Protective Effect of Ellagic Acid Against Inflammatory Injury Induced byHigh Concentration of LPS in Ovarian Granulosa Cells in vitro
LU Qing-Mei, PAN Zhi-Ren, WEN Xiao-Yan, LIU Bin, ZHANG Xi-Lu, CHEN Yi, XIAO Yang, ZHAO Jia-Fu*
Key Laboratory of Genetic Breeding and Reproduction of Highland Mountain Animals of Ministry of Education/Key Laboratory of Animal Genetic Breeding and Reproduction of Guizhou Province/School of Animal Science, Guizhou University, Guizhou 550025, China
Abstract：Proliferation, apoptosis and steroid hormone synthesis of ovarian granulosa cells (OGCs) play important roles in follicle development, lipopolysaccharide (LPS) -induced inflammatory responses and oxidative stress have been recognized as important factors affecting ovarian function. As a natural polyphenolic compound, ellagic acid (EA) exhibits multiple biological activities, including antioxidant, anti- cancer, anti-inflammatory, and anti-viral properties. In this study, OGCs were isolated and cultured bycollecting ovarian tissues from healthy and sexually mature Guizhou black goats (Capra hircus). The effects of EA and LPS on the survival rate of OGCs were detected by CCK-8 assay, and the relative expression of genes related to the synthesis of inflammation factors and steroid hormones were detected by qRT-PCR. The content of estradiol (E2), progesterone (P4) and related antioxidant indexes in the cell supernatant were detected by ELISA to evaluate the functional changes of OGCs. The results showed that the concentration of EA at 1 μmol/L had the best effect on the survival rate of OGCs, after 24 h of LPS treatment, the half maximalinhibitory concentration (IC50) for OGCs was 200 μg/mL. qRT-PCR results showed that, compared with the control group, the inflammatory factors in the LPS group, interleukin-1β (IL-1β), IL-6 and tumor necrosis factor α (TNF- α) were extremely significantly higher in relative expression (P<0.01); cytochrome P450 family 11 subfamily A1 member (CYP11A1), CYP19A1, 3β -hydroxysteroid dehydrogenase (3β -HSD) and steroidogenic acute regulatory protein (STAR), steroid hormone synthesis genes were extremely significantly reduced in expression (P<0.01). Compared with the LPS group, the relative expression of IL-1β and TNF- α was extremely significantly lower in the LPS+EA group (P<0.05), IL-6 had a tendency to decrease; CYP11A1 and CYP19A1 expression was extremely significantly up-regulated (P<0.01), and the results of ELISA showed that the secretion of E2 in the LPS group was significantly reduced (P<0.05), and the secretion of P4 was extremely significantly reduced (P<0.01), malondialdehyde (MDA) concentration was significantly increased (P<0.05), while the concentrations of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and reactive oxygen species (ROS)extremely significantly increased (P<0.01); while E2 and P4 were significantly up-regulated (P<0.05) and GSH-Px, SOD and ROS concentrations were extremely significantly decreased (P<0.01) in the LPS+EA group compared with the LPS group, and the concentration of MDAtended to decrease. Exogenous addition of EA could serve to alleviate LPS-induced inflammatory responses and oxidative damage in OGCs and could moderate the functional disorders of OGCs caused by LPS to some extent. This study revealed the potential mechanism of ellagic acid in protecting OGCs from inflammatory responses and oxidative damage, which provides a theoretical basis for the development of safe and effective natural anti-inflammatory antioxidants. Meanwhile, it also provides new ideas for research on improving the reproductive performance of livestock and coping with reproductive system-related diseases.
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