Protopanaxadiol improves endometriosis associated infertility and miscarriage in sex hormones receptors-dependent and independent manners

Abstract

Background: Patients with endometriosis (EMs) have high risks of infertility and spontaneous abortion. How to remodel the fertility of patients with EMs has always been the hot spot and difficulty in the field of reproductive medicine. As an aglycone of ginsenosides, protopanaxadiol (PPD) possesses pleiotropic biological functions and has high medicinal values. We aimed to investigate the effect and potential mechanism of PPD in the treatment of EMs-associated infertility and spontaneous abortion. Methods: The EMs mice models were constructed by allotransplantation. The pregnancy rates, embryo implantation numbers and embryo resorption rates of control and EMs were counted. RNA sequencing, qRT-PCR, enzyme linked immunosorbent assay (ELISA) and FCM analysis were performed to screen and confirm the expression of endometrial receptivity/decidualization-related molecules, inflammation cytokines and NK cell function-related molecules in vitro and/or in vivo. The SWISS Target Prediction, STRING and Cytoscape were carried out to predict the potential cellular sensory proteins, the protein-protein interaction (PPI) network between sensory proteins and fertility-related molecules, respectively. Micro-CT detection, liver and kidney function tests were used to evaluate the safety. Results: Here, we observe that PPD significantly up-regulates endometrial receptivity-related molecules (e.g., Lif, Igfbp1, Mmps, collagens) and restricts pelvic inflammatory response (low levels of IL-12 and IFN-γ) of macrophage, and further remodel and improve the fertility of EMs mice. Additionally, PPD increases the expression of decidualization-related genes and Collagens, and promotes the proliferation, residence, immune tolerance and anagogic functions of decidual NK cells (low levels of CD16 and NKp30, high levels of Ki67, VEGF, TGF-β) in pregnant EMs mice, and further triggers decidualization, decidual NK cell-mediated maternal-fetal immune tolerance and angiogenesis, preventing pregnant EMs mice from miscarriage. Mechanically, these effects should be dependent on ESRs, PGR and other sensory proteins (e.g., AR). Compared with GnRHa (the clinic first-line drug for EMs), PPD does not lead to the decline of serum estrogen and bone loss. Conclusion: These data suggest that PPD prevents EMs-associated infertility and miscarriage in sex hormones receptors-dependent and independent manners possibly, and provides a potential therapeutic strategy with high efficiency and low side effects to remodels the fertility of patients with EMs.

Keywords: GnRHa; decidualization; endometrial receptivity; endometriosis; protopanaxadiol.

Figure 1

EMs mice model with endometrial transplantation displays impaired fertility and elevated inflammation. (A) On the day 4, peritoneal fluids and uterus of the normal BalB/C female mice (Ctrl) and / or EMs mice were collected, or the rest of Ctrl and / or EMs mice were randomly mated with healthy male adult mice, respectively, and then the data of pregnancy rate, the number of embryos, and the absorption rate were counted on day 18. (B&C) FCM was used to evaluate the expression of IFN-γ in CD45⁺F4/80⁺/CD11b⁺ Mφ macrophages of PF (n=8). (D) mRNA expression levels of Lif and Igfbp1 in eutopic endometrium of Ctrl and EMs mice (n=10). (E) The pregnancy rates, number of implanted embryos and the embryo absorption rate of Ctrl mice (n=11) and EMs mice (n=16) at the gestation of day 13.5 were quantified in (F&G), and the absorption sites were indicated by the red triangle. Mean ± SEM, **p<0.01, ***p<0.001.

Figure 2

PPD regulates hormones receptors expression and restricts the growth of ectopic lesions of EMs mice. (A) The EMs model of BALB/C mice were intraperitoneally injected with control vehicle (5 % DMSO, every days), PPD (45 mg/kg, every 4 days), and GnRHa (0.5 µg, every day) from day 4 to day 32. (B-D) On day 32, the number (B) and weight of ectopic lesions (C) were analyzed. The serum concentrations of E2 were measured by ELISA (D) (n=5). (E) mRNA expression levels of Pgr, Esr1 and Esr2 in eutopic endometrium of EMs mice (n=8). Mean ± SEM, NS, no significant difference, **p<0.01, ***p<0.001.

Figure 3

PPD promotes the endometrial receptivity possibly in hormone receptor dependent and independent manners. (A-C) mRNA-seq was performed to evaluate the differential expression genes in HESCs after treatment with or without PPD (40 µM) for 48 h. Gene Ontology (GO) enrichment and KEGG pathway enrichment analyzed have been shown. (D) qRT-PCR was used to evaluate the expressions of MMPs and collagens in HESCs after treatment with or without PPD (40 µM) for 48 h. (E) HESCs were treated by DMSO (1‰), or medroxyprogesterone acetate (MPA, 1 µM) combining with estradiol (E2, 1 nM), or PPD (40μM) for 48 h, and RT-PCR was used to measure the expression level of endometrial receptivity-related genes. (F) The mRNA level of endometrial receptivity-related genes in endometrium of EMs mice treated by control vehicle (5‰DMSO, every day), PPD (45 mg/kg, every 4 days), and GnRHa (0.5 µg, every day) (n=8). (G) PPI network of predicted sensory proteins of PPD (ESR1 and ESR2), PGR, endometrial receptivity-related molecules and collagens were obtained by STRING database and Cytoscape. (H) Top 15 predicted sensory proteins of PPD (get from SWISS Target Prediction website, showed in Table 1) involved in the regulation of endometrial receptivity and ECM remodeling, including AR, CYP19A1, HSD11B1, etc. (I) HESC were pre-treated with RU486 (1 nM or 10 nM) for 48h, then treated with or without PPD (40 µM) for 48h, or treated with DMSO (1 %) as control. Then, the mRNA level of PGR, IGFBP1 and LIF were analyzed by RT-PCR. Mean ± SEM, NS, no significant difference, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 4

