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Abstract

OBJECTIVE:

Selfheal has been used for many years in hyperprolactinemia induced galactorrhea, menstrual disorders, and dysgenesis with satisfactory curative effect. However, its mechanism is still unclear. This study intended to investigate the effect of selfheal extract on hyperprolactinemia in vivo and in vitro, in order to elucidate its mechanism of anti-hyperprolactinemia.

PATIENTS AND METHODS:

Hyperprolactinemia rat model was established. High dose (28.8 g/(kg $\cdot$ d)), middle dose (14.4 g/ (kg $\cdot$ d)), and low dose (7.2 g/(kg $\cdot$ d)) of selfheal extract were used to treat the model to observe impact on serum estradiol (E2), progesterone (P), prolactin (PRL), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) levels. Three cell lines MMQ, GH3, and PC12 were applied to investigate selfheal extract effect on PRL secretion, dopamine D2 receptor, and dopamine transporter (DAT).

RESULTS:

High and middle dose of selfheal extract significantly reduced PRL level in hyperprolactinemia rat compared with model group ( $P<$ 0.01). Compared with normal control, 5 mg/ml and 10 mg/ml selfheal extract obviously inhibited PRL secretion in MMQ cells that high expressed D2 receptor after 24 hours ( $P<$ 0.01), but did not affect PRL secretion in GH3 cells lack of D2 receptor. 8 mg/ml selfheal extract markedly suppressed D2 receptor and DAT expression in PC12 cells that strongly expressed D2 receptor and DAT ( $P<$ 0.01).

CONCLUSIONS:

Selfheal extract treated hyperprolactinemia through dopamine D2 receptor with significant effect.

Keywords

Selfheal extract hyperprolactinemia PRL dopamine D2 receptor DAT

1. Introduction

Hyperprolactinemia refers to peripheral blood prolactin abnormal elevation (PRL $>$ 25 ng/ml) caused by various reasons. It is featured as scanty menstruation, amenorrhea, galactorrhea, infertility, and habitual abortion [1]. Hyperprolactinemia incidence was 0.4%. As a type of disease seriously impaired women’s physical and mental health, its incidence in women with dysgenesis reached 9–17% [2, 3]. Generally, hyperprolactinemia can be induced by hypothalamus – pituitary lesion, pituitary tumor, paspertin, chlorpromazine, cimetidine, or hypothalamus – pituitary dysfunction. Among them, pituitary prolactinoma was a common cause of hyperprolactinemia [4].

At present, bromocriptine, cabergoline, and pergolide were mainly used for hyperprolactinemia treatment in clinic. They were all dopamine D2 receptor agonist that can inhibit PRL secretion, restore menstruation and fertility, and thus were widely used to treat HPRL with remarkable effect. However, these drugs were expensive and with certain side effects. Bromocriptine can decline PRL level significantly, and also can restrain tumor size and restore gonad function. But it also had side effects, such as dizziness, orthostatic hypotension, and gastrointestinal tract reaction. Specially, about 12% of the patients can’t tolerate its effective treatment quantity [5, 6]. Traditional Chinese medicine had definite therapeutic effect in the treatment of hyperprolactinemia. Selfheal has been used for many years in hyperprolactinemia induced galactorrhea, menstrual disorders, and dysgenesis with satisfactory curative effect. It was reported that selfheal compound preparation can significantly reduce the high PRL level in hyperprolactinemia and regulate endocrine disorder [7]. However, there was still lack of experimental research about selfheal treating hyperprolactinemia. MMQ cell line was derived from rat pituitary adenoma cells with large amount of dopamine D2 receptor expression; GH3 cell line was from rat pituitary prolactinoma cells lack of dopamine D2 receptor expression; PC12 cells were from rat pheochromocytoma PC12 cells highly expressed dopamine D2 receptor and dopamine transporter (DAT). Since they can be used to observe whether through dopamine D2 receptor pathway to inhibit PRL secretion, these three cells were often used to study hyperprolactinemia as in vitro cell model. This study intended to investigate the effect of selfheal extract on hyperprolactinemia in vivo and in vitro, in order to elucidate its mechanism of anti-hyperprolactinemia.

