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

OBJECTIVE

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 HO 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- groups transfected with 3 types of si- plasmids. A control + si-NC group was also set up, in which HLE-B3 cells not treated with HO were transfected with si-NC empty plasmids. The gene and protein expression levels in the si-NC and si- groups were determined by RT-qPCR and Western blot. Two si- 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- parallel groups, and the control + si-NC group were determined by Western blot. A si- 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- 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 significantly upregulated in ARC samples ( < 0.05) and enriched in the AMPK pathway. 2) Compared with cells not treated with HO, 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, knockdown suppressed AMPK pathway activity, with all the si- groups showing a significantly decreased p-AMPK/AMPK ratio compared to that of the si-NC group ( < 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 < 0.05). Compared to those in the si-NC group, these indicators were improved in the si- group (all < 0.05). However, compared with the findings before AICAR treatment, these effects were antagonized after AICAR treatment in the si- group (all < 0.05).

CONCLUSION

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