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The efficacy and mechanism study of Yiru Tiaojing Granule on treating hyperprolactinemia in vitro and in vivo

Yuanyi Wei1 #, Xian Wang1,2 #, Zhiling Yu3, Wenqi Zhou4, Lili Wang1, Fei Qin1, Chunxia Wang1 *, Lianbing Hou1 *

Affiliation

1. Department of Pharmacy, Nanfang Hospital, Southern Medical University, Guangzhou, China

2. Guangzhou Pharmaceutical Co., Ltd, Guangzhou, China

3. School of Chinese Medicine, Hong Kong Baptist University, Hong Kong, China

4. First School of Clinical Medicine, Southern Medical University, Guangdong, China

# These authors contributed equally to this work

* Correspondence

Dr. Chunxia Wang (email: wangcx@smu., Tel.: +86 02062787463) or prof. Lianbing Hou (email: hlianbing@, Tel.: +86 02066142175), Department of Pharmacy, Nanfang Hospital, Southern Medical University, 1838 North Guangzhou Avenue, Guangzhou, Guangdong 510515, China

Abstract

Yiru Tiaojing Granule (YRTJ), a traditional Chinese medicine formula, is used to treat hyperprolactinemia (HPRL). This study was conducted to evaluate the mechanism of action and pharmacological activity of YRTJ on prolactin (PRL) secretion. The animal model of HPRL was induced by metoclopramide. Dopamine D2 receptor (D2DR) in HPRL rat models was analyzed by immunohistochemistry. The biochemical parameters, including follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), progesterone (P), testosterone (T) and PRL were measured by ELISA. Furthermore, the expression of PRL and D2DR was analyzed by Western boltting. The YRTJ significantly decreased the PRL level in the HPRL rat model, and increased the E2, LH and P levels. The high-, and medium-dose of YRTJ reduced D2DR expression in brain (p < 0.001) and produced a similar effect on bromocriptine (p < 0.001). YRTJ-medicated serum reduced (p < 0.001) PRL expression in MMQ cells in a concentration-dependent manner, but had no effects on GH3 cells. The level of D2DR in MMQ cells was also increased dose-dependently (p < 0.05). In addition, the protein kinase A (PKA) and cyclic AMP (cAMP) in MMQ cells were significantly attenuated dose-dependently by treatment with high- and medium-dose YRTJ-medicated serum (p < 0.05) and bromocriptine-medicated serum (p < 0.01). The results suggested that YRTJ was effective against HPRL and the activation of D2DR, which was related to the cAMP/PKA signal pathway, might be the potential mechanism.

Key words: Yiru Tiaojing Granule; Hyperprolactinemia; MMQ cell; GH3 cell; Mechanism

Abbreviations

D2DR: dopamine D2 receptor;

DAT: dopamine transport protein;

E2: estradiol;

FSH: follicle-stimulating hormone;

HPRL: hyperprolactinemia;

LH: Luteinizing Hormone;

MCP: metoclopramide;

P: progesterone ;

PRL: prolactin;

T: testosterone;

YRTJ: Yiru Tiaojing Granule

PKA: protein kinase A

1. Introduction

Hyperprolactinemia is caused by dysfunction of hypothalamus-pituitary-gonadal axis, which is characterized by consistently higher PRL level than normal. The prevalence of HPRL is more in females as compared to males. The incidence of HPRL ranges from 0.4% in an unselected adult population to as high as 9-17% in women with reproductive diseases [1]. Prolactinomas, which accounts for 25-30% of pituitary tumors, are the most common cause of chronic HPRL [2]. In addition, HPRL is also a common adverse effect of antipsychotic therapy, with an incidence of about 40–70% in schizophrenic population [3, 4].

Bromocriptine is used in the treatment of HPRL, however, it is associated with several side effects, including vomiting, nausea, headache, constipation, dizziness, faintness, depression, postural hypotension, digital vasospasm, and nasal stuffiness [5]. Moreover, the level of serum PRL increases in 75% of the patients after the discontinuation of the treatment [6]. Some studies have reported the clinical use of bromocriptine if raised PRL level is not regressed after the reduction of antipsychotic doses [1, 7]. However, recently, it was found that cabergoline was more effective in reducing persistent HPRL, amenorrhea/oligomenorrhea, and galactorrhea than bromocriptine [5]. Many adverse effects are common to both cabergoline and bromocriptine; however, the former is associated with lower incidences of these effects[8]. Thus, cabergoline is a treatment of choice for HPRL in European countries and USA. Most of the patients often cannot tolerate the standard treatment; therefore, there is a critical need for an alternative treatment.Clinical studies have reported that Yiru Tiaojing Granule (YRTJ), which is composed of Radix Paeoniae alba, Rhizoma Curculiginis, Radix Morinda officinalis and Radix Glycyrrhizae, has therapeutic potential in alleviating the symptoms of HPRL and reducing the PRL level, and is associated with an effective rate of 94.7% [9].