2. Materials and methods

2.1 Animals

Sixty adult female SD rats at 8 weeks and 180 $\pm$ 20 g were provided by Weifang Medical College (laboratory animal production license: SYXK 2002–0012). Animal qualified number 0064357, animal experimental facility occupancy permit SYXK 2014-0028. Experimental animals were fed in the standard SPF environment with normal lighting, noise, temperature, and ventilation.

Rats were used for all experiments, and all procedures were approved by the Animal Ethics Committee of Weifang People’s Hospital.

2.2 Drugs and reagents

Selfheal medicinal materials were provided by the Chinese herbal medicine co., LTD. and identified by Professor Chen from the Hubei University of Chinese Medicine. Medicinal materials species and quality conformed to the stipulation of “Chinese pharmacopoeia” version 2010. Selfheal extract was prepared according to the following method: selfheal medicinal material was soaked in eight times amount of water for 1 h after cleaning. It was boiled in high heat for 30 min, and then boiled in slow fire for 45 min. The liquid was collected after filtration. Then the dregs were boiled again as before and the liquid got in two times were concentrated to 3.38 g/mL.

ELISA kits for estradiol, progesterone, prolactin, follicle stimulating hormone, and luteinizing hormone were got from Boster (batch number: 201305647). Methanesulfonic acid was from Gedeon Richter Plc (batch number: 10076). Metoclopramide injection was purchased from Wuxi 7th pharmaceutical co., LTD. (batch number: 1006125). Western blot detection kit was from Shanghai Xin Yu Biotech co., LTD. (batch number: 20140506).

2.3 Modeling

50 mg/kg metoclopramide injection was subcutaneous injected to the back of the rat twice a day for seven consecutive days [8].

Table 1
Selfheal extract impact on hormone level in hyperprolactinemia rat ( $\bar{x}$ $\pm$ s, $n=$ 10)

Group	PRL (pg/mL)		E ${}_{2}$ (pmol/L)		P (ng/mL)		FSH (IU/L)		LH (mIU/mL)
Normal	203.48	$\pm$ 7.614	3.602	$\pm$ 0.35	1.225	$\pm$ 0.07	0.968	$\pm$ 0.07	1.749	$\pm$ 0.12
Model	429.25	$\pm$ 32.48 ${}^{2)}$	1. 415	$\pm$ 0.16 ${}^{2)}$	0.549	$\pm$ 0.18 ${}^{2)}$	0.359	$\pm$ 0.04 ${}^{2)}$	0.954	$\pm$ 0.05 ${}^{1)}$
Bromocriptine	218.45	$\pm$ 12.3 ${}^{4)}$	2.649	$\pm$ 0.24 ${}^{4)}$	0.743	$\pm$ 0.04 ${}^{3)}$	0.727	$\pm$ 0.03 ${}^{3)}$	1.633	$\pm$ 0.06 ${}^{3)}$
High dose of selfheal	221.33	$\pm$ 12.4 ${}^{4)}$	2.673	$\pm$ 0.18 ${}^{4)}$	0.892	$\pm$ 0.06 ${}^{3)5)}$	0.645	$\pm$ 0.03 ${}^{3)}$	1.727	$\pm$ 0.24 ${}^{3)5)}$
Middle dose of selfheal	247.46	$\pm$ 14.8 ${}^{4)}$	2.428	$\pm$ 0.41 ${}^{3)}$	0.824	$\pm$ 0.05 ${}^{3)}$	0.694	$\pm$ 0.03 ${}^{3)}$	1.61	$\pm$ 0.05 ${}^{3)}$
Low dose of selfheal	331.37	$\pm$ 44.31	1.754	$\pm$ 0.52	0.552	$\pm$ 0.13	0.527	$\pm$ 0.08	1.031	$\pm$ 0.13

${}^{1)}P<$ 0.05, ${}^{2)}P<$ 0.01, compared with normal control; ${}^{3)}P<$ 0.05, ${}^{4)}P<$ 0.01, compared with model group; ${}^{5)}P<$ 0.05, ${}^{6)}P<$ 0.01, compared with positive group.