PPD alleviates pro-inflammatory cytokines production of macrophage in endometriotic milieu. (A) The PPI network of top 15 predicted sensory proteins of PPD and cytokines secreted by Mφ. (B&C) The expression level of inflammatory factors in CD45⁺F4/80⁺/CD11b⁺ Mφ of peritoneal fluids (PF) treated by the control (5 % DMSO, every day, n=15), PPD (45 mg/kg, every 4 days, n=18), and GnRHa (0.5 µg, every day, n=18) was evaluated by FCM. (D&E) FCM was used to measure the expression level of IFN-γ, and IL-12 in U937 cells co-cultured with or without PPD-pretreated (40 µM) HESCs for 48 h (n=6). Mean ± SEM, NS, no significant difference, *p<0.05, **p<0.01, ***p<0.001.

Figure 5

PPD prevents the risk of spontaneous abortion of EMs mice. (A) After 28 days of treatment with Ctrl (5 % DMSO, every days), PPD (45 mg/kg, every 4 days), and GnRHa (0.5 µg, every day), these female EMs mice were mated with healthy adult male mice. (B-D) The pregnancy rates, number of embryos implanted and the embryo absorption rate of the control group, the PPD group, and the GnRHa group at the gestation of day 13.5 were quantified in (C&D), and the absorption sites were indicated by the red triangle (n=16). Mean ± SEM, NS, no significant difference, **p<0.01, ***p<0.001.

Figure 6

PPD promotes decidualization by triggering multiple biological processes. (A-C) mRNA-seq was performed to evaluate the differential expression genes in decidual stromal cells (DSCs) after treatment with or without PPD (40 µM) for 48 h. GO enrichment (B) and KEGG pathway enrichment (C) analyzed have been shown. (D) qRT-PCR was used to evaluate the expressions of LIF, IGFBP1, MMPs and collagens in DSCs after treatment with or without PPD (40 µM) for 48 h. (E) The mRNA expressions of LIF, IGFBP1, and MMPs in uterus of normal pregnancy mice or EMs pregnancy mice of the control group, the PPD-treated EMs pregnancy mice group, and the GnRHa-treated EMs pregnancy mice group (n=12). Mean ± SEM, NS, no significant difference, *p<0.05, **p<0.01, ***p<0.001.

Figure 7

PPD increases the residence, immune and angiogenic functions of decidual NK cells. (A) The STRING tool was used to obtain PPI between predicted sensory proteins of PPD and cytokines produced by NK cells, and the PPI network was shown. (B) Flow cytometry gating strategy for identifying the CD45⁺ leukocytes and CD45⁺CD3^-CD49a⁺dNK cells within the CD45⁺CD3^- gate, and quantitative analysis of the proportions of all leukocytes and dNK cells in the decidua of the Ctrl normal mice (n=20), the EMs mice treated by control vehicle (n=27), PPD (n=32), or GnRHa (n=19). (C) dNK cells separated for human decidual tissues, were treated with different concentrations of PPD (0, 10, 20, or 40 µM, n=18-30). Mean ± SEM, NS, no significant difference, *p<0.05, **p<0.01, ***p<0.001.

Figure 8

PPD does not cause bone loss with a good safety. (A) After 28 days for treatment, the bodyweight of the mice was recorded. (B) The H&E staining of the kidneys and livers, and the TRAP staining and ALP staining on mice femurs. (C) The serum biochemical index, and the levels of TRAP and ALP in the serum had been detected (n=6-9). (D) Three-dimensional micro-CT images of the trabecular microstructure of distal tibia metaphysis at the transaxial and axial view. (E) The BMD, BV, TV, BV/TV, trabecular thickness, and trabecular number of EMs mice in the control group, the PPD group and the GnRHa group were detected by Micro-CT (n=10). Mean ± SEM, NS, no significant difference, *p<0.05, **p<0.01.

Figure 9

Schematic roles of PPD in remodeling fertility and preventing the risk of spontaneous abortion of EMs. Patients with endometriosis (EMs) have increased risks for infertility and spontaneous abortion. As one of two metabolites of ginsenoside (the main components extracted from ginseng), Protopanaxadiol (PPD) significantly up-regulates endometrial receptivity-related molecules (e.g., Lif, Igfbp1, Mmps, collagens) and restricts pelvic inflammatory response (low levels of IL-12 and IFN-γ) of macrophage, and further remodel and improve the fertility of EMs mice (upper part of figure). Additionally, PPD increases the expression of decidualization-related genes (e.g., Lif, Igfbp1, Mmps) and Collagens, and promotes the proliferation, residence, immune tolerance and anagogic functions of decidual NK cells (low levels of CD16 and NKp30, high levels of Ki67, VEGF, TGF-β and CXCL10) in pregnant EMs mice, and further triggers decidualization, decidual NK cell-mediated maternal-fetal immune tolerance and angiogenesis, preventing pregnant EMs mice from miscarriage. Mechanically, these effects should be dependent on ESRs, PGR and other sensory proteins. Therefore, the potential therapeutic value of PPD in EMs-related infertility and miscarriage should be emphasized due to high efficiency and low side effects.