Quercetin Alleviates Endoplasmic Reticulum Stress-Induced Apoptosis in Buffalo Ovarian Granulosa Cells

Abstract

Endoplasmic reticulum (ER) stress plays a crucial role in granulosa cell (GCs) apoptosis, which is the main cause of follicular atresia. Quercetin (QC), a plant-derived flavonoid, has antioxidant, anti-inflammatory, and other biological properties. However, whether QC can alleviate the effects of ER stress on buffalo GCs remains unknown. In this study, we constructed an ER stress model in buffalo GCs by using tunicamycin (TM) and pre-treated with QC to explore the effect of QC on cells under ER stress. Apoptosis was detected by Annexin fluorescein 5 isothiocyanate (V-FITC), and the expressions of mRNA and related proteins involved in ER stress and apoptosis were detected via real-time polymerase chain reaction and Western blot. The results revealed that ER stress can cause apoptosis in GCs, whereas QC pre-treatment can prevent apoptosis caused by ER stress. After pre-treatment with QC, the expression levels of ER stress-related genes and proteins significantly decreased, pro-apoptotic genes were significantly down-regulated, and anti-apoptotic genes were significantly up-regulated. Furthermore, the results of Chop gene overexpression suggested that QC alleviated ER stress via the PERK/CHOP signaling pathway. In this study, we preliminarily elucidated that QC alleviates ER stress-induced apoptosis in buffalo GCs, and the results suggest a novel strategy for delaying follicular atresia by inhibiting GCs apoptosis.

Keywords: apoptosis; buffalo granulosa cells; endoplasmic reticulum stress; quercetin.

Figure 1

Buffalo GCs culture and identification: (A) (a): The morphology of buffalo GCs was observed under the microscope. (B) Identification of buffalo GCs by cellular immunofluorescence. (b): Cell nucleus were stained by DAPI (blue), (c): Cell nucleus and cell membranes were stained by FSHR (green), (d): Merge of (b,c).

Figure 2

Construction of an ER stress model: (A) Cells were treated with TM at the indicated dose of 0–5 µg/mL for 24 h and 48 h. Ten thousand cells were treated with Annexin V-FITC and PI reagent and analyzed on a fluorescence-activated cell sorting (FACS) instrument. Percentages of Annexin V-positive/PI-negative and Annexin V-positive/PI-positive cells are shown. (B) q-PCR analysis of Perk, Ire1, Atf6, Chop, and Atf4 mRNA expression in GCs after TM treatment. (C) Western blotting was performed to detect the protein level with the indicated antibodies against ATF4, CHOP, BCL-2, Caspase9, and BAX. The asterisks refer to the level of significance (** p < 0.01, *** p < 0.001). Values are presented as means ± SD for 3 biological replicates.

Figure 3

The effect of QC on TM-induced apoptosis in GCs. control: control group; TM + QC: pre-treated with QC, and then treated with TM; TM: TM alone treatment group; QC: QC alone treatment group: (A) The effects of different concentrations of QC treatment on cell viability for 24, 48, and 72 h. (B) The GCs were pretreated with QC for 8 h, and then treated with 2.5 µg/mL TM for 40 h, and the apoptosis rate was detected by flow cytometry. (C) Statistics on apoptosis. (D) The intensity of intracellular ROS was examined using fluorescence microscopy. The higher the fluorescence intensity, the stronger the intracellular ROS activity. (E) Fluorescence Intensity Statistics. The asterisks refer to the level of significance (* p < 0.05, ** p  <  0.01).Values are presented as means ± SD for 3 biological replicates.

Figure 4

Gene expression changes in GCs after QC treatment: (A) The mRNA expression level of Bax, Bcl2, Caspase3, and Caspase9 in different treatment groups was examined by q-PCR. (B) The expression levels of proteins in different treatment groups were detected by Western blotting. (C) The mRNA expression of Atf4, Chop, Perk, and Eif2a in different treatment groups was examined by q-PCR. The asterisks refer to the level of significance (* p < 0.05, ** p < 0.01). Values are presented as means ± SD for 3 biological replicates.

Figure 5

Effects of activation of Chop gene. control: blank control group; pcDNA3.1: transfected with pcDNA3.1 plasmid group; pcDNA3.1-CHOP: transfected with Chop overexpression plasmid group; pcDNA3.1-CHOP + QC: pretreated with QC, then transfected with Chop overexpression plasmid group: (A) The expression level of the Chop gene after transfection of pcDNA3.1-CHOP plasmid. (B) After transfection of pcDNA-CHOP plasmid, cell apoptosis was evaluated by flow cytometry. (C) Statistics of apoptotic cells. (D) After transfection of pcDNA-CHOP plasmid, the intensity of ROS in cells was detected by fluorescence microscope. The higher the fluorescence intensity, the higher the intracellular ROS content. (E) Fluorescence density statistics. The asterisks refer to the level of significance (** p < 0.01). Values are presented as means ± SD for 3 biological replicates.

Figure 5

Effects of activation of Chop gene. control: blank control group; pcDNA3.1: transfected with pcDNA3.1 plasmid group; pcDNA3.1-CHOP: transfected with Chop overexpression plasmid group; pcDNA3.1-CHOP + QC: pretreated with QC, then transfected with Chop overexpression plasmid group: (A) The expression level of the Chop gene after transfection of pcDNA3.1-CHOP plasmid. (B) After transfection of pcDNA-CHOP plasmid, cell apoptosis was evaluated by flow cytometry. (C) Statistics of apoptotic cells. (D) After transfection of pcDNA-CHOP plasmid, the intensity of ROS in cells was detected by fluorescence microscope. The higher the fluorescence intensity, the higher the intracellular ROS content. (E) Fluorescence density statistics. The asterisks refer to the level of significance (** p < 0.01). Values are presented as means ± SD for 3 biological replicates.