Rubicon, a Key Molecule for Oxidative Stress-Mediated DNA Damage, in Ovarian Granulosa Cells

Abstract

Aging drives excessive ovarian oxidative stress (OS), impairing fertility and affecting granulosa cells (GCs), which are involved in folliculogenesis. This study aims to clarify the relationship between OS and autophagy in GCs and to identify compounds that enhance OS resistance. We identified Rubicon, an autophagy suppressor, as a key mediator of DNA damage in GCs under OS. Hydrogen peroxide (H₂O₂) compromised cell viability via DNA damage in the human GC cell line, HGrC1, without affecting autophagic activity. However, autophagy activation increased OS resistance in HGrC1 cells, and vice versa. Among clinically safe materials, trehalose, a disaccharide, protected cells as an autophagy activator against H₂O₂-induced cytotoxicity. Trehalose significantly increased autophagic activity, accompanied by reduced Rubicon expression, compared to other carbohydrates. It also reduced the expression of DNA damage-responsive proteins and the production of reactive oxygen species. Rubicon knockdown mitigated OS-induced DNA damage, while Rubicon overexpression enhanced DNA damage and decreased HGrC1 cell viability. Trehalose enhanced OS resistance by activating autophagy and suppressing Rubicon in a bidirectional manner. As Rubicon expression increases in aged human ovaries, trehalose may improve ovarian function in patients with infertility and other OS-related diseases.

Keywords: Rubicon; autophagy; granulosa cell; infertility; oxidative stress; trehalose.

Figure 1

H₂O₂ treatment reduced cell viability due to DNA damage but did not affect autophagy in HGrC1 cells. (A) Cell viability assessed using the WST-1 assay in HGrC1 cells treated with various concentrations of H₂O₂ for 24 h. The Y-axis represented absorbance normalized to the control (set as 1), and the X-axis indicated H₂O₂ concentration (µM). (B) Reactive oxygen species (ROS) levels of HGrC1 cells treated with H₂O₂ treatment at either 0 or 125 µM for 2 h. The Y-axis shows fluorescence intensity normalized to the average value at 0 µM of H₂O₂. The X-axis indicates H₂O₂ concentration (µM). (C) Western blotting (WB) analysis of DNA damage-responsive proteins in HGrC1 cells treated with 0 or 125 µM H₂O₂ for 24 h. WBs were shown as follows: p-ATM, ATM, p-p95, p95, γH2AX, and Actin. Actin was used as the control. (D–F) Quantification of protein expression levels based on the Western blot results shown in (C). The graphs represented the relative ratios of p-ATM/ATM (D), p-p95/p95 (E), and γH2AX/Actin (F) under the indicated conditions. All values were normalized to the control group (set as 1). (G) Autophagy flux assay in HGrC1 cells. Cells were treated with H₂O₂ (µM) for 24 h, followed by chloroquine (CQ, 100 µM) for 2 h before the harvest. (H) WB analysis of autophagy-related proteins in HGrC1 cells treated with 0, 125, or 250 µM H₂O₂ for 24 h. Western blots were shown as follows: p-mTOR, mTOR, UVRAG, Beclin1, Rubicon, ULK1, ATG3, ATG5, ATG14, p62, and Actin. Results were obtained from at least three independent experiments. Significant difference tests were also performed. Data are expressed as the mean ± S.E. * p < 0.05, ** p < 0.01, ns: not statistically significant.

