Icariin interacts with IGFBP3 to alleviate diabetic cataract through PI3K/AKT signaling pathway

Abstract

Diabetic cataract (DC), a well-recognized complication in diabetic patients, can progress to blindness if not adequately managed, with currently limited therapeutic strategies. Icariin (ICA), a natural compound derived from Epimedium, has been demonstrated exhibiting anti-inflammatory and anti-oxidant, but its impact on diabetic cataracts remains elusive. In this study, we used both in vitro SRA01/04 cells and in vivo SD rats' model to explored the protective effects of ICA in cataract formation. Following network pharmacology, proteomic and surface plasmon resonance (SPR) analyses further demonstrated that ICA interacts with insulin-like growth factor-binding protein-3 (IGFBP3) and modulates oxidative stress as well as apoptosis via the PI3K/AKT signaling pathway. These findings collectively demonstrated that ICA could alleviate high glucose-induced oxidative stress and cell apoptosis in vitro and in vivo, suggesting that ICA is a potent natural compound with protective effects in DC, offering an effective therapeutic approach for the disease management.

Keywords: Medicine; Molecular biology; Molecular medicine.

Graphical abstract

Figure 1

ICA alleviates diabetic cataracts in diabetic SD rats (A) Measurements of changes in body weight in SD rats in no-diabetic rats treated with normal saline (Ctrl), diabetic rats treated with normal saline (HG), and diabetic rats treated with normal saline + ICA (HG + I) during 12 weeks of continuous feeding. (B) Measurements of changes in blood glucose in SD rats in Ctrl, HG, and HG + I groups during 12 weeks of continuous feeding. (C) Photograph of the lens of rats in Ctrl, HG, and HG + I groups. (D) Statistic analysis of (C). (E) Hematoxylin and eosin (H&E) stains of eyes in diabetic rats in Ctrl, HG, and HG + I groups, Scale bars, 200 μm. Data are representative of three independent experiments. The data are presented as the mean ± SEM. n = 15. ∗p < 0.05 and ∗∗p < 0.01.

Figure 2

ICA alleviates high glucose-induced oxidative stress and apoptosis in SRA01/04 cells (A) The level of ROS in SRA01/04 cells stimulated by DMEM (Ctrl), high glucose DMEM (HG), and high glucose DMEM + ICA (HG + I) for 48 h, Scale bars, 100 μm. (B) Statistic analysis of (A). (C) The levels of CAT, GPx, SOD, and MDA in SRA01/04 cells by Ctrl, HG, and HG + I treatment for 48 h. (D) Flow cytometry was performed to analyze the effect in SRA01/04 cells by Ctrl, HG, and HG + I treatment for 48 h. (E) Statistic analysis of (D). (F) Immunoblot analysis of the apoptosis-related proteins in SRA01/04 cells by Ctrl, HG, and HG + I treatment for 48 h. (G) Statistic analysis of (F). Data are representative of three independent experiments. The data are presented as the mean ± SEM. n = 3. ∗p < 0.05 and ∗∗p < 0.01.

Figure 3

ICA enhances the PI3K/AKT signal pathway in SRA01/04 cells (A) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the differentially expressed proteins upregulated in the HG + I group compared to the HG group. (B) Immunoblot analysis of the indicated proteins in SRA01/04 cells stimulated by DMEM (Ctrl), high glucose DMEM (HG), and high glucose DMEM + ICA (HG + I) for 48 h. (C) Statistic analysis of (B). Data are representative of three independent experiments. The data are presented as the mean ± SEM. n = 3. ∗p < 0.05 and ∗∗p < 0.01.

Figure 4

Inhibiting the PI3K/AKT pathway can reverse the protective effect of icariin on SRA01/04 cells (A) Immunoblot analysis of the indicated proteins in SRA01/04 cells stimulated by high glucose DMEM (HG), high glucose DMEM + ICA (HG + I), and high glucose DMEM + ICA + LY294002 (HG + I + LY294002) for 48 h. (B) Statistic analysis of (A). (C) The level of ROS in SRA01/04 cells stimulated by HG, HG + ICA, and HG + I + LY294002 for 48 h, Scale bars, 100 μm. (D) Statistic analysis of (C). (E) The levels of CAT, GPx, SOD, and MDA in SRA01/04 cells stimulated by HG, HG + I, and HG + I + LY294002 for 48 h. (F) Flow cytometry was performed to analyze the effect in SRA01/04 cells stimulated by HG, HG + ICA, and HG + ICA + LY294002 for 48 h. (G) Statistic analysis of (F). (H) Immunoblot analysis of the apoptosis-relative proteins in SRA01/04 cells stimulated by HG, HG + I, and HG + I + LY294002 for 48 h. (I) Statistic analysis of (H). Data are representative of three independent experiments. The data are presented as the mean ± SEM. n = 3. ∗p < 0.05 and ∗∗p < 0.01.

Figure 5

IGFBP3 is a target for ICA (A) Volcano plot (HG + I vs. HG) of differentially expressed proteins; proteins enriched in signaling pathways (upregulated and downregulated) are labeled. (B) Heatmap shows protein expression in every group. (C) Immunoblot analysis of the IGFBP3 in SRA01/04 cells stimulated by DMEM (Ctrl), high glucose DMEM (HG), and high glucose DMEM + ICA (HG + I) for 48 h. (D) IGFBP3 expression in aqueous humor from patients with transparent crystalline lens (Ctrl, n = 13, female = 7, male = 6), age-related cataract (ARC, n = 59, female = 42, male = 17), and diabetic cataract (DC, n = 28, female = 17, male = 11). (E) The hydrogen bond between ICA and IGFBP3 are shown by yellow dashed lines, and the residues (blue) of the interactions and Icariin (yellow) are displayed as sticks. (F) At 40–70 ns, the average RMSD is 0.4906 and the standard deviation is 0.0488; analysis of hydrogen bond formation in the ICA and IGFBP3 complex. (G) The affinity and binding kinetics of ICA for IGFBP3 measured by SPR. Serial dilutions (2-fold) from 7.8 μM compounds in injected into the captured IGFBP3 protein. Kinetic data from one representative experiment were fit to a 1:1 binding model. The profiles are shown for IGFBP3. Data are representative of three independent experiments. The data are presented as the mean ± SEM. n = 3. ∗p < 0.05 and ∗∗p < 0.01.

Figure 6

ICA alleviates oxidative stress and apoptosis in diabetic SD rats (A) Immunoblot analysis of the indicated proteins in the lens of SD rats in Ctrl, HG, and HG + I groups; 3 lenses were surgically harvested and pooled to extract protein for immunoblot assay. (B) Statistic analysis of (A). (C) The levels of CAT, GPx, SOD, and MDA in the lens of SD rats in Ctrl, HG, and HG + I groups; 3 lenses were surgically harvested and pooled to extract protein for immunoblot assay. (D) Immunoblot analysis of the apoptosis-relative proteins in the lens of SD rats in Ctrl, HG, and HG + I groups; 3 lenses were surgically harvested and pooled to extract protein for immunoblot assay. (E) Statistic analysis of (D). Data are representative of three independent experiments. The data are presented as the mean ± SEM. n = 15. ∗p < 0.05 and ∗∗p < 0.01.