Abstract

   Anemarrhenae rhizome and Phellodendri cortex have historically been
   used for the treatment of precocious puberty (PP) in oriental medicine.
   Our study aimed to evaluate the effect of APE, a mixture of the
   extracts from these herbs, against danazol-induced PP in female rats.
   The offspring were injected danazol to establish the PP model, and then
   treated with APE daily, and observed for vaginal opening. At the end of
   the study, the levels of gonadotropic hormones, such as estradiol,
   follicle-stimulating hormone, and luteinizing hormone, were determined
   by ELISA. Moreover, the mRNA expression of GnRH, netrin-1, and UNC5C in
   hypothalamic tissues was determined by real-time PCR. Network
   pharmacological analysis was performed to predict the active compounds
   of APE and their potential actions. APE treatment delayed vaginal
   opening in rats with PP. In addition, APE treatment reduced LH levels
   and suppressed UNC5C expression. Gene set enrichment analysis revealed
   that the targets of APE were significantly associated with GnRH
   signaling and ovarian steroidogenesis pathways. In conclusion, APE may
   be used as a therapeutic remedy to inhibit the activation of the
   hypothalamic–pituitary–gonadal axis.

   Keywords: Anemarrhenae rhizome, Phellodendri cortex, precocious
   puberty, danazol, network pharmacology

1. Introduction

   Precocious puberty is an endocrine disorder characterized by the onset
   of secondary sexual characteristics before the age of eight years in
   girls and nine years in boys [[42]1]. The incidence of precocious
   puberty in girls is higher than that in boys [[43]2]. Precocious
   puberty is classified into three major types: central precocious
   puberty (CPP) that is gonadotropin-dependent, peripheral precocious
   puberty that is gonadotropin-independent, and normal variant puberty
   [[44]3]. The early activation of the hypothalamic–pituitary–gonadal
   axis (HPGA) leads to the release of gonadotropins that initiate the
   development of secondary sexual characteristics and accelerate bone
   maturation in individuals with CPP [[45]4]. Therefore, CPP can be
   distinguished from the other two types of precocious puberty by hormone
   test and bone age determination [[46]1].

   The incidence and prevalence of CPP in Korea were investigated based on
   the national registry data from the Health Insurance Review and
   Assessment Service. Between 2008 and 2014, 37,890 girls and 1220 boys
   were diagnosed with CPP nationwide. The incidence rate (per 100,000
   children) of CPP during this period was 262.8 for girls and 7.0 for
   boys. The overall prevalence of CPP during the period 2008–2014 was
   410.6 for girls and 10.9 for boys. Moreover, this epidemiologic study
   showed that the annual incidence of CPP among Korean children rapidly
   increased by the year during 2008–2014 in both girls (from 89.4 to
   415.3) and boys (from 1.6 to 14.7) [[47]5].

   Until now, gonadotrophin-releasing hormone analog (GnRHa) is the only
   effective treatment for CPP among the currently available, which
   maintains the stable level of GnRH to reduce the steroid hormones to
   the prepubertal level. Several synthetic peptide drugs, such as
   leuprolide, triptorelin, and goserelin, have been used clinically
   [[48]6,[49]7]. GnRHa continuously stimulates the anterior pituitary
   through GnRH receptors. The long-term continuous stimulation of the
   anterior pituitary downregulates its responsiveness, which suppresses
   the production of luteinizing hormone (LH) and follicle-stimulating
   hormone (FSH) [[50]8,[51]9]. In this way, administration of GnRHa
   reduces the secretion of gonadal sex steroids to the prepubertal levels
   in individuals with CPP. However, various common side effects of GnRHa
   have been reported, such as injection site reactions, sterile abscess,
   pain, bruising, and nausea [[52]10]. In addition, there are no clear
   evidence of the effect of GnRHa treatment on increasing the adult
   height of girls with CPP aged between 6 and 8 years [[53]7,[54]8].