2.4 Grouping and administration

Sixty rats were randomly equally divided into 6 groups, including normal control, model group, positive drug bromocriptine group, low, middle and high dose of selfheal extract groups. All the rats were prepared as hyperprolactinemia model except normal control. Dosage of administration was calculated according to body surface area [9]. Distilled water was gave to the stomach in normal control and model group. Selfheal extract high, middle and low dose were gave at 28.8 g/ (kg $\cdot$ d) (10 times of clinical dose), 14.4 g/ (kg $\cdot$ d) (5 times of clinical dose), and 7.2 g/(kg $\cdot$ d) (2.5 times of clinical dose). Bromocriptine was gave at 0.421 mg/(kg $\cdot$ d). 2 ml drugs or distilled water was gave once a day for 30 consecutive days.

2.5 Cell culture

MMQ, GH3, and PC12 cell lines derived from rat were cultured in F12 medium containing 5% fetal bovine serum and 12.5% horse serum and maintained in 37 ${{}^{\circ}}$ C, saturated humidity, 5% CO ${}_{2}$ . The cells were passaged every 4 days and changed medium every other day.

4.0 $\times$ 10 ${}^{6}$ /ml MMQ cell suspension in logarithmic phase were seeded in 96-well plate at 200 $\mu$ l/well. After 24 h, 1, 2, 4, 5, 8, and 10 mg/ml selfheal extracts together with 35 $\mu$ l medium were added to the well, respectively. After 12 h, 24 h, and 48 h incubation, four wells were selected to detect PRL ELISA and Western blot with four repetitions. Also, after 1, 2, 4, 5, 8, and 10 mg/ml selfheal extracts acted on GH3 and PC12 cells for 24 hours, GH3 cells culture medium was collected to determine PRL secretion and synthesis, and PC12 cells were collected to test dopamine D2 receptor and dopamine transporter (DAT) expression.

2.6 Sample detection

2.6.1 Rat serum hormone detection

After 30 days, the blood was extracted from the eyeball to detect rat serum prolactin, estradiol, progesterone, luteinizing hormone, and follicle-stimulating hormone levels by ELISA. PRL content in MMQ and GH3 cells were also tested by ELISA according to the manual (Boster).

2.6.2 Western blot

Western blot was applied to test PRL protein expression in MMQ and GH3 cells, and D2 receptor and DAT protein levels in PC12 cells.

2.6.3 Statistical analysis

ANOVA and Student-Newman-Keuls method were used for data comparison. The data was presented as $\overline{X}\pm SD$ . $P<$ 0.05 was considered with statistical difference.

3. Results

3.1 Selfheal extract impact on hormone level in hyperprolactinemia rat

PRL level significantly increased ( $P<$ 0.01), while serum $E_{2}$ , P, FSH, and LH level obviously declined ( $P<$ 0.05) in hyperprolactinemia rat compared with normal control. High and middle doses of selfheal extract significantly reduced PRL level ( $P<$ 0.01), while markedly elevated serum $E_{2}$ , P, FSH, and LH level in different extent ( $P<$ 0.05). Its effect was similar with bromocriptine (Table 1).

Table 2
Selfheal extract impact on PRL secretion in MMQ cells at different time point

Selfheal extract (mg/ml)		Drug time (hr)
	12	24	36	48
0	1.68 $\pm$ 0.01	1.82 $\pm$ 0.01	1.76 $\pm$ 0.02	1.43 $\pm$ 0.01
1	1.69 $\pm$ 0.01	1.64 $\pm$ 0.03	1.60 $\pm$ 0.01	1.41 $\pm$ 0.01
5	1.72 $\pm$ 0.02	1.57 $\pm$ 0.02 ${}^{2)}$	1.53 $\pm$ 0.01 ${}^{1)}$	1.39 $\pm$ 0.02
10	1.75 $\pm$ 0.01	1.41 $\pm$ 0.01 ${}^{2)}$	1.48 $\pm$ 0.03 ${}^{2)}$	1.45 $\pm$ 0.01

${}^{1)}P<$ 0.05 , ${}^{2)}P<$ 0.01, compared with 0 mg/ml group.

Figure 1.

