Effects of Deer Antler Base Polypeptide on Hormone-induced Mammary Hyperplasia in Mice

Abstract

The objective of this study is to evaluate the effects and therapeutic mechanism of deer antler base polypeptide on mammary hyperplasia in female mice. A trail was conducted on seventy mice which were divided into seven groups including the groups of control, mock, Chinese traditional medicine, and Western medicine, as well as high-dose, medium-dose, and low-dose of polypeptide from deer antler. A combination of oestrogen-progestin induced the mammary hyperplasia. We used an intraperitoneal injection in a 30 d trial and used intragastric administration to treat the disease for a further four weeks. The body weight, organ coefficient, serum hormone level and oxidation level, as well as the pathological section of breast tissue were assessed. Statistical analysis was performed using factor analysis of variance. Compared with the mock group, the high-dose group could increase the serum T-SOD, and GSH-PX activities and thymus coefficient (P < 0.01), decrease the serum MDA concentration and the coefficient of ovary and uterus (P < 0.01). In addition, the high-dose group had reduced levels of E₂, PRL, and improved P, and LH as confirmed by ELISA to regulate hormone secretion to normal (P < 0.01). The pathological section of breast tissue produced by HE staining showed that the high-dose group reduced the number of inflammatory cells, glandular secretion, the hyperplasia and dilatation of the duct epithelium, and recovered the atrophic alveolar. The results revealed that deer antler base polypeptide has positive effects in the treatment of HMG and the prevention of breast cancer.

Introduction

Deer antler base is a bone tissue which ossifies in the following year and fracture in male Sika Deer (Cervus nippon Temminch) or Red Deer (Cervus elaphus Linnaeus) where it is removed by sawing off the antlers (Zhang et al., 2006; Jiang et al., 2014). It is a type of deer bone which is an edible medicine and its efficacy is closed to some other antler types (Wu et al., 2013). Deer antler base has been recorded in the Chinese medical classical Shen Nong Ben Cao Jing some 2000 years ago and is believed to nourish liver, regulate the kidney, invigorate the spleen, strengthen bones and muscles, and promote blood flow. Deer antler base contains protein, polypeptide, amino acids, polysaccharides, cholesterol, phosphatidylcholine, etc., and contains a lot of calcium, phosphorus, zinc, iron, manganese, copper, and other trace elements, and is rich in protein substances as its the main pharmaceutical ingredients. Modern medical research results proved that deer antler base improves immunity, offers anti-inflammatory and anti-fatigue benefits, and exerts a significant influence on treatment of early breast carbuncle (Zha et al., 2013). Protein polypeptide obtained from hydrolysing deer antler base by signal enzyme could promote phagocyte function, increase the efficacy of T lymphocyte, and enhance immunity (Wang et al., 2008). The synergistic effects of deer antler base polypeptide on a good anti-fatigue function arising through enhancing the adrenal function and increasing the content of hepatic glycogen has been proved (Shi et al., 2011). In recent years, the treatment of deer antler base on HMG has gradually increased.

Hyperplasia of the mammary gland (HMG) is one of most common benign diseases of the breast in middle-aged women (Marchant and Douglas, 2002). Clinical research showed that the mammary epithelium and mesenchyma cells undergo hyperplasia caused by ovarian malfunction leading to hormone disorders, hyperstimulation, and the augmentation of water and sodium stasis in the connective tissue resulting in lobular oedema as the primary causes of the disease. It is observed that there are several morphological types of hyperplasic lesions in the mouse mammary gland including the hyperplasitic alveolar nodules and ductal hyperplasias, which is considered as a preneoplastic lesion (Medina, 2002). Thus, it tends to have carcinogenic effects, although it is not an inflammation or cancer (Wang et al., 2011). It was noted that within a few years the incidence of illness had increased quickly, ranking first among mastopathies (Das et al., 2015). The curative medicines used are currently drugs such as Tamoxifen and Chinese traditional medicine preparations. The effectiveness of drugs could reach 90%, but there were too many side effects and hidden dangers, such as menstrual disorders and uterine bleeding (Henry et al., 2014). However, Chinese traditional medicine has the advantages of lasting effects and fewer side effects. So this study is important in that it tries to block the development of HMG to cancer.