   In Chinese medicine, some medicinal plants have been used as an
   alternative therapy for the treatment of CPP, including Anemarrhenae
   asphodeloides rhizome (Ji-Mo) and Phellodendri cortex (Huang Bai)
   [[55]11,[56]12,[57]13,[58]14,[59]15]. The combination treatment with
   the Western and Oriental medicines may be an effective strategy for CPP
   treatment. Many research and clinical trials have been conducted to
   evaluate the effects of herbal medicines on CPP [[60]15,[61]16,[62]17].
   In this study, we investigated the effect of the extract of
   Anemarrhenae asphodeloides rhizome and Phellodendri cortex, abbreviated
   as APE, against CPP in an animal model represented by delayed vaginal
   opening and reduced release of gonadotropic hormones against
   danazol-induced precocious puberty in female rats. Simultaneously,
   network pharmacological analysis was used to explore the active
   compounds of APE and their potential in alleviating precocious puberty
   symptoms.

2. Results

2.1. Cytotoxicity Test of APE on GT1-7 Cells

   GT1-7 is an immortalized mature mouse hypothalamic GnRH neuronal cell
   line. GT1-7 cells can be used as an in vitro model of GnRH-secreting
   neurons in the hypothalamus [[63]18]. The cytotoxic effect of APE on
   GT1-7 cells was evaluated using cell viability assay. As shown in
   [64]Figure 1, cell viability was not significantly affected after
   treatment with 10–100 μg/mL APE. Thus, APE up to a concentration of 100
   μg/mL was not cytotoxic to GT1-7 cells.

Figure 1.

   [65]Figure 1
   [66]Open in a new tab

   Cytotoxicity test of APE on GT1-7 cells. GT1-7 cells were seeded in a
   96-well plate and then treated with APE at 10–100 μg/mL to evaluate its
   cytotoxic effect.

2.2. Effect of APE on Vaginal Opening

   Female laboratory rats were injected danazol on PD 5 to establish the
   PP model. To precisely define the onset of puberty, vaginal opening was
   chosen as a reliable sign of sexual maturation. Vaginal opening was
   observed in danazol-induced PP rats on PD 29, five days earlier than in
   the vehicle group ([67]Figure 2). Vaginal opening was significantly
   delayed in the APE-treated group to PD 32. Thus, APE showed an
   inhibitory effect on PP in female rats.

Figure 2.

   [68]Figure 2
   [69]Open in a new tab

   APE delayed vaginal opening in danazol-treated female rats. (a)
   Schematic representation of the experimental design. (b) Treatment with
   the APE extract delayed vaginal opening. (c) The representative images
   of vagina of rats in different experimental groups on postnatal day 29.
   * p < 0.05, compared to the PP group.

2.3. Body Weight, Body Length, and ALP Level

   There was no significant difference in body weight gain among different
   experimental groups ([70]Figure 3a). Accelerated growth, which results
   in a restricted final stature, is an important sign of PP. The results
   revealed that, compared with the other groups, treatment with APE in
   the long term slightly reduced the body length of rats ([71]Figure 3b).
   We also measured the serum ALP level, a marker of bone maturation
   [[72]19], to evaluate the effect of APE on regulating the growth rate.
   Only leuplin showed an inhibitory effect to restore the serum ALP
   concentration to the basal level; however, APE treatment did not change
   the serum ALP level in the PP model ([73]Figure 3c).

Figure 3.

   [74]Figure 3
   [75]Open in a new tab

   Effect of APE on the growth rate of experimental rats. (a) The body
   weight gain in rats of different groups on PD 39. (b) Effect of APE on
   body length of rats in the PP model. (c) The serum ALP levels in rats
   of different groups. * p < 0.05; ** p < 0.01, compared to the PP group.

2.4. Organ Index of Uterus, Pituitary, and Hypothalamus

   The organ index of the uterus did not vary significantly among
   different experimental groups. The pituitary index was decreased after
   treatment with APE for the long term, as shown in [76]Figure 4. In
   addition, treatment with leuplin reduced the hypothalamus index
   compared with that of the PP group. Leuplin is a sustained-release
   injectable formulation of leuprorelin acetate that is commonly used for
   the treatment of hormone-dependent diseases such as prostate cancer,
   premenopausal breast cancer, transgender hormone therapy, and early
   puberty [[77]8].

