Gsk-3β-dependent Nrf2 Antioxidant Response Modulates Ferroptosis Of Lens Epithelial Cells

Objective:At present,the most effective treatment for age-related cataract(ARC)is surgical intervention,but there are still many problems(such as postoperative complications),and its pathogenesis has not been fully identified as the main limiting bottleneck for exploring prevention and treatment drugs.Studies have shown that the pathogenesis of ARC is mainly based on oxidative damage of lens epithelial cells(LECs)and oxidative damage-induced cellular ferroptosis is one of the leading causes of agerelated diseases.In addition,nuclear factor erythroid 2-related factor 2(Nrf2)is an important regulator of the antioxidant stress pathway,and Nrf2 regulates ferroptosis by binding to the antioxidant response element(ARE)of its downstream gene to mediate transcription abnormalities of multiple key genes in the ferroptosis pathway.Meanwhile,glycogen synthase kinase-3β(GSK-3β)can regulate the protein degradation level of Nrf2.Therefore,this research focuses on whether GSK-3β affects Nrf2 activity and LECs antioxidant capacity by regulating the Nrf2 protein degradation pathway,then causes abnormal expression of ferroptosis related genes,reduction of intracellular glutathione(GSH),accumulation of Fe2+ and lipid peroxides in cells and induces ferroptosis in LECs,ultimately promotes the occurrence and development of ARC.Methods:(1)Ferroptosis occurred in lens epithelial cells of age-related cataract:Firstly,slit lamp microscopy was used to observe the lens of ARC patients and 24-month-old C57BL/6J mice(aged mice);After the lens capsules of ARC patients and 24-month-old mice were obtained,H&E staining was used to detect the arrangement,morphology and number of LECs,transmission electron microscopy,western blot and immunofluorescence staining were used to detect changes in the level of LECs ferroptosis.Then,the mouse cataract model induced by oxidative stress was constructed by tail vein injection of sodium iodate(NaIO3,50mg/kg);We used a stereo microscope to observe whether the mouse model has cataract and obtained the mouse lens capsules;H&E staining was used to detect the arrangement,morphology and number of LECs;Lillie staining,western blot and immunofluorescence staining were used to detect alterations in the level of LECs ferroptosis.In addition,2.5mM NaIO3 treatment of human lens epithelial cell line HLEB3 cells was used for 24h to construct a cell model;HLE-B3 cells were pretreated with ferroptosis inhibitors,apoptosis inhibitors and necrotizing apoptosis inhibitors for 2 hours,and cell activity was detected by CCK-8 experiment,then the main death form of the cell model was determined;The levels of oxidative damage and ferroptosis in cell models were detected by ROS kit,MDA kit,FerroOrange staining,GSH/GSSG kit and western blot,to construct an oxidative stress-induced LECs model in vitro.(2)Regulatory effect of Nrf2 on ferroptosis in lens epithelial cells:Western blot was used to detect the protein expression levels of Nrf2 and its downstream gene HO-1 of antioxidant damage pathway in ARC patients,aged mice,oxidative stress-induced lens of mouse cataract models and oxidative stress-induced LECs model.After the lens capsules of systemic Nrf2 knockout mice and littermate wild-type mice injected with NaIO3 was obtained,Lillie staining and immunofluorescence staining were used to detect levels of ferroptosis in LECs.Then the expression level of Nrf2 in HLE-B3 cells was downregulated by cell transfection technology and up-regulated by pretreatment of Nrf2 agonist(tBHQ);In the oxidative stress-induced LECs model,the levels of oxidative damage and ferroptosis of LECs were detected by ROS kit,FerroOrange staining and western blot.(3)Regulatory effect of GSK-3β-dependent Nrf2 protein degradation pathway on ferroptosis in lens epithelial cells:Western blot was used to detect the protein expression changes of Nrf2,Keapl and GSK-3β at the protein level in ARC patients,aged mice,oxidative stress-induced mouse cataract model lens capsule and oxidative stress-induced LECs model;RT-PCR was used to detect the expression changes of Nrf2 and Keap1 at the RNA level in the oxidative stress-induced ferroptosis model of LECs.HLE-B3 cells were pretreated in vitro with the GSK-3β inhibitor(SB216763,10 μM)for 2 hours,then oxidative stress-induced LECs model ferroptosis levels and protein expression levels of Nrf2 and GSK-3β were detected by