Hyperplasia of mammary gland (HMG) is a pathologic hyperplasia of the mammary gland lobules induced by an imbalance of estrogen and progesterone [1]. It is related to menstrual cycle, lactation, occupation, sex hormone use, diet, and stress [2,3]. The prevalence of HMG has increased in recent years, which is notable since it is associated with cancer. Indeed, HMG may be confused with early breast cancer. A recent study reported that traditional Chinese medicine (TCM) can significantly attenuate endocrine dyscrasia and inhibit HMG development [4]. Radix bupleuri, also named Chaihu in Chinese, is a common TCM herbal drug used to regulate endocrine disorders [5].

In this study, we evaluated the effects of Radix bupleuri extract (RPE) in a rat model of HMG.

Material

Three batches of Radix bupleuri were collected from Bozhou City, Anhui Province of China in July 2014. Taxonomic identification was performed by Professor Gan Gu of Zhejiang Pharmaceutical College, China. A voucher specimen of herbarium (No. RBE 201405031) was deposited in the College of Pharmacy, Zhejiang Pharmaceutical College, China for future reference. Aqueous RBE was obtained by steeping the dried plant three times in water at 60 °C for 1 h each time before drying in an oven and freeze-drying the last extract obtained. The yield was 66.67 %; 1 g powder was roughly equivalent to 1.5 g crude sample.

Animals

Virgin female Wistar rats weighing 200–240 g and Kunming female mice (18–22 g) were provided by the Heilongjiang University of Traditional Chinese Medicine (certificate No. SYXK 2006-0004). The animals had free access to feed and water and were allowed to acclimatize for at least 1 week before the study. The animal experiment was approved by Animal Care and Use Committee of Heilongjiang University of Traditional Chinese Medicine (approval ref no. 20120907) and was carried out in compliance with the Directive 2010/63/EU on the handling of animals used for scientific purposes [6].

Animal groups

Rats were treated with estrogen (0.5 mg/kg) intramuscularly for 25 days, followed by progestogen (5 mg/kg) for another 5 days to induce HMG. The rats were randomly divided into 6 groups of 10 rats each: control (untreated); HMG; HMG + positive control (RPSJC 800 mg/kg); and three HMG + RBE groups (200, 400, or 800 mg/kg doses). From the 31st day, rats in the control and HMG groups received distilled water by gavage, those in the RPSJC group were treated with RPSJC, and the RBE groups received RBE. Treatments were given orally once daily for 4 weeks.

Cotton pellet-induced granuloma test

Sterile cotton pellets (10 mg) were subcutaneously implanted in the groin of anesthetized mice (18–22 g). Ten animals were used for every treatment. The animals received 200, 400, or 800 mg/kg of RBE; RPSJC (800 mg/kg); or saline (10 ml/kg). These treatments were given once a day through an oral cannula over 7 consecutive days. On the 8th day, the mice were sacrificed, and the cotton pellet removed, dried overnight at 60 °C, and weighed. The increase in weight was determined and used for further calculation.

HMG assessment

The nipple heights of all rats were measured after the treatment. Levels of serum estradiol (E2), progesterone (P), prolactin (PRL), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) were determined using enzyme-linked immunosorbent assay kits (Shenzhen Xin-Bo-Sheng Biological Technology Co Ltd, China). The uterus and ovary indices were calculated as organ weight divided by body weight.

Histopathologic assessment

Mammary gland tissues from each group were fixed in 10 % buffered formalin, embedded in paraffin, cut into 4-µm-thick sections, stained with hematoxylin-eosin (H-E) and Masson’s trichrome (M-T) and examined under a light microscope. HMG severity was based on four parameters (gland alveolus hyperplasia, lobules, vessel shape and thickness, and secretion).

Statistical analysis

Values are expressed as mean ± SEM. Multiple group comparisons were performed using one-way analysis of variance (ANOVA) followed by Dunnett’s test to detect differences. P < 0.05 was considered significant in all cases.

Cotton pellet-induced granuloma

RBE significantly and dose-dependently decreased the dried weight of the cotton pellet granuloma. The inhibitory values for 200, 400, and 800 mg/kg of RBE were 17.69, 21.82, and 27.38 %, respectively. Notably, RPSJC had a lower inhibitory effect than the 200 mg/kg RBE dose. These results suggest that RBE effectively suppressed granulomatous tissue formation during chronic inflammation ().

Effect of RBE on nipple height in rats

As shown in , rat nipple height was significantly decreased by both RPSJC (800 mg/kg) and RBE (800 mg/kg) compared with the HMG group (p < 0.01) ().

Effect of RBE on serum sex hormone levels in rats

After injection of estrogen and progestogen in HMG model rats, E2, PRL, and FSH levels significantly increased, while P and LH levels decreased. RBE (800 mg/kg) treatment significantly decreased E2 PRL, and FSH compared with the HMG group (all p < 0.01), while it significantly increased P and LH levels (p < 0.01) ().

Effect of RBE on uterus and ovary index in rats

The uterus index of HMG rats was significantly increased compared with untreated control animals (p < 0.01). This was significantly attenuated by both RPSJC (p < 0.01) and high-dose RBE (p < 0.05). The ovary index in the high-dose RBE group was not significantly different compared with untreated control animals ().

Effect of RBE on mammary gland morphology

Control rats did not have any evidence of histologic abnormalities or proliferative lesions; they also had fewer acinars and no mammary duct secretion (A and B). Conversely, HMG rats exhibited intense hyperplasia of the gland alveolus and lobule, secretion, vessel arborization, lymphoplasia, and plasma cells (C and D). At week 4, RPSJC-treated group rats showed significantly decreased hyperplasia, less secretion, and fewer lymphocytes (E and F). RBE-H (800 mg/kg) markedly alleviated the degree of HMG by limiting hyperplasia in the gland alveolus and lobules and decreasing secretion and lymphocytes (G and H).

There is evidence that chronic inflammation induced by persistent chemical, bacterial, or viral agents is a risk factor for cancer [7-10]. Inflammation is an important process required to fight microbial infections, heal wounds, and maintain tissue homeostasis, but it can also lead to cancer [11-13]. Several recent studies suggested that inflammation has an important role in all phases of tumor development, including tumor initiation, tumor promotion, invasion, metastatic dissemination, and immune system evasion [14]. Specifically, the development of breast cancer is related to inflammation. Epidemiological studies have revealed that non-steroidal anti-inflammatory drug use can decrease the risk of developing breast cancer [15,16].

The inflammatory granuloma is a characteristic feature of chronic inflammatory processes. Dried weight of pellets correlates with the amount of granulomatous tissue; therefore, the results of the present study show that RBE-H exerts strong anti-inflammatory activity in a mouse model of chronic inflammation.

In the present study, RBE effectively inhibited chronic proliferative inflammatory processes and in a rat model of HMG. The mechanism might involve regulating endocrine and immune functions to balance hormone levels and attenuate pathologic mammary gland proliferation.

RBE is a potentially useful treatment for HMG. Therefore, further studies are required to develop Radix bupleuri for clinical use.