Figure 4.

   [78]Figure 4
   [79]Open in a new tab

   Effect of APE on the organ index of uterus, pituitary, and hypothalamus
   of experimental rats. * p < 0.05, compared to the PP group.

2.5. Effect of APE on Serum Hormone Levels in Rats

   The onset of PP is marked by the activation of the HPGA, which leads to
   an increase in gonadotropic hormones, such as E2, FSH, and LH. There
   was no significant change in the serum E2 level between the APE-treated
   group and the non-treated group. Neither leuplin or APE affected the
   level of FSH in rats with PP. The results revealed that treatment with
   APE only suppressed LH secretion in the early stages of PP ([80]Figure
   5).

Figure 5.

   [81]Figure 5
   [82]Open in a new tab

   Effect of APE on serum gonadotropic hormone levels in experimental
   rats. ** p < 0.01; *** p < 0.001, compared to the PP group.

2.6. Effect of EIF on Hypothalamic GnRH, Netrin-1, and UNC5C mRNA Expressions

   The hypothalamic tissues were collected from the brains of experimental
   rats on PD 29 to evaluate the mRNA expression of GnRH, netrin-1, and
   UNC5C. In the hypothalamus of rats with PP, the expression levels of
   netrin-1 and its receptor UNC5C were enhanced, which resulted in the
   release of GnRH [[83]20]. As shown in [84]Figure 6, treatment with APE
   reduced the expression of GnRH, netrin-1, and UNC5C. The mRNA level of
   UNC5C was notably decreased after APE treatment.

Figure 6.

   [85]Figure 6
   [86]Open in a new tab

   Effect of APE on mRNA expression of hypothalamic GnRH, netrin-1, and
   UNC5C. * p < 0.05; ** p < 0.01, compared to the PP group.

2.7. Identifying Bioactive Compounds and Targets of APE

   We employed TCMSP to construct the herb–compound–target network
   [[87]21]. A total of 54 compounds was identified from Anemarrhenae
   rhizome and Phellodendri cortex. Oral bioavailability and drug-likeness
   thresholds (≥30% and 0.18%, respectively) were applied to screen the
   compounds having potential in vivo medicinal effects. We found 15
   compounds that met the threshold and identified 104 relevant targets
   for the selected compounds. The list of compounds and their data are
   shown in [88]Table 1.

Table 1.

   List of compounds from Anemarrhenae rhizome and Phellodendri cortex.
   Name of Compound Pubchem ID Structure OB (%) DL (%)
   Niloticin 44559946 graphic file with name plants-11-00023-i001.jpg
   41.41427 0.81833
   Stigmasterol 5280794 graphic file with name plants-11-00023-i002.jpg
   43.82985 0.75665
   β-sitosterol 222284 graphic file with name plants-11-00023-i003.jpg
   36.91391 0.75123
   Corbisterol 12303924 graphic file with name plants-11-00023-i004.jpg
   37.42312 0.75103
   Palmatine 19009 graphic file with name plants-11-00023-i005.jpg
   64.60111 0.64524
   Isocorypalmine 440229 graphic file with name plants-11-00023-i006.jpg
   35.76844 0.59227
   Asperglaucide 10026486 graphic file with name plants-11-00023-i007.jpg
   58.0163 0.51972
   Beta-Anhydroicaritin 14583584 graphic file with name
   plants-11-00023-i008.jpg 45.41193 0.43786
   Dehydrotanshinone IIA 128994 graphic file with name
   plants-11-00023-i009.jpg 43.76229 0.40019
   Phellopterin 98608 graphic file with name plants-11-00023-i010.jpg
   40.18556 0.27878
   n-cis-feruloyltyramine 6440659 graphic file with name
   plants-11-00023-i011.jpg 118.3477 0.26399
   Kaempferol 5280863 graphic file with name plants-11-00023-i012.jpg
   41.88225 0.24066
   Coumaroyltyramine 13939145 graphic file with name
   plants-11-00023-i013.jpg 112.9016 0.20234
   Skimmianine 6760 graphic file with name plants-11-00023-i014.jpg
   40.13655 0.19638
   Magnograndiolide 5319198 graphic file with name
   plants-11-00023-i015.jpg 63.70888 0.18833
   [89]Open in a new tab

   OB: oral bioavailability; DL: drug likeness.