In the current study, deer antler base polypeptide extracted by double enzymolysis in a laboratory was used on HMG model mice induced by exogenous hormone dosage. Then mice arranged in different groups were separately treated with low, medium, and high doses of polypeptide, and the positive control group was used as a basis for comparison. The treatment effects were determined by evaluating mice body mass, E2, P, PRL, LH, T-SOD, MDA, GSH-PX content in serum, organ index, and breast tissue pathological section in HE stain. The anti-oxidant, anti-inflammatory and the regulation of hormones therewith should be analysed as essential to understanding possible mechanisms of action of deer antler base polypeptide on HMG.

Materials and Methods

Ethics statement    This experiment was approved by the Animal Ethics Committee of Liaoning (Permit No.: 201331004-2). All procedures related to the mice and their care conformed to internationally accepted principles in the Guidelines for Keeping Experimental Animals issued by the Government of China.

Materials    Deer antler base stemming from the first year of Male Sika Deer antler shedding, in powder form, were provided by Liaoning Xifeng deer farm, polypeptide (011523) appeared as a pale yellow powder after laboratory extraction, Tamoxifen (TMXF) citrate was purchased from Yangtze River Pharmaceutical Group Co., Ltd, (Jiangsu, China), Rupixiao (RPX) were purchased from Hongyao Pharmaceutical Co., Ltd, (Shenyang, China), estradiol benzoate injections, and progesterone injections, were purchased from Ningbo Second Hormone Factory, (Ningbo, China), All other chemicals used were of analytical grade.

Deer antler base powder hydrolysis procedure    Deer antler base powder was mixed by magnetic stirrer, the pH and temperature regulated, then enzymatic hydrolysis was undertaken then we killed the enzyme (in a 95°C water bath for 10 min). We centrifuged the samples at 4 500 rpm for 15 min, before collecting the supernatant and twice centrifuging it in pellet form. We then combined the supernatant with deer antler base polypeptide to form a pale yellow powder (coarse product) obtained through low-temperature cryodesiccation (the yield was about 5.1%) (Pi et al., 2015).

The deer antler base polypeptide content is 65.10% used in this experiment and contains l7 kinds of nitrogen acid, which contains seven kinds of essential amino acids, the results are illustrated in Table 1.

Table 1. Amino acid constitutes of deer antler base polypeptide

name	Asp	Thr	Ser	Glu	Gly	Ala	Cys
content (mg/g)	4.92	6.269	4.934	9.135	4.88	10.217	1.065
name	Ile	Leu	Tyr	Phe	Lys	His	Arg
content (mg/g)	6.484	21.716	4.052	17.138	8.879	3.097	11.195

Animals    Female non-pregnant Kunming white mice (7–8 weeks old) weighing about 22–26 g were purchased from the Experimental Animal Resources Centre of Liaoning Province. The animals were maintained under standard conditions of humidity, temperature (25 ± 2°C) and light (12 h light/12 h dark). They were fed a standard rat pelleted diet and had free access to water. The animal experiments were conducted according to the guidelines for the care and use of laboratory animals in our University (Yang et al., 2015).

Experimental design

Preparation of models    72 mice were fasted before the experiment and for 16 h afterwards in appropriate conditions. 10 of them were injected with 0.2 mL physiological saline once per day for 30 d as a control group. Under the same conditions, others were set-up as the HMG model and injected abdominally with estradiol benzoate to 0.5 mg/kg (body mass), once per day for 25 d, then changed to a regime injecting progesterone 4 mg/kg (body mass) once daily for 5 d. During this period, redness gradually appeared on the mice nipple, and a portion of the areola. After 30 d of intraperitioneal injections, we selected one mouse at random. Breast tissues stained in HE were prepared by paraffin-section method to confirm the success, or otherwise, of the treatment (Yu et al., 2005).