Figure 2

Alternation of autophagic status affected cell viability in HGrC1 treated with H₂O₂. (A–D) Cell viability of HGrC1 cells with H₂O₂ (µM) co-treated with Tat-Beclin1 (T-B1) (µM), an autophagy activator, (A), or autophagy inhibitors, bafilomycin A1 (Baf, nM) (B), chloroquine (CQ, µM) (C), or wortmannin (Wort, nM) (D) for 24 h. The Y-axis represented absorbance normalized to the control (set as 1). (E) Autophagy flux assay in HGrC1 cells treated with T-B1 for 24 h. Cells were treated with T-B1 (µM) for 24 h, followed by chloroquine (CQ, 100 µM) for 2 h before the harvest. (F) The graphs showed the expression levels of LC3-II, which were normalized to that of Actin in the treated HGrC1 cells in (E). Expression levels were evaluated as the median of three independent experiments. Statistical significance was assessed using the Mann–Whitney U test. (G) Western blotting analysis of LC3-II in HGrC1 cells treated with wortmannin (nM) for 24 h. Actin was used as an internal control. Results were obtained from at least three independent experiments. Significant difference tests were also performed. Data are expressed as the mean ± S.E. * p < 0.05.

Figure 3

Effects of Trehalose treatment on autophagy-related proteins in HGrC1 cells. (A) Western blotting (WB) analysis evaluating LC3 expression levels in response to various autophagy activators in HGrC1 cells. The cells were treated for 24 h with Torin1 (1 nM), T-B1 (40 µM), Rapamycin (Rapa, 1 µM), Resveratrol (Resv, 100 µM), Curcumin (CCM, 10 µM), and Trehalose (Tre, 100 mM). (B) Cell viability assessed using the WST-1 assay in HGrC1 cells with various concentrations of Tre for 24 h. The Y-axis represented the mean fluorescence intensity, and the X-axis showed the Tre concentration (mM). (C) WB analysis of LC3 expression in HGrC1 cells treated with 100 mM maltose (Mal), 100 mM Tre, or 200/100 mM glucose (Glu) (mM) for 24 h. Actin was used as the control. The control cells were cultured in a medium only. (D) WB analysis of autophagy flux in HGrC1 cells treated with Trehalose (Tre) for 24 h. Cells were exposed to 100 mM Tre for 24 h, followed by chloroquine (CQ, 100 µM) treatment for 2 h before harvest. (E) Quantification of LC3-II expression based on the WB data shown in (D), demonstrating the effect of Tre and CQ on autophagy flux. (F) WB analysis of autophagy-related proteins in HGrC1 cells treated with 100 mM Tre for 24 h. WB were shown as follows: Rubicon, Beclin1, LC3, p62, UVRAG, Ulk1, TFEB, ATG3, ATG5, ATG14, and Actin. (G) WB analysis of Rubicon expression in HGrC1 cells treated with 100 mM Maltose (Mal), 100 mM Trehalose (Tre), or 200/100 mM Glucose (Glu) for 24 h. Actin was used as the loading control. Control cells were cultured in medium only. (H) Quantification of Rubicon expression based on the Western blot data shown in (G), comparing its levels across different treatment conditions. Results were obtained from at least three independent experiments. Significant difference tests were also performed. Data are expressed as the mean ± S.E. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Figure 4

Trehalose-enhanced oxidative stress resistance in HGrC1 cells. (A) Cell viability assessed using the WST-1 assay in HGrC1 cells with various concentrations of H₂O₂ in the presence of 100 mM trehalose (Tre) for 24 h. The Y-axis represented the mean fluorescence intensity, and the X-axis showed the H₂O₂ concentration (µM). (B) ROS levels of HGrC1 cells treated with H₂O₂ treatment at either 0 or 125 µM for 2 h. The Y-axis shows fluorescence intensity normalized to the average value at 0 µM H₂O₂. The X-axis indicates H₂O₂ concentration (µM). (C) WB analysis of DNA damage-responsive proteins in HGrC1 cells treated with 125 µM H₂O₂ in the presence or absence of 100 mM Tre for 24 h. Western blots were shown as follows: p-ATM, ATM, p-p95, p95, γH2AX, and Actin. Actin was used as the control. (D–F) Quantification of protein expression levels based on the Western blot results shown in (C). The graphs represented the relative ratios of p-ATM/ATM (D), p-p95/p95 (E), and γH2AX/Actin (F) under the indicated conditions. All values were normalized to the control group (set as 1). (G) Immunofluorescence staining of γH2AX in HGrC1 cells treated with 250 µM H₂O₂ in the presence or absence of 100 mM Tre for 24 h. Scale bar, 200 µm. (H) The graphs showed the fluorescence intensity of γH2AX based on the images in (G). The X-axis showed H₂O₂ concentration (µM), and the Y-axis represented the fluorescence intensity. Results were obtained from at least three independent experiments. Significant difference tests were also performed. Data are expressed as the mean ± S.E. * p < 0.05, ** p < 0.01.

