Transcriptomic analysis reveals that the anti-PCOS effects of Zishen Qingre Lishi Huayu recipe may involve pathways and genes related to autophagy, steroidogenesis, oxidative stress, and inflammation in granulosa cells

Abstract

Zishen Qingre Lishi Huayu recipe (ZQLHR) is a Chinese medicine compound composed of nine herbs for the treatment of polycystic ovary syndrome (PCOS). It is used to nourish kidneys, clear heat, reduce dampness and dissipation blood stasis by promoting diuresis and blood circulation, dredging the meridians and harmonizing menstruation in the treatment of PCOS. Several clinical studies have shown that ZQLHR is effective in the treatment of PCOS, but the underlying mechanism remains unclear. In this study, we researched on the effects and mechanism of action of ZQLHR during treatment of human granulosa cells (hGCs) obtained from PCOS patients in order to provide a scientific basis for the clinical application of ZQLHR in the treatment of PCOS, emphasize the importance of some genes that have been reported to play a role in the pathogenesis or therapeutic mechanisms of PCOS from the perspective of disease treatment, and identify some new genes and signaling pathways that may play an important role in the treatment of PCOS. KGN cells (a granulosa cell-like tumor cell line) were subjected to a cell counting kit-8 assay to explore the appropriate intervention concentration and duration of ZQLHR. Treated with or without ZQLHR (ZQLHR and control groups), the hGCs obtained from PCOS patients were sequenced using RNA sequencing, and the genes thus detected were further analyzed through Kyoto encyclopedia of genes and genomes enrichment analysis, gene set enrichment analysis, and individuation gene analysis. These genes were also compared with PCOS-related genes in other databases. To further verify the authenticity of the differentially expressed genes between the two groups, the expression of eight randomly selected vital genes and three proteins of interest was verified through real time quantitative polymerase chain reaction and Western blot experiment respectively. The best intervention concentration and duration for ZQLHR to promote the proliferation of KGN cells were 0.2% and 48h respectively in this experiment. Multiple signaling pathways and 55 focus differentially expressed genes, both related to autophagy, steroidogenesis, oxidative stress-related longevity, inflammation, and complications of PCOS, may play an important role in the therapeutic mechanism of action of ZQLHR. The expression of eight genes is consistent with the result of RNA sequencing, and the expression of three proteins of interest is the same as expected. The promotion of hGCs proliferation upon treatment with ZQLHR may be a manifestation of ZQLHR in the treatment of PCOS patients. The positive effects of ZQLHR against PCOS may involve pathways and genes related to autophagy, steroidogenesis, oxidative stress-related longevity, and inflammation in hGCs. Some components of ZQLHR applied for the treatment of PCOS may also be effective for the treatment of some complications of PCOS.