ctrl group（P<0.01）;the Zn2+ level was increased dramatically in the Cis+ZnSO₄ group compared to the Cis group（P<0.01）.4.The results of flow cytometry and the Western Blot method showed that the protein expression of Bax,cleaved Caspase3 was increased（P<0.01）.In contrast,the protein expression of Bcl2 was decreased（P<0.05）,and the apoptosis rate of GCs was increased in the Cis group compared with the Ctrl group;compared with the Cis group,the Bax,cleaved Caspase3 protein expression was decreased（P<0.01）,while Bcl2 protein expression was increased（P<0.05）.The apoptosis rate of GCs was reduced.5.Compared with the Ctrl group,ROS levels were increased in the Cis group（P<0.01）;compared with the Cis group,the Cis+ZnSO₄ group ROS levels were reduced in the Cis+ZnSO₄ group compared with the Cis group（P<0.01）.6.Compared with the Ctrl group,MMP was reduced in the Cis group（P<0.01）;compared with the Cis group,MMP was increased in the Cis+ZnSO₄group（P<0.01）.The expression of p-PI3K/PI3 K,p-AKT/AKT,and p-GSK3β/GSK3β was significantly lower in the Cis group compared to the Ctrl group（P<0.01）;the expression of p-PI3K/PI3 K,p-AKT/AKT and p-GSK3β/GSK3β was significantly higher in the Cis+ZnSO₄ group compared to the Cis group（P<0.01）.Conclusions1.Zn2+ can increase E2 secretion and decrease FSH secretion;it can protect tissue interstitium and blood vessels from damage,reduce excessive follicular atresia,maintain follicular development,and play a protective role on the structure and function of cisplatin-injured prematurely failing ovaries.2.Zn2+ can reduce apoptosis by decreasing ROS production and MMP reduction in GCs with cisplatin-induced injury.3.Zn2+ can play a protective role against cisplatin-induced premature ovarian failure by activating the PI3K/AKT/GSK3β signaling pathway,inhibiting oxidative stress,and reducing apoptosis of GCs.