Figure 5

Effects of Rubicon knockdown on autophagy activity and oxidative stress resistance. (A) WB analysis of Rubicon in HGrC1 cells to evaluate the levels of Rubicon knockdown using siRNA. Cells were treated with each siRNA at a final concentration of 10 nM for 48 h. siNC represented the negative control, while siRub1, siRub2, and siRub3 corresponded to siRNAs targeting Rubicon. (B) Quantification of protein expression levels based on the Western blot results shown in (A). The graphs represented the relative expression of Rubicon under the indicated conditions. All values were normalized to the control group (set as 1). (C) Autophagy flux assay in HGrC1 cells, which were introduced with siNC, siRub2, or siRub3. After 48 h of siRNA transduction, cells were treated with 100 µM chloroquine (CQ) for 2 h before the harvest. Actin was used as an internal control. (D) Cell viability assessed using the WST-1 assay in HGrC1 cells, which were introduced with siNC, siRub2, or siRub3, with various concentrations of H₂O₂. The Y-axis represented the mean fluorescence intensity, and the X-axis showed the H₂O₂ concentration (µM). (E) WB analysis of DNA damage-responsive proteins in HGrC1 cells, which were introduced with siNC, siRub2, or siRub3, treated with 250 µM H₂O₂ for 24 h. Western blots were shown as follows: Rubicon, p-ATM, ATM, p-p95, p95, γH2AX, and Actin. Actin was used as the control. (F–H) Quantification of protein expression levels based on the Western blot results shown in (E). The graphs represented the relative ratios of p-ATM/ATM (F), p-p95/p95 (G), and γH2AX/Actin (H) under the indicated conditions. All values were normalized to the control group (set as 1). Results were obtained from at least three independent experiments. Significant difference tests were also performed. Data are expressed as the mean ± S.E. * p < 0.05, ** p < 0.01.

Figure 6

Enhancement of the oxidative stress by Rubicon overexpression. (A) HGrC1 cells were transfected with the Rubicon plasmid, and Rubicon expression levels were assessed using pEGFP-Rub. The numbers indicate the amount of plasmid (ng) used for transfection, and Actin served as the loading control. (B) For overexpression experiments (OE), 2500 ng of pEGFP-Rub was used, and the Rubicon/Actin ratio was compared with that of the control group. (C) Cell viability was evaluated across various H₂O₂ concentrations in both the Rubicon-OE and control groups. The Y-axis represented the mean fluorescence intensity, while the X-axis indicates the H₂O₂ concentration (µM). (D) Western blot analysis was performed to assess DNA damage-responsive proteins in HGrC1 cells transfected with pEGFP-Rub and treated with 250 µM H₂O₂ for 24 h. Blots for Rubicon, p-ATM, ATM, p-p95, p95, γH2AX, and Actin (loading control) are shown. (E–G) Quantitative analysis of the Western blot data in (D) was presented as the relative ratios of p-ATM/ATM (E), p-p95/p95 (F), and γH2AX/Actin (G). Values were normalized to the control group (set as 1). Significant difference tests were also performed. Data are expressed as the mean ± S.E. * p < 0.05.

Figure 7

Comparison of Rubicon expression in human ovaries pre- and post-menopause. (A) WB analysis of Rubicon in human ovarian tissue. The left four lanes represent premenopausal samples, while the right four lanes correspond to postmenopausal samples. (B) The graphs showed the expression levels of Rubicon, which were normalized to that of Actin. PreMP means premenopausal group, and PostMP means postmenopausal group. Significant difference tests were also performed. Data are expressed as the mean ± S.E. * p < 0.05.