In order to verify the clinical potential of YRTJ, we conducted clinical trials to study its effects and mechanisms. Since PRL is secreted by lactotrophs, whose secretive and proliferative activities are strongly inhibited by the stimulation of D2DR [10], most anti-HPRL agents reduce the PRL secretion through this mechanism. Thus, we tested the effects of YRTJ on the expression of serum hormones and D2 receptor in HPRL animal model. Additionally, the effects on the expression of PRL and D2 receptor and PRL secretion were also detected in vitro using serum pharmacological methods. This method, was considered to be more reliable and suitable for the in vitro experiments of the traditional Chinese medicine [11,12]. Results of the study have shown that YRTJ exerted its anti-HPRL activity via the activation of D2DR.

2. Materials and methods

2.1 Plant materials

The YRTJ granules were purchased from Guangzhou ZhiXin Pharmaceutical Co. and authenticated by Professor Chuanming Liu, School of Chinese Medicine, Southern Medical University, Guangzhou, China. Voucher specimens (130401, 130402, 130401 and 130501 for Radix Paeoniae alba, Rhizoma Curculiginis, Radix Morinda officinalis and Radix Glycyrrhizae, respectively) were deposited in Southern Medical University, Guangzhou 510515, P.R. China.

2.2 Preparation of YRTJ

The Chinese medicine YRTJ (batch no. 20130605) was supplied by Nanfang Hospital, Southern Medical University. It composed of four herbs, namely, Rhizoma Curculiginis 27.2%, Radix Paeoniae alba 27.2%, Radix Morinda officinalis 22.7% and Radix Glycyrrhizae 22.7%. The component herbs were surface-cleaned, mixed and submerged into octuple volumes of distilled water. After the first decoction for 3 h, the suspension was filtered and the filtrate was collected. For second decoction of 2 h, 4x distilled water (v/v) was added to the herb mix for a second decoction of 2 h, and the filtrate was collected again after filtration. The residual herb mix was decocted for the third time under the same condition as the second decoction. The filtrates from all the three decoctions were combined and stewed for 12 h, and then condensed to a relative density of 1.20- 1.25 (80℃).

The qualitative and quantitative assays of paeoniflorin, curculigoside, liquiritin and monotropein (the major components of YRTJ) were performed to check the basic quality of the YRTJ using thin-layer chromatography (TLC) and HPLC [13].

2.3 Cell culture

MMQ cell-line was purchased from Cell Culture Centre, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. The cell-line was cultured in F12 medium containing 2.5% fetal bovine serum (FBS) and 15% horse serum (HS). The MMQ cell-line expresses PRL and dopamine D2 receptor.

GH3 cell-line was purchased from Cell Culture Centre of Sun Yat-sen University. The cell-line was cultured in F12K medium containing 2.5% FBS and 15% HS. The GH3 cell-line expresses only PRL.

2.4 HPRL animal model

The research was conducted strictly in accordance with the Guidelines for the Care and Use of Laboratory Animals of Southern Medical University (Permit Number: 4402102095, Date: 2013/03/26). Virgin female Sprague–Dawley rats from the Animal Experiment Center of Southern Medical University Ltd with regular 4-day oestrous cycles and weighing 180~220 g were used for the in vivo experiment. Animals were housed in a room with controlled temperature (25℃) and humidity under a 12 h light/12 h dark cycle with free access to food and water. Vaginal smears were examined daily to determine the starting point of the experiment, and blood samples were collected in the dioestrum stage of the oestrous cycle.

The rats (N = 80) were given intraperitoneal injection of MCP (75 mg/kg, twice-daily) for 15 days. In addition, 12 untreated rats were used as controls. According to the requirements of statistical testing, we examined the serum PRL levels of 36 rats randomly at the 15th day after treatment to confirm the successful establishment of HPRL rat model.

2.5 Effects of YRTJ on serum hormones in HPRL ratmodel

The HPRL rats were randomly divided into 5 groups (13 per group). Rats in the negative control group were administered 0.9% saline (2 mL); rats in the YRTJ groups were orally administered with low-dose YRTJ (1.67 g/kg/day), medium -dose YRTJ (3.33 g/kg/day) and high-dose YRTJ (6.67 g/kg/day) doses of YRTJ. Rats in the positive control group were orally administered with bromocriptine (purity>98%, 1 mg/kg/day, Guangzhou Pharmaceutical Co., Ltd, China).

After intragastric administration of drugs for 30 days, eight rats from each group were randomly selected for blood sample collection from abdominal aorta after anesthesia. Six serum hormones (PRL, E2, FSH, LH, P, and T) were detected by ELISA, following the manufacturers’ instructions. The PRL concentration was determined with a standard curve of known PRL concentrations.

