Regulatory Role of the AKT1/AMPK Pathway in Oxidative Stress of Lens Epithelial Cells in Cataract Patients

Abstract

To elucidate the regulatory role and the underlying molecular mechanisms of serine/threonine protein kinase 1 (AKT1) in the lens epithelium of patients with age-related cataract (ARC).

Methods 

1) Differentially expressed genes in ARC were screened using bioinformatics analysis (Genecard and GEO database GSE213546), and key genes were identified through functional enrichment analysis (KEGG and GO). 2) An oxidative stress model of lens epithelial cells was established by treating HLE-B3 cells with 200 μmol/L H2O2 for 24 h. RT-qPCR and Western blot were performed to assess AKT1 gene and protein expression changes. 3) Model cells were randomly divided into a si-NC group transfected with si-NC plasmids and 3 parallel si-AKT1 groups transfected with 3 types of si-AKT1 plasmids. A control + si-NC group was also set up, in which HLE-B3 cells not treated with H2O2 were transfected with si-NC empty plasmids. The AKT1 gene and protein expression levels in the si-NC and si-AKT1 groups were determined by RT-qPCR and Western blot. Two si-AKT1 parallel groups demonstrating the most significant changes in expression levels were selected for further experiments. The protein expression levels of AMPK and phosphorylated AMPK (p-AMPK) in the si-NC group, the two selected si-AKT1 parallel groups, and the control + si-NC group were determined by Western blot. A si-AKT1 parallel group demonstrating significant changes in p-AMPK/AMPK values was selected and treated with Acadesine (AICAR), an AMPK agonist, to verify the role of the AMPK pathway. Western blot was performed to determine Bcl-2 and Bax protein levels in the si-NC group, the control + si-NC group, and the si-AKT1 group before and after the administration of AICAR. Flow cytometry was performed to measure apoptosis and reactive oxygen species (ROS) levels, while ELISA kits were used to assess the levels of superoxide dismutase (SOD), malondialdehyde (MDA), and reduced glutathione (GSH).

Results 

1) Through bioinformatics analysis, 78 differentially expressed genes were identified, with AKT1 significantly upregulated in ARC samples (P < 0.05) and enriched in the AMPK pathway. 2) Compared with cells not treated with H2O2, AKT1 mRNA and protein expression increased in the oxidative stress model cells. 3) The p-AMPK/AMPK ratio was higher in the si-NC group than that in the control + si-NC group. In contrast, AKT1 knockdown suppressed AMPK pathway activity, with all the si-AKT1 groups showing a significantly decreased p-AMPK/AMPK ratio compared to that of the si-NC group (P < 0.05). Compared with the control + si-NC group, the si-NC group exhibited elevated ROS and MDA levels, increased apoptosis rate, reduced SOD and GSH levels, downregulated Bcl-2, and upregulated Bax (all P < 0.05). Compared to those in the si-NC group, these indicators were improved in the si-AKT1 group (all P < 0.05). However, compared with the findings before AICAR treatment, these effects were antagonized after AICAR treatment in the si-AKT1 group (all P < 0.05).

Conclusion 

The oxidative stress-related gene AKT1 may be a key pathogenic factor in cataract, and AKT1 induces oxidative stress and apoptosis in lens epithelial cells by modulating AMPK pathway activity.

Keywords: Age-related cataract, Oxidative stress, Serine/Threonine kinase 1, Protein kinase