The present study demonstrates that the administration of a hot water extract of BCB can increase the number of newborn pups as compared with sham-treated mice and can improve the number of implantation sites in pregnant mice treated with RU486.
BCB contains the 11 herbs; Cuscutae Semen, Dioscoreae Rhizoma, Rubi Fructus, Ginseng Radix, Lycii Fructus, Angelicae Gigantis Radix, Perillae Folium, Amomi Fructus, Artemisiae Argyi Folium, Zingiberis Rhizoma Crudus, and Zizyphi Fructus. Among those, Cuscutae Semen is often used to treat female infertility in traditional medicine and has been shown to significantly increase the number of ovulating ovaries in mice . Cuscutae Semen, Dioscoreae Rhizoma, and Rubi Fructus are the main herbs used to tonify kidneys, and Rubi Fructus is also used to treat female infertility [35, 36]. Furthermore, Rubi Fructus has been reported to improve female gonadal function imbalance and follicle numbers and sizes [37, 38]. In addition, Dioscoreae Rhizoma has been shown to prevent abortion, reinforce essences, and tonify kidneys . Moreover, Ginseng Radix and Lycii Fructus have effects that include the reinforcing of Qi and Yin and improve embryo implantation and the endometrial environment [40, 41]. Angelicae Gigantis Radix is also an effective treatment for infertility due to its ability to nourish blood [42, 43]. Perillae Folium and Amomi Fuctus are representative herbal medicine that may be used to reduce the risk of abortion under threatening conditions . Artemisiae Argyi Folium is also a representative herbal medicine and used to treat female infertility and gonadal function imbalance . These previous findings strongly suggest BCB has therapeutic potential in the context of poor endometrial receptivity.
Decidualization is a series of changes in uterus during the early stages of pregnancy that are necessary for placenta formation and fetal development [46–48]. Decidualization occurs only near the embryos in mice, whereas this process occurs periodically, regardless of the presence or absence of an embryo in humans . During decidualization, uterine epithelium falls apart and is followed by extensive proliferation and angiogenesis in the subepithelial stroma . The signal transduction required for successful decidualization is generated by two types of cell–cell interactions: embryo–epithelial and epithelial–stromal . COX-2 and epidermal growth factor (EGF) family members including heparin binding-EGF (HB-EGF) are activated in luminal epithelium surrounding blastocyst and induce activation of Bmp2 (bone morphogenic protein 2) and Wnt4 (wingless-related mouse mammary tumor virus integration site 4) resulting in stromal cell growth [46, 50]. COX-2 deletion suppresses implantation and decidualization as well as fertilization in the COX-2 deficiency mouse model . In the present study, we also demonstrated that BCB can prevent the RU486-induced down-regulation of COX-2 in the RU486-treated mouse uterus, which suggests BCB-induced COX-2 may be involved in implantation and decidualization.
Nitric oxide (NO) is a key mediator of various physiological functions including vascular functions and inflammatory responses [51, 52], and has been reported to play a crucial role during implantation and pregnancy establishment [53–55]. In addition, it has been reported that NO may regulate the growth and development of preimplantation embryos [56–58]. NO is generated by three isoforms of nitric oxide synthase (NOS). Up-regulation of cytokine-inducible NOS (iNOS) and endothelial NOS (eNOS) have been found in pregnant rodent uteri [59, 60]. In this study, we demonstrated that BCB administration increased the expression of iNOS in RU486-treated mouse uteri, suggesting that BCB-induced iNOS may enhance implantation and decidualization.
NF-κB induces COX-2 expression in various cells including gingival fibroblasts and endometrial stromal cells [61–63], and the expression of pro-inflammatory cytokines in uterus throughout the estrous cycle is necessary for embryo receptivity and successful blastocyst implantation . NF-κB dimers are sequestered in the cytoplasm by binding with IκBα and hence inactivated . Activation of NF-κB occurs via IκBα degradation, which is initiated by IκB kinase complex-induced phosphorylation of IκBα. NF-κB activation is known to be crucial for development beyond the 2-cell stage of mouse embryos . Furthermore, the deregulation of IκBα has been associated with the aging of mouse and bovine oocytes [67–70]. In the present study, we observed BCB administration induced the phosphorylation of IκBα in uterus tissues, suggesting that BCB might improve implantation rates via the NF-κB/IκBα activation pathway.
ROS have beneficial effects on biological functions but can also produce pathological conditions within the female reproductive system . ROS concentration may also have major effects on the implantation and fertilization of eggs . Recently, Qin et al. (2016) reported that dehydroepiandrosterone improves endometrium receptivity and enhance embryo implantation by inhibiting the generation of intracellular ROS in endometrial stromal cells , and Nicol et al. (2000) reported glucose 6-phosphate dehydrogenase prevented oxidative stress-induced embryopathies . In concert with those findings, in this study, BCB showed strong scavenging activities against DPPH and the superoxide anion radicals, and inhibited ROS production in CHO-K1 cells treated with H2O2. These results suggest that the therapeutic effect of BCB against infertility may be due to antioxidant activity.