Preparation of experimental mouse    The mice of control group received equal amounts of 0.5 mL physiological saline once per d. The successful preparation of the model mice were divided randomly into six groups, with each group consisting of ten animals. Every mouse received equal amounts of 0.5 mL physiological saline once per day as the mock group. According to the body surface ratio and dose conversion between experimental animals and humans,group I animals were pre-treated with 200 mg/(kgod) RPX as the positive Chinese medicine group,and group II animals were pre-treated with 3.9 mg mg/(kg•d) TMXF as the positive western medicine group. Group III-V animals were treated separately with deer antler base polypeptide 75 mg/(kg•d), 125 mg/(kg•d), and 200 mg/mg/(kg•d) for four consecutive weeks from day 2 onwards. The experimental period extended for eight weeks with a total of seven groups. These experiments were performed in accordance with government and international standards.

Detection of mice body mass    The seven groups of animals were weighed with an electronic balance after the end of the drug treatment, and the data recorded.

Detection of serum T-SOD, GSH-PX, and MDA contents    At the end of the first 24 h, seven groups of blood samples (0.2 mL per mouse) in each group were collected and allowed to clot at 4°C for 20 minutes; serum was separated by centrifugation and stored at −20°C. The levels of T-SOD, MDA, and GSH-PX in the blood were detected using commercially available kits operated according to the manufacturer's instructions. (Nanjing, Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China) and the results expressed as U/mL, nmol/mL, and µmol/L.

Detection of serum E₂, P, PRL, and LH concentrations    At the end of the first 24 h, seven blood samples (0.2 mL per mouse) in each group were collected and allowed to clot at 4°C for 20 min, serum was separated by centrifugation and stored at −20°C. The levels of estradiol (E₂), progesterone (P), prolactin (PRL), and luteinising hormone (LH) in the blood were evaluated using E₂, P, PRL, and LH ELISA kits (R&D Systems Co., Ltd, China) according to manufacturers' instructions: the results were expressed as pg/mL, pmol/L, pg/mL, and pg/mL.

Detection of organ coefficient    Organs examined included thymus spleen, thymus, ovary, and uterus that had been taken to remove the fat tissues after weighing upon sacrificing the animals. We calculated the organ coefficient as follows:   

Detection of breast tissue pathological section    The second full breast on the chest had been cut to the maximum depth of its basal part. We allowed a 4% formaldehyde solution to settle and observed the breast tissue morphology in paraffin-embedded tissues stained by HE under a visible-light microscope.

Statistical analysis    SPSS17.0 software (SPSS Inc., Chicago, USA) was used for statistical evaluation, and data were expressed as mean ± standard deviation. Factor analysis of variance was used to determine the differences among groups. A P value < 0.05 was considered statistically significant (Altman and Bland, 1995).

Results and Disscussion

Changes in body mass and organ coefficient    The body weight and organ coefficient levels accurately reflect the state of immunity and the changes in the ovary and uterus could indicate the effect of endometrial growth (Liu et al., 2011). The results are illustrated in Table 2. We observed that, compared with the control group, the thymus coefficient, spleen coefficient and body mass had been reduced significantly, while the ovary and uterus quality had increased in the mock group (^aP < 0.01). The thymus coefficient and body mass of the medium, and high-dose groups were significantly higher than those in mock group after treatment (^eP < 0.01); furthermore the TMXF and RPX groups had a significant difference only in spleen coefficient (^eP < 0.01); in all treated groups, the body mass significantly increased (^eP < 0.01), but the body mass decreased slightly in the high-dose group. The RPX group had a significant difference only in ovary coefficient (^eP < 0.01), which indicated the effects were unsuitable; however the uterus coefficient in the high-dose group could reach a normal level. The mass of the mice in deer antler base polypeptide groups, compared to the control group, showed no significant differences (P > 0.05). The effects of inhibited weight were thus indicated. In conclusion, the results revealed that the high doses of the deer antler base group had the best effect when treating HMG.