2.8. Pathway Enrichment Analysis of APE

   Gene set enrichment analysis (GSEA) was performed on the KEGG database
   to identify potential pathways of the active compounds from
   Anemarrhenae rhizome and Phellodendri cortex [[90]22,[91]23]. The GnRH
   signaling and ovarian steroidogenesis pathways were chosen to predict
   the protein targets related to the therapeutic effect of APE on PP. We
   found that the numbers of related targets for GnRH signaling and
   ovarian steroidogenesis pathways were five each, which was
   significantly associated with both pathways ([92]Table 2). The targets
   of APE active compounds in GnRH signaling and ovarian steroidogenesis
   pathways were visualized using KEGG Mapper ([93]Figure 7).

Table 2.

   Significant enrichment pathways and their target genes related to the
   therapeutic effect of APE on PP.
   Pathway Overlap p-Value Adjusted p-Value Targets (Gene Symbol)
   GnRH signaling 5/93 0.00012 0.00059 MAPK8; JUN; PRKACA; MAPK14; CALM1
   Ovarian steroidogenesis 5/49 5.49 × 10^−0.6 4.45 × 10^−5 ALOX5; INSR;
   AKR1C3; PRKACA; PTGS2
   [94]Open in a new tab

Figure 7.

   [95]Figure 7
   [96]Open in a new tab

   The target genes of APE active compounds in the GnRH signaling and
   ovarian steroidogenesis pathways. (a) The GnRH signaling pathway. (b)
   The ovarian steroidogenesis pathway in theca-interstitial cells and
   granulosa cells. The orange-colored box represents a predicted target
   gene of APE active compounds.

2.9. Herb–Compound-Target Network of APE

   We constructed and visualized herb–compound–target network of APE using
   Cytoscape [[97]24], as shown in [98]Figure 8. The network consisted of
   121 nodes and 412 edges, in which nodes denote herbs or active
   compounds or protein targets (2, 15, and 104, respectively), and edges
   denote a herb–compound or compound–target relationship (16 and 396,
   respectively). Among the targets related to GnRH signaling and ovarian
   steroidogenesis pathway, the protein targets showing the highest degree
   were PTGS2, MAPK14, PRKACA, and CALM1 (11, 9, 7, and 5, respectively).
   Those targets are expected to be cumulatively affected by the compounds
   of APE. Among the active compounds of APE, kaempferol, β-sitosterol,
   palmatine, and isocorypalmine were found to be closely connected to
   targets involved in the GnRH signaling and ovarian steroidogenesis
   pathway (8, 4, 4, and 4, respectively, as a number of compound–target
   interactions). These compounds are predicted to be active compounds
   that mainly contribute to the effect of APE on PP.

Figure 8.

   [99]Figure 8
   [100]Open in a new tab

   Compound–target network of APE. The targets genes of the GnRH signaling
   and ovarian steroidogenesis pathways are presented by colored nodes.
   Genes directly associated with PP are bordered in the network. The size
   of each node is proportional to its interaction potential.

3. Discussion

   The results of in vivo study indicated that APE treatment delayed
   vaginal opening in rats with PP by inhibiting the activation of HPGA.
   APE inhibited the increase in the pituitary index and LH serum level at
   the onset of PP. Moreover, APE reduced mRNA expression of UNC5C in
   hypothalamic tissues, which is involved in the regulation of GnRH
   release. According to the previous references on the TCMPS database, 15