Western blot detection of selfheal extract impact on PRL expression in MMQ cells after 24 h.

3.2 Selfheal extract impact on PRL secretion in MMQ and GH3 cells

Compared with normal control, 5 mg/ml and 10 mg/ ml selfheal extract obviously inhibited PRL secretion in MMQ cells after 24 and 36 h ( $P<$ 0.005). Western blot showed that 4 mg/ml and 8 mg/ml selfheal extract markedly suppressed PRL expression in MMQ cells ( $P<$ 0.01) (Fig. 1, Tables 2 and 3). However, different doses of selfheal extract presented no significant impact on PRL secretion and expression in GH3 cells (Fig. 2, Table 4).

Table 3
Selfheal extract impact on PRL expression in MMQ cells after 24 h

Selfheal extract (mg/ml)	0	1	2	4	8
PRL expression	0.85 $\pm$ 0.04	0.79 $\pm$ 0.09	0.76 $\pm$ 0.08	0.39 $\pm$ 0.04 ${}^{2)}$	0.28 $\pm$ 0.03 ${}^{2)}$

${}^{1)}P<$ 0.05 , ${}^{2)}P<$ 0.01, compared with 0 mg/ml group.

Figure 2.

Western blot detection of selfheal extract impact on PRL expression in GH3 cells after 24 h.

3.3 Selfheal extract impact on dopamine expression in PC12 cells

It was showed that after 24 h treatment, 4 mg/ml and 8 mg/ml selfheal extract markedly increased dopamine D2 receptor and DAT expression in PC12 cells ( $P<$ 0.05 or $P<$ 0.01) (Figs 3 and 4, Tables 5 and 6).

Table 4
Selfheal extract impact on PRL expression in GH3 cells after 24 h

Selfheal extract (mg/ml)	0	2	4	8
PRL secretion (ng/ml)	1.42 $\pm$ 0.04	1.44 $\pm$ 0.06	1.43 $\pm$ 0.07	1.41 $\pm$ 0.06
PRL expression	0.11 $\pm$ 0.01	0.12 $\pm$ 0.01	0.13 $\pm$ 0.02	0.10 $\pm$ 0.03

${}^{1)}P<$ 0.05, ${}^{2)}P<$ 0.01, compared with 0 mg/ml group.

Table 5

Selfheal extract impact on dopamine D2 receptor expression in PC12 cells

Selfheal extract (mg/ml)	0	2	4	8
Dopamine D2 receptor expression	0.14 $\pm$ 0.02	0.18 $\pm$ 0.01	0.20 $\pm$ 0.01 ${}^{1)}$	0.25 $\pm$ 0.01 ${}^{2)}$

${}^{1)}P<$ 0.05 , ${}^{2)}P<$ 0.01, compared with 0 mg/ml group.

Table 6

Selfheal extract impact on DAT expression in PC12 cells

Selfheal extract (mg/ml)	0	2	4	8
Dopamine D2 receptor expression	0.22 $\pm$ 0.01	0.24 $\pm$ 0.02	0.31 $\pm$ 0.01 ${}^{1)}$	0.34 $\pm$ 0.01 ${}^{2)}$

${}^{1)}P<$ 0.05 , ${}^{2)}P<$ 0.01, compared with 0 mg/ml group.

Figure 3.

Western blot detection of selfheal extract impact on dopamine D2 receptor expression in PC12 cells.

Figure 4.

Western blot detection of selfheal extract impact on DAT expression in PC12 cells.

4. Discussion

The malfunction of liver can lead to menstrual disorders and breast milk abnormal secretion. Hyperprolactinemia, also called “galactorrhea” in Chinese medicine, is mainly related to imbalance of Chong and Ren Channels, breast collaterals obstacle, or liver Qi stagnation. Selfheal can stimulate the menstrual flow and regulate Chong and Ren balance. Selfheal compound preparations can significantly inhibit high PRL level, thus improve ovarian function by regulating the hypothalamus – pituitary gonadal axis balance and recover estradiol level to cure hyperprolactinemia [7]. In this study, selfheal extract got from high-temperature decoction and filtration can effectively suppress exorbitant PRL and regulate hormone disorder to treat hyperprolactinemia.