Table 2. Changes of organ coefficient and body mass (g) in control and experimental mice.

Groups	Thymus coefficient	Spleen coefficient	Uterus coefficient	Ovary coefficient	Body mass
Mock	0.053±0.009a	0.320±0.025a	0.565±0.052a	0.068±0.006a	32.30±1.18a
Control	0.115±0.044	0.408±0.032	0.407±0.043	0.048±0.005	34.61±0.27
Test group-I	0.098±0.006f	0.381±0.044e	0.506±0.066f	0.047±0.006e	35.16±1.29e
Test group-II	0.091±0.009	0.407±0.046e	0.417±0.051e	0.043±0.004e	35.57±1.29e
Test group-III	0.113±0.016e	0.372±0.026f	0.540±0.096a	0.054±0.010e	35.97±1.19e
Test group-IV	0.123±0.048e	0.409±0.071e	0.488±0.052e	0.046±0.012e	34.81±0.84e
Test group-V	0.153±0.004e	0.425±0.057e	0.403±0.086e	0.042±0.008e	33.92±1.29e

Notes: Each value is expressed as mean ± SD for ten mice in each group.

Test group-I, RPX group; Test group-II, TMXF group; Test group-III, low dose of deer antler base polypeptide; Test group-IV, medium dose of deer antler base polypeptide; Test group-V, high dose of deer antler base polypeptide.

Statistical differences: ^aP < 0.01, ^bP < 0.05, ^eP < 0.01, ^fP < 0.05 and NS - not significant.

a, b as compared with Control group; e, f as compared with Mock group.

Changes in serum T-SOD, GSH-PX, and MDA    T-SOD and GSH-PX in serum are two enzymes reflecting the ability of the anti-oxide free radical. MDA is one of the most important products of membrane lipid peroxidation and showed the extent to which cells are attacked by the radical (Chen et al., 2015). The serum concentrations: T-SOD, GSH-PX, and MDA are presented in Fig. 1. The serum T-SOD and GSH-PX in the mock group decreased and the MDA level increased significantly compared with that in the control group (^aP < 0.01). The results showed that all treated groups had higher T-SOD and GSH-PX counts than those of the mock group (P < 0.05), indicating the cells anti-oxidant response to the body. Among all groups, the counts in TMXF, and the high-dose group were significantly higher than those in the mock group (^eP < 0.01), but showed no difference from the control group (P > 0.05). The results indicated that the TMXF, and high-dose groups had the best ability to generate anti-oxidant free radicals. Except for RPX and the low-dose group, all treated groups had lower MDA counts than those in the mock group (^eP < 0.01), but the high-dose group data were closest to the control group data. In conclusion, the results suggested that the high doses given to the deer antler base group could improve their antioxidant capacity, reducing the degree of membrane lipid peroxidation.

Fig. 1.

Changes of serum T-SOD, GSH-PX, MDA levels in control and experimental mice.

Notes: Each value is expressed as mean ± SD for ten mice in each group.

Test group-I, the RPX group; Test group-II, the TMXF group; Test group-III, low dose of deer antler base polypeptide; Test group-IV, medium dose of deer antler base polypeptide; Test group-V, high dose of deer antler base polypeptide.

Statistical significance: ^aP < 0.01, ^bP < 0.05, ^eP < 0.01, ^fP < 0.05 and NS - not significant. a, b as compared with Control group; e, f as compared with Mock group.

Changes in serum E₂, P, PRL, and LH    The serum E₂, P, PRL, and LH, reflect the merisis of the body cells and tissues, which is mainly relevant to the cause of mammary hyperplasia (Liu et al. 2012; Mol and Lantinga 1999). All the serum hormone concentration changes in each group are illustrated in Fig. 2. In the mock group, the E₂ and PRL concentrations were higher than those in the control group, and the P and LH concentrations were lower (^aP < 0.01). The P and LH concentrations in all treated groups showed higher levels than those in the mock group throughout the trial. However, the P and LH concentrations had significant differences from the control group (^bP < 0.05) and the TMXF, and high-dose groups showed significant differences compared with the mock group (^eP < 0.01), and no difference to the control group, thus evincing efficacious treatment. Compared with the TMXF group, the high-dose group showed better effects and was closer to the normal level thus indicating that its treatment effects were optimal. The E₂ and PRL concentrations in all treated groups decreased significantly, but had significant differences compared with the control group (^aP < 0.01), apart from in the high-dose group (P > 0.05) while it differed from the mock group (^eP < 0.01). In conclusion, the results indicated that a high dose could induce the best effect on regulating the hormone level.

