Cai's prescription inhibits granulosa cell apoptosis through ARHGAP4 on poor ovarian responders

Abstract

Purpose: Poor ovarian response (POR) is a big challenge for in vitro fertilization. The traditional Chinese medicine, Cai's Prescription of Tonifying Kidney and Strengthening Vitals (Cai's Prescription) has yielded satisfactory results for POR treatment clinically, but systematic scientific research of Cai's Prescription is not well reported. This study aimed to investigate the clinical effect of Cai's Prescription on poor ovarian responders and its biological mechanism.

Methods: Serum was collected from poor ovarian responders, and IL-1β, INFγ, FSH, E₂ and AMH levels were analyzed by ELISA. Ovarian antral follicles were identified and counted using transvaginal ultrasound. The embryo quality grading were done on day 3 after retrieval. We used high-throughput sequencing of granulosa cells to investigate the gene transcription patterns of ovarian granulosa cells in poor ovarian responders after Cai's Prescription pretreatment. The expression level of ARHGAP4 was analyzed by quantitative real-time PCR and western blot. The effects of ARHGAP4 for granulosa cells were analyzed by CCK-8 assay, annexin-V and PI staining, ELISA and western blot. The effects of Cai's Prescription on the expression of PI3K-Akt pathway and apoptosis were analyzed by western blot.

Results: In this study, we found that Cai's Prescription pretreatment had the tendency to improve the ovarian reserve function and could increase the number of high quality embryos for poor ovarian responders. Through high-throughput sequencing of mRNA in granulosa cells, we discovered ARHGAP4, which is a member of GTPase-activating proteins (GAPs) may be a candidate target for POR treatment. ARHGAP4 was significantly increased in poor ovarian responders and can be recovered after Cai's Prescription pretreatment. Mechanically, combining the cell line model and clinical tissue samples, we found that ARHGAP4 can accelerate cell apoptosis and inflammation response in granulosa cells via PI3K-Akt signaling pathway. In addition, Cai's Prescription pretreatment for three months significantly reduced the high level of ARHGAP4 in poor ovarian responders.

Conclusion: This study shows that the traditional Chinese medicine, Cai's Prescription yielded satisfactory results for poor ovarian responders clinically and ARHGAP4 may be a candidate target for POR treatment.

Keywords: ARHGAP4; Apoptosis; Poor ovarian response; Traditional Chinese medicine.

Fig. 1

Analysis of gene expression data in normal and poor ovarian responders. (A) Volcano plot comparing the differentially expressed genes detected by high-throughput sequencing in granulosa cells between normal and poor ovarian responders. (B-C) KEGG enrichment analyses of the differentially expressed genes in granulosa cells between normal and poor ovarian responders. (B) The top 20 upregulated KEGG pathways. (C) The top 20 downregulated KEGG pathways. (D-E) Gene Ontology (GO) functional enrichment analyses of the differentially expressed genes in granulosa cells between normal and poor ovarian responders. (D) The top 20 upregulated GO enrichment scores. (E) The top 20 downregulated GO enrichment scores

Fig. 2

The effects of Cai’s Prescription for poor ovarian responders. (A) The antral follicle count (AFC) of poor ovarian responders with or without Cai’s Prescription treatment. (B-D) Anti-Mullerian hormone (AMH), follicle-stimulating hormone (FSH) and E₂ levels were quantified via ELISA from serum in poor ovarian responders with or without Cai’s Prescription treatment. (E-F) The number of retrieved oocytes and high-quality embryos obtained from poor ovarian responders with or without Cai’s Prescription treatment. Boxes represent median and interquartile range; whiskers represent minimum and maximum

Fig. 3

Analysis of gene expression data in poor ovarian responders with or without Cai’s Prescription treatment. (A) Volcano plot comparing the differentially expressed genes detected by high-throughput sequencing in granulosa cells from poor ovarian responders with or without Cai’s Prescription treatment. (B-C) KEGG enrichment analyses of the differentially expressed genes in granulosa cells from poor ovarian responders with or without treatment with Cai’s Prescription. (B) The top 20 upregulated KEGG pathways. (C) The top 20 downregulated KEGG pathways. (D-E) Gene Ontology (GO) functional enrichment analyses of the differentially expressed genes in granulosa cells from poor ovarian responders with or without Cai’s Prescription treatment. (D) The top 20 upregulated GO enrichment scores. (E) The top 20 downregulated GO enrichment scores

Fig. 4

Cai’s Prescription downregulates ARHGAP4 level in granulosa cells from poor ovarian responders. (A) The mRNA levels of 3 members from ARHGAP family in granulosa cells from normal, poor ovarian responders and poor ovarian responders after Cai’s Prescription treatment. Data are mean ± SD for three independent experiments. (B) The protein level of ARHGAP4 in granulosa cells as described in A

Fig. 5

ARHGAP4 regulates cell proliferation and apoptosis in vitro. (A) The expression level of ARHGAP4 was detected by western blot in KGN cells transfected with pCMV-tag 3A-ARHGAP4 or ARHGAP4 siRNA. (B) CCK-8 assay showed that ARHGAP4 overexpression significantly decreased the ability of KGN cell proliferation, and downregulation of ARHGAP4 showed inverse effect. Data are mean ± SD for three independent experiments. (C) Flow cytometry analysis of annexin-V and propidium iodide (PI) staining of apoptotic KGN cells with ARHGAP4 overexpression or downregulation

Fig. 6

ARHGAP4 regulates inflammation and AMH level. IL-1β (A), INFγ (B) and AMH (C) levels were quantified via ELISA from the supernatant of KGN cells with indicated treatments. Data are mean ± SD for three independent experiments

Fig. 7

ARHGAP4 regulates cell apoptosis through PI3K-Akt pathway. (A) The protein level of Bax was increased, while phosphorylation levels of AKT, S6 were decreased in KGN cells with ARHGAP4 overexpression. (B) The protein level of Bax was decreased, while phosphorylation levels of AKT, S6 were increased in KGN cells with ARHGAP4 downregulation

Fig. 8

Cai’s Prescription regulates PI3K-Akt pathway. The protein levels of Bax, Cleaved Caspase3, AKT, S6, and phosphorylation of AKT, S6 were analyzed in granulosa cells from normal, POR and POR after Cai’s Prescription treatment patients