PRL is a peptide hormone that is one of the hormones secreted by the pituitary gland. It is mainly regulated by pituitary portal system, and double controlled by PRL release inhibiting factor (PIF) and PRL releasing factor (PRF). In general, PRL release is restrained by neurotransmitter dopamine. PIF inhibitory regulation plays the dominant role which is mainly exerted by dopamine. It can suppress PRL secretion by combining with dopamine D2 receptor on the surface of prolactin cells [10, 11]. On the other hand, thyroid-stimulating hormone controls the PRL release stimulus signal and promotes PRL production. Therefore, in the hypothalamus – pituitary gonad axis, several factors may weaken the PIF inhibitory factor regulation, such as hypothalamus dopamine synthesis retardation, dopamine transmission, or dopamine receptor binding, leading to PRL secreted by hypothalamus greatly elevated and hyperprolactinemia occurrence [12, 13, 14, 15].

At present, bromocriptine, cabergoline, and pergolide were used to treat hyperprolactinemia in clinic. They were all dopamine D2 receptor agonist that can bind with dopamine D2 receptor on pituitary prolactin cells to produce similar effect with dopamine. It can decrease cAMP content in hypothalamus and inhibit protein kinase A (PKA) production to reduce the protein phosphorylation level in cells, thereby inhibiting PRL secretion and restoring patient’s menstruation and fertility [16, 17, 18]. This study used three cell lines MMQ, PC12, and GH3, to discuss the mechanism of selfheal extract in treating hyperprolactinemia. MMQ cell line was derived from rat pituitary adenoma cells with high levels of dopamine D2 receptor expression; GH3 cell line was from rat pituitary prolactinoma cells lack of dopamine D2 receptor expression; PC12 cells were from rat pheochromocytoma PC12 cells highly expressed dopamine D2 receptor and DAT. High and middle dose of selfheal extract can obviuosly inhibit PRL secretion in MMQ cells, but did not affect PRL secretion and expression in GH3 cells. Meanwhile, it can significantly enhance dopamine D2 receptor and DAT expression in PC12 cells, suggesting that selfheal extract suppress PRL secretion through dopamine D2 receptor. However, the limitation in our study still exists that the facilitating effect of selfheal extract on dopamine D2 receptors requires further elucidation. For instance, additional experiments in PC12 cell lines also include the validation of the promoting role of selfheal extract on the agonistic activity of dopamine D2 receptor by using propylbutyldopamine [19] and counteraction of selfheal extract towards dopamine D2 receptor antagonist, such as haloperidol [20].

Taken together, our study demonstrated that selfheal extract contributed to a therapeutic effect on hyperprolactinemia via promoting PRL secretion and increasing dopamine D2 receptor and DAT expressions.

Conflict of interest

None.

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The mechanism of selfheal extract in treating hyperprolactinemia

Abstract

OBJECTIVE:

PATIENTS AND METHODS:

RESULTS:

CONCLUSIONS:

Keywords

1. Introduction

2. Materials and methods

2.1 Animals

2.2 Drugs and reagents

2.3 Modeling

Table 1 Selfheal extract impact on hormone level in hyperprolactinemia rat ( x ¯ ± s, n = 10)

2.5 Cell culture

2.6 Sample detection

2.6.1 Rat serum hormone detection

2.6.2 Western blot

2.6.3 Statistical analysis

3. Results

3.1 Selfheal extract impact on hormone level in hyperprolactinemia rat

Table 2 Selfheal extract impact on PRL secretion in MMQ cells at different time point

Table 3 Selfheal extract impact on PRL expression in MMQ cells after 24 h

Table 4 Selfheal extract impact on PRL expression in GH3 cells after 24 h

Conflict of interest

References

Table 1
Selfheal extract impact on hormone level in hyperprolactinemia rat ( $\bar{x}$ $\pm$ s, $n=$ 10)

Table 2
Selfheal extract impact on PRL secretion in MMQ cells at different time point

Table 3
Selfheal extract impact on PRL expression in MMQ cells after 24 h

Table 4
Selfheal extract impact on PRL expression in GH3 cells after 24 h