Fig. 2.

Changes of serum E₂, P, PRL, and LH content in control and experimental mice.

Notes: Each value is expressed as mean ± SD for ten mice in each group.

Test group-I, the RPX group; Test group-II, the TMXF group; Test group-III, low dose of deer antler base polypeptide; Test group-IV, medium dose of deer antler base polypeptide; Test group-V, high dose of deer antler base polypeptide.

Statistical significance: ^aP < 0.01, ^bP < 0.05, ^eP < 0.01, ^fP < 0.05 and NS - not significant. a, b as compared with Control group; e, f as compared with Mock group.

Observation of breast tissue pathological sections    The breast tissue pathological section can reflect the changes of the structure and morphology of the body directly (Yang, 2006). All results were presented in Fig. s 3. In the control group, via microscopy, the mice mammary tissue lobule had no proliferation and expansion, and no hyperplasia and atrophy of the glandular duct epithelium. There were no pathological changes of congestion and inflammatory cell invasion. There were a few secretions in the alveolar region, and the cell was healthy (Fig. 3A). In the mock group, there were some dilated cells and powder-dye secretion of the gland duct lumen, hyperplasia of the glandular duct epithelium, atrophy of the alveolar epithelium, and a inflammatory cell invasion seen in the interstitial areas with reduced connective tissue. HMG had been proved to be successful (Fig. 3B). All treated groups were better than the mock group. Among the two control groups, the RPX had less inflammatory cell invasion and oedema was found in the interstitial area. The alveolar was normal (Fig. 3C). The number of atrophic alveolar cells that had been found in the TMXF group was less than that in the RPX group, but more secretions in the lumen of the gland occurred than in the mock group (Fig. 3D). In all deer antler based-groups, a noticeable effect was not found only in the low-dose group with dilated ducts, a few powder dye secretions in the gland duct lumen, and a little inflammatory cell invasion in the interstitial region compared to the mock group (Fig. 3E). The breast tissues of the medium-, and high-dose groups returned to normal, although there was a little atrophic alveolar in the medium-dose group, the alveolar epithelial and lobule had no difference (Fig. 3F). In the high-dose group, the alveolar and gland duct had no abnormalities, there was a few powder dye secretions in alveolar areas and no inflammation, which was the closest to the control group's pathological features (Fig. 3G).

Fig. 3.

Observation of mice breast tissue pathology in different group

Breast tissue is the target organ of the gonadal hormone, and its physiological and pathological changes are closely related to the regulation of hormones (Wang et al., 2008). The competition of hormone receptors on the target organs induce disorders of the hypothalamus-adenohypophytic system and destroy the balance of breast tissue proliferation and involution if the serum oestrogen is present in excessive amounts (Marinelli et al., 2004). Therefore, reducing the sensitivity of breast tissue to hormones and taking the anti-oestrogen therapy are important treatments. The active ingredient of Chinese herbal medicine was chosen for its certain advantages, such as low, or no side effects (Liu et al., 2012). Here, we investigated the therapeutic effect of different doses of polypeptide of deer antler base on mice of mammary hyperplasia. The results indicated that the high-dose group possessed optimal immune-enhancing activity and hormone regulation ability compared with all other positive groups, indicating the synergetic effect in treating HMG.

