Abstract:

Objective To discuss the improvement effect of short-chain fatty acids (SCFAs) on the injury of ovarian granulosa cells in polycystic ovary syndrome (PCOS), and to clarify its possible mechanism. Methods The ovarian granulosa cells were extracted from the follicular fluid of PCOS patients and non-PCOS patients， respectively. Real-time fluorescence quantitative PCR （RT-qPCR） and Western blotting methods were used to detect the expression levels of tumor necrosis factor-α （TNF-α）， interferon γ （IFN-γ）， interleukin （IL）-6， and IL-18 mRNA and proteins in the two kinds of cells； Western blotting method was used to detect the microtube-associated protein 1 light chain 3 （LC3）-Ⅱ/LC3-Ⅰ ratios and ubiquitin-binding protein p62 expression levels in two types of cells. The human ovarian granulosa cells KGN were treated with three kinds of SCFAs sodium acetate （NaA）， sodium propionate （NaP）， and sodium butyrate （NaB） at different concentrations （0， 6， 12， 24， and 48 mmol·L^-1）， respectively. Cell counting kit-8 （CCK-8） method was used to detect the proliferation activities of normal KGN cells after treated with three kinds of SCFAs. The human ovarian granulosa cells KGN were taken and divided into control group， lipopolysaccharide （LPS） group （1 mg·L^-1 LPS）， NaA+LPS group （48 mmol·L^-1 NaA+1 mg·L^-1 LPS）， NaP+LPS group （48 mmol·L^-1 NaP+1 mg·L^-1 LPS）， and NaB+LPS group （48 mmol·L^-1 NaB+1 mg·L^-1 LPS）. CCK-8 method was used to detect the proliferation activities of the cells in various groups； enzyme-linked immunosorbent assay （ELISA） was used to detect the levels of estradiol （E2） and progesterone （P） in supernatant of the cells in various groups； RT-qPCR method was used to detect the expression levels of TNF-α， IFN-γ， IL-6， and IL-18 mRNA in the cells in various groups； Western blotting method was used to detect the the expression levels of TNF-α， IFN-γ， IL-6， and IL-18 proteins and LC3-Ⅱ/LC3-Ⅰ ratios and p62 protein expression levels in the cells in various groups. Results Compared with non-PCOS patients， the mRNA and protein expression levels of TNF-α， IFN-γ， IL-6， and IL-18 in the ovarian granulosa cells of PCOS patients were significantly increased （P<0.05）， the LC3-Ⅱ/LC3-Ⅰ ratio was increased （P<0.05）， and the p62 protein expression level was decreased （P<0.05）. Compared with control group， there were no significant differences in the proliferation activities of the KGN cells in various groups after treated with different concentrations （6， 12， 24， and 48 mmol·L^-1） of NaA， NaP， and NaB （P>0.05）. Compared with LPS group， the proliferation activities of the cells in NaA+LPS group， NaP+LPS group， and NaB+LPS group were increased （P<0.05）， the levels of E2 and P in the cell supernatant were increased （P<0.05）， the expression levels of TNF-α， IFN-γ， IL-6， and IL-18 mRNA in the cells were decreased （P<0.05）， the LC3-Ⅱ/LC3-Ⅰ ratio was decreased （P<0.05）， and the p62 protein expression level was increased （P<0.05）. Conclusion The levels of inflammatory factors are increased in the ovarian granulosa cells of PCOS patients and induce cell autophagy. SCFAs can improve the inflammatory-induced autophagic injury of ovarian granulosa cells and increase the cell proliferation activity.

Key words: Polycystic ovary syndrome, Ovarian granulosa cells, Short-chain fatty acids, Inflammation, Cell autophage, Inflammatory factor