2.6 Immunohistochemistry

After intragastric administration of drugs for 30 days, brain tissues of the rats were isolated, cleaned and soaked in formalin. Following this, the brain tissues were embedded in paraffin and were sectioned. The paraffin sections were deparaffinized in xylene, rehydrated in graded alcohols, and incubated with 3% hydrogen peroxide to block the endogenous peroxidase activity. The sections were stained with primary anti-D2DR antibody (1:1000 dilutions) at 4℃ overnight. After incubation with a biotinylated secondary antibody at room temperature for 40 min, the slides were stained with 3, 3-diaminobenzidine (DAB). Finally, the sections were counterstained with Mayer’s hematoxylin, differentiated, dried, and sealed. Images were taken in five randomly selected fields on each slide to determine the average optical density value (IOD/area). The optical density was analyzed by Image - Pro Plus 6.0 software.

2.7 Serum Preparation

After 7 days of acclimation, 25 SD rats (200±20 g) were randomized into 5 groups: control group (administered with distilled water); bromocriptine group [administered with 4 mL of bromocriptine (0.6 μg/mL per day)], and three YRTJ-medicated serum groups orally administered with 16.7 mg/kg, 33.3 mg/kg or 66.7 mg/kg of YRTJ, respectively, twice–a-day for 7 days. Before the last administration, rats were kept on fasting for 12 h but were provided with free access to water. Two hours after the last administration, the rats were anaesthetized with chloralhydrate and blood was obtained from the abdominal aorta aseptically. The serum was obtained by centrifugation at 3000 rpm/min for 15 min. YRTJ-medicated serum, bromocriptine-medicated serum and blank control serum were filtered through 0.22 μm filter membranes to remove bacteria, and then stored at -80℃ for further analysis.

2.8 Effects of YRTJ-medicated serum on PRL secretion

The MMQ cells were seeded in a 96-well plates (1×105 cells/well). After starving the groups for 12 h, the YRTJ-medicated serum (5%) from each group was added to the wells (3 wells per group). The plates were cultured for 48 hours, and were centrifuged. The supernatants for each group was collected, and PRL concentrations were determined using a PRL ELISA kit.

2.9 Western blot analysis

After 48 h of the treatment with medicated serum from each group, the MMQ and GH3 cells were harvested and washed twice with ice-cold PBS and were added with cell lysis buffer. The lysates were centrifuged (13,000 rpm) at 4℃ for 20 min. The protein concentration was determined by bicinchoninic acid assay (BCA). The supernatants with 5× loading buffer were boiled at 100℃ for 10 min and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)with 10% gels. The separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% non-fat milk for 1 h at room temperature and were incubated overnight with primary antibodies (PRL antibody; D2DR antibody) at 4℃. The following day, the blots were incubated with secondary antibody at room temperature followed by three times of 5-min washing with tris-buffered saline and Tween 20 (TBST). The membrane was scanned by in vivo imaging systems. To calculate the fold changes, the densities of protein bands were determined by Quantity One software.

2.11 Effect of haloperidol and YRTJ-medicated serum co-treatment on PRL expression in MMQ cells

MMQ cells were collected to examine the expression of PRL after the co-treatment with high-dose YRTJ group serum and 100μm haloperidol. The PRL protein was detected by western bolt.

2.12 Analysis of cAMP levels and protein kinase A (PKA) activity

MMQ cells were seeded on a 6-well plates (1×106/ml). After starved for 12 h, the YRTJ-medicated serum (5%) from each group was added to the wells for another 48 hours. The cells were treated with 0.1 M HCl after removing the culture media and incubated for 10 min to confirm cell lysis. The cell lysates were centrifuged at room temperate and the supernatant was used directly in the assay. The intracellular cyclic AMP level was measured by the Cyclic AMP EIA Kit (Cayman) according to the manufacturer's instructions.In addition, cellular proteins in each group were collected using lysis buffer. A nonradioactive PKA activity assay kit (Enzo Life Sciences) was used to measure PKA activity in the samples.

3. Results

3.1 Effects of YRTJ on serum hormone in rat model of HPRL

The PRL level in the model group was significantly higher than the other groups (p < 0.001), which suggested that the HPRL model was successfully established. After treatment with the YRTJ granule for 30 days, the metoclopramide MCP-induced elevated PRL level was significantly reduced in a dose-dependent manner (p < 0.001 versus model). There was no significant difference between the effects of high-dose bromocriptine and YRTJ granule. Moreover, bromocriptine was more effective in regulating E2 (p < 0.001 versus model), T (p < 0.01 versus model), and LH (p < 0.01 versus model) than YRTJ. However, high- or medium-dose of YRTJ E2 (p < 0.001 versus model), Pg (p < 0.05 versus model), T (p < 0.01 versus model), and LH (p < 0.01 versus model). These findings suggested that YRTJ can effectively inhibit the secretion of PRL and regulate sex hormone levels in HPRL rat models (Fig. 1 and Table 1).

Fig.1. HPRL rat models were prepared by injecting metoclopramide (MCP, 75 mg/kg, twice-daily) for 15 days, followed by treatment with or without bromocriptine or YRTJ at different doses for 30 days. Untreated animals served as control. (A) Serum PRL; (B) T; (C) LH; (D) P; (E) E2; and (F) FSH were measured.Data is expressed as mean±SEM (N=3) and analyzed using one-way ANOVA. * p ................
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