Growing evidence has shown that the oxy-radical produced by the enzyme system and non-enzyme system could induce cell damage and lead to cell metabolism and function disorder or even death through lipid peroxidation decomposition products. In recent years, the study found that the oxy-radical increased in patients with mammary hyperplasia, therefore, the activity of T-SOD and GSH-PX are used to measure the ability to remove oxy-radicals and assess anti-oxidant recovery performance (Karki et al., 2014). In this experiment, the activity of serum GSH-PX was measured as it could protect the cell membrane structure and function without interference and damage by peroxide. Judging from our results, high doses made the serum MDA content decrease after treatment, and the GSH-PX activity increased, indicating that the body peroxide levels had fallen and the state of cells attacked by radicals had improved. Rising T-SOD activity enhanced the ability to remove oxy-radicals from the body; however, the activity of T-SOD in high-dose groups was higher than in the control group. The result is consistent with the previous studies of Liu (Liu et al., 2013). It was related to the function of deer antler base polypeptide in enhancing the ability of antioxidants (Huang et al., 2013; Liu et al., 2010; Li et al., 2008). The results indicated that deer antler base polypeptide catalysed the oxy-radical's disproportionate reaction specifically through enhancing T-SOD activity to scavenge the peroxide free radical associated with inflammatory processes, resulting in a strong anti-inflammatory effect to repair damaged mammary gland cells timeously. The result showed that the coefficient of the thymus increased and the coefficients for the uterus and ovary decreased. It proved that the immunity of the organism had improved, the inflammation was relieved and endometrial growth had recovered to normal. The inhibitory effect of deer antler base polypeptide on mice body mass might have been due to the fact that the protein contains many kinds of bioactive factors affecting bidirectional regulation of cell proliferation, with a biphasic dose effect, and also associated with double molecular weight of non-regulatory proteins. The two-way coordination explains why deer antler base polypeptide could both promote animal growth and development and inhibit the growth of animals (this is consistent with the report of Ma Guangli et al.) (Ma and Wang, 2010).

When PRL in serum had increased abnormally, it would directly stimulate the breast tissue, or promote the synthesis of E₂ and inhibit the secretion of P in luteal phase further inducing excessive breast tissue proliferation. The P in serum could resist the abnormal proliferation of target tissue and protect the structure of target organs (Cheskis, 2004). In synergy with follicle oestrogen, LH was regulated by luteinizing hormone release in the hypothalamus which stimulated the secretion of ovarian oestrogen (Wang et al., 1976). Therefore, the levels of E₂, P, PRL, and LH were an important indicator able to measure the secretion of pituitary hormones. Judging from our results, the high-dose group of deer antler base polypeptide regulated the secretion of oestrogen and progesterone through decreased content of E₂ and PRL and increased that of P and LH to make the alveolar resume normal behaviour, meanwhile, abating the continuous stimulation of oestrogen and proliferation of mammary epithelial cells and interstitial cells, reducing connective tissue water-sodium retention and relieving oedema.

We observed pathological tissue sections by microscopy: inflammatory cell invasion had improved, the alveolar and duct cavity areas returned to normal. Anti-oestrogen therapy reduced the sensitive of mammary tissue to abnormal stimulation, protected the structure and function of mammary tissue, and the pharmacological effects were positively correlated with dose. The morphology of the high-dose group was close to that of the control group, and had the best effect.

The therapeutic effect of deer antler base polypeptide on HMG is desirable. The reason for it may be related to its enhancing the phototrophic function of phagocytes. Compared with other drugs, through the coordination of a variety of physiological functions, and cross-complementation, deer antler base polypeptide could promote blood flow combined with anti-inflammatory and analgesic actions thus making further research worthwhile (Wang and Yao, 2012).

Conclusions

The results revealed that deer antler base polypeptide could effectively regulate the ovarian endocrine system, enhance the immune system, and improve the morphological changes of hyperplasia of the mammary gland (HMG) induced by hormones. Deer antler base polypeptide has positive effects on the treatment of HMG and the prevention of breast cancer.

Acknowledgements    This research was performed under the auspices of the Food Science College and Animal Experimental Centre of Shenyang Agricultural University.

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