ROS kit,MDA kit,FerroOrange staining,western blot and immunofluorescence co-localization.Then SB216763 was injected intraperitoneally(10mg/kg,once a day,3 days before each injection of NaIO3)into mice,and the lens capsules of the mouse oxidative stress induced cataract model were obtained.The levels of LECs ferroptosis were detected by H&E staining,Lillie staining and immunofluorescence staining.Results:(1)Compared with mild ARC patients,the arrangement of LECs in patients with severe ARC was more disordered,and mitochondria showed a typical ferroptosis change;The protein levels of TfR1 and FTH1 increased,while the levels of SLC40A1 and GPX4 decreased.Compared with control group,aged mice also showed similar experimental results to the patients with severe ARC described above,and these results demonstrated that ferroptosis occurred in LECs of ARC and worsened with exacerbation of cataract.After tail vein injection of NaIO3,white cloudiness appeared in the mouse lens,Fe2+accumulation in LECs increased,proteins levels associated with ferroptosis changed,suggesting that the oxidative stress induced cataract model by was successfully constructed in vivo using NaIO3.The results of CCK-8 showed that after pretreatment of HLE-B3 cells with ferroptosis inhibitors had the best effect on cell activity salvage,indicating that ferroptosis was the main form of cell death in this model;In addition,the levels of ROS,MDA and Fe2+ in NaIO3 group were significantly increased,the levels of GSH in cells were significantly reduced,proteins levels associated with ferroptosis changed,indicating that we successfully constructed the oxidative stress-induced LECs model in vitro using NaIO3.(2)In ARC patients,aged mice,oxidative stress-induced mouse cataract model lens capsules and oxidative stress-induced LECs model,the protein expression levels of Nrf2 and HO-1 decreased significantly,indicating decreased Nrf2 protein levels lead to a decrease incellular antioxidant capacity.In the oxidative stress-induced cataract model,the level of Fe2+ in the cytoplasm of LECs of Nrf2 knockout mice was significantly higher than that in the WT group,and the protein expression of key genes in ferroptosis was more obvious,indicating that the loss of Nrf2 exacerbates the ferroptosis of LECs in vivo.In oxidative stress-induced LECs model,transfection of si-Nrf2 intensified the expression of key proteins of ferroptosis in cells caused by NaIO3,while tBHQ alleviated the changes in expression of key proteins of ferroptosis in cells and the increase of intracellular ROS and Fe2+ levels caused by NaIO3.The above experiments results indicated that Nrf2 plays an important regulatory role in regulating oxidative stress-induced LECs ferroptosis.(3)The results of lens capsules of ARC patients,aged mice,oxidative stress-induced mouse cataract model lens capsules and oxidative stress-induced LECs model showed a downward trend of Nrf2 and Keapl at the protein levels,but there was no significant change in the expression of Nrf2 at the RNA level in the oxidative stress-induced LECs ferroptosis model,suggesting that degradation of Nrf2 protein increased and was nonKeap1-mediated.Meanwhile the protein expression levels of GSK-3β increased.HLE-B3 cells were pretreated with SB216763(GSK-3β inhibitor)and the results showed that SB216763 significantly inhibited the accumulation of intracellular ROS,MDA and Fe2+caused by NaIO3,and also inhibited the expression changes of key proteins in ferroptosis.Then,SB216763 was injected into mice and significantly inhibited the death of LECs,the accumulation of Fe2+ in cells and the expression changes of ferroptosis key proteins caused by NaIO3.The above experiments results showed the regulatory effect of GSK-3βdependent Nrf2 protein degradation pathway on ferroptosis in lens epithelial cells.Conclusion:This study shows that during the aging process of the body,the LECs are stimulated to oxidative stress and activate GSK-3β,thus increase Nrf2 protein degradation.Nrf2 protein expression levels decreased,and Nrf2 binds to ARE of its downstream genes regulating their expression,the expression of SLC7A11,GPX4 and SLC40A1 downregulated,meanwhile the expression of TfR1 and FTH1 up-regulated,resulting in decreased intracellular glutathione levels and increase of Fe2+ levels,further promotes the accumulation of reactive oxygen species and lipid peroxides,exacerbates LECs ferroptosis,ultimately contributes to the occurrence of ARC.