Alpha-Lipoic Acid Ameliorates Impaired Steroidogenesis in Human Granulosa Cells Induced by Advanced Glycation End-Products

Abstract

Background: Ovarian granulosa cells (GCs) are essential for follicular development. Ovarian advanced glycation end-products (AGEs) accumulation is related to GCs dysfunction. Alpha-lipoic acid (ALA) illustrates therapeutic capabilities for infertility-related disorders. Therefore, this study assessed the effects of ALA on AGEs-induced GCs hormonal dysfunction.

Methods: The study was conducted from October 2021 to September 2022 at the Department of Medical Genetics, Shiraz University of Medical Sciences. Isolated GCs (n=50) were divided into control, human glycated albumin (HGA), HGA+ALA, and ALA treatments. Steroidogenic enzymes and AGE receptor (RAGE) genes were assessed by qRT-PCR. Steroid hormones and RAGE protein were evaluated using ELISA and Western blotting. Data were analyzed using GraphPad Prism software (ver. 9), and P<0.05 was considered significant.

Results: Our findings showed that HGA treatment significantly (P=0.0001) increased RAGE (by 140.66%), STAR (by 117.65%), 3β-HSD (by 165.68%), and 17β-HSD (by 122.15%) expression, while it decreased CYP19A1 (by 68.37%) expression. RAGE protein level (by 267.10%) was also increased in HGA-treated GCs. A significant decrease in estradiol (by 59.66%) and a slight and sharp elevation in progesterone (by 30.40%) and total testosterone (by 158.24%) levels was also observed. ALA treatment ameliorated the HGA-induced changes in steroidogenic enzyme mRNA levels (P=0.001) and steroid hormone secretion (P=0.010).

Conclusion: This work shows that ALA therapy likely corrects hormonal dysfunctions caused by AGEs in luteinized GCs. This effect is probably achieved by decreased RAGE expression. Clinical research is needed to understand how AGEs and ALA interact in the ovary, which might lead to a more targeted ovarian dysfunction therapy.

Keywords: Advanced glycation end-products; Alpha-lipoic acid; Gonadal steroid hormones; Granulosa cells; Humans.

Figure 1

A schematic view of the cell culture and study procedures is shown (BioRender.com). HGA: Human glycated albumin; ALA: Alpha-lipoic acid; GCs: Granulosa cells

Figure 2

The effects of non-toxic concentrations of ALA on the viability of human luteinized GCs are shown using MTT assay. The experiments were performed in duplicate. The results were reported as mean±SEM (n=5). ****P=0.0001 represents significant differences between experimental groups. ALA: Alpha-lipoic acid; GCs: Granulosa cells; MTT: 3–4, 5-dimethylthiazol-2-yl-2, 5-diphenyltetrazolium bromide

Figure 3

The effects of HGA (400 µg/mL) with/without ALA (100 µM) are shown on the expression of STAR (A), 3β-HSD (B), 17β-HSD (C), and CYP19A1 (D) genes in human luteinized GCs. Quantitative real-time PCR was done for steroidogenic enzyme gene expression levels after 48 hours incubation. The experiments were performed in duplicate. The results were reported as mean±SEM (n=8). *P=0.022, ***P=0.001, and ****P=0.0001 represent significant differences between experimental groups. ALA: Alpha-lipoic acid; HGA: Human glycated albumin; GCs: Granulosa cells; STAR: Steroidogenic acute regulatory protein; 3β-HSD: 3β-hydroxysteroid dehydrogenase; 17β-HSD: 17β-hydroxysteroid dehydrogenase; CYP19A1: Cytochrome P450 aromatase

Figure 4

The effects of HGA (400 µg/mL) with/without ALA (100 µM) are shown on E2 (A), P4 (B) and total T (C) release by human luteinized GCs. After 48 hours incubation, cell culture media were collected for ELISA assay of released sex steroid hormones. The experiments were performed in duplicate. The results are reported as mean±SEM (n=20). **P=0.002 and ****P=0.0001 represent significant differences between experimental groups. ALA: Alpha-lipoic acid; HGA: Human glycated albumin; GCs: Granulosa cells; E2: Estradiol; P4: Progesterone; T: Testosterone

Figure 5

The effects of HGA (400 µg/mL) with/without ALA (100 µM) are shown on RAGE gene expression (A) and RAGE protein level (B) in human luteinized GCs. Quantitative real-time PCR was done for levels of RAGE gene expression after 48 hours incubation (n=8). Western blots and densitometry readings of RAGE protein bands were done using an enhanced chemiluminescence kit and X-ray film (n=5). The band intensity of RAGE protein was analyzed by densitometry readings/intensity ratio using ImageJ software and was normalized to the corresponding GAPDH value. The experiments were performed in duplicate. The experiments were reported as mean±SEM. ***P=0.001 and ****P=0.0001 represent significant differences between experimental groups. ALA: Alpha-lipoic acid; HGA: Human glycated albumin; GCs: Granulosa cells; RAGE: Receptor for advanced glycation end-products

Figure 6

Schematic diagram represents the relationship between ALA and HGA in human luteinized GCs. HGA (as an AGEs representative) affects steroidogenesis by up-regulating STAR, 3β-HSD, and 17β-HSD (upward red arrows), and down-regulating CYP19A1 (downward red arrows). ALA inhibits the expression of the RAGE gene and its protein. Moreover, it restores the effect of HGA on STAR, 3β-HSD, 17β-HSD, and CYP19A1 (green arrows). ALA: Alpha-lipoic acid; HGA: Human glycated albumin; AGEs: Advanced glycation end-products; GCs: Granulosa cells; STAR: Steroidogenic acute regulatory protein; 3β-HSD: 3β-hydroxysteroid dehydrogenase; 17β-HSD: 17β-hydroxysteroid dehydrogenase; CYP19A1: Cytochrome P450 aromatase; RAGE: Receptor for advanced glycation end-products