Effect of Boschniakia Rossica Extract on BMP-5 and ActRIIA in Mice Model of Kidney-Yang Decient

Background Boschniakia rossica is a famous Chinese traditional drug and mainly distributed in Korea, Japan, Russia, and China. The dried whole grass of Boschniakia rossica is commonly used in traditional Chinese medicine to treat kidney deciency, yang deciency, waist and knee aches and constipation. In order to quantify the active ingredients in B. rossica chinensis and body weight, clinical features and serum SOD and MDA activities, cAMP, cGMP, and testosterone (T), follicle-stimulating growth hormone (FSH), mouse bone morphogenetic protein-5 (BMP-5), mouse activin receptor IIA (ActRIIA) were measured to elucidate the mechanism underlying its treatment of kidney yang deciency potential. Methods Extracts of B. rossica were evaluated by HPLC-MS analyses. Kidney-yang deciency syndrome was induced in mouse with hydrocortisone (10 mg/Kg, 15d) and treated with B. rossica extract. The animals were treated orally with 3 doses of B. rossica extract: 1000, 500 and 250 mg/Kg, for 15 days. Body weight and clinical characteristics were recorded daily. For biochemical analyses, the following tests were performed: superoxide dismutase (SOD) activity, malondialdehyde (MDA) activity, testosterone (T) content, follicle growth hormone (FSH) content, cyclic adenosine monophosphate (cAMP) content, mouse bone morphogenetic protein-5 (BMP-5) content and activin receptor IIA (ActRIIA) content. Results Our study found that all the mice except the blank group showed signicant systemic depletion caused by hydrocortisone. Low T content, low cAMP activity and high cGMP content were consistent with the physiological and biochemical indexes of kidney-yang deciency syndrome. After oral gavage, the pathological conditions were improved in both the Positive group and the B. rossica groups.


Background
Dried whole grass of B rossica was often used as a supplement or vitality drug to enhance renal function and sexual dysfunction in China [1]. The extract of B. rossica has a variety of pharmacological activities, including anti-oxidation, anti-in ammatory, anti-tumor, and improving animal memory [2][3]. This study was mainly to study the therapeutic effect of extract of B. rossica on mice with kidney yang de ciency syndrome.
Modern studies have shown that the pathogenesis of KYDS is mainly characterized by different degrees of dysfunction of the hypothalamic-pituitary-target gland axis (adrenal gland, thyroid gland and gonad), which is characterized by diarrhea, cold limbs, decreased mobility, unresponsiveness, loss of appetite, Weight loss, sputum and polyuria [4][5][6]. Activin has a certain in uence on the regulation of gonadal hormone in the hypothalamic-pituitary-target gland axis, and may affect the formation of bone protein, Page 3/12 which providing more scienti c explanation for the kidney yang de ciency syndrome. Activin A is a member of the transforming growth factor-beta superfamily, which has endocrine and paracrine effects and that is at the heart of male reproductive biology and health [7].
Bone morphogenetic protein (BMP) is a secreted protein, which belonging to the transforming growth factor-beta (TGF-beta) superfamily. They play an important role in bone formation, heart and cartilage formation and neurodevelopment [8][9][10][11]

Preparation of sample solutions and standard solutions
The grass B. rossica was dried and pulverized, passed through a 60-mesh screen to reserve. Accurately weighed 1.00 g dried powder of B. rossica, added 50 ml of methanol in an Erlenmeyer ask, ultrasonically extracted for 30 min, and make up the weight with methanol. A 5 ml of the ltrate was evaporated and dried, dissolved in a 10 ml volumetric ask with a chromatographic methanol and diluted to the mark, then ltered through a microporous membrane (0.22 µm), and the solution was stored in a refrigerator at 4 ℃ to reserve.

Chromatographic conditions
A Klimail 100-5 C18 column (250 mm × 4.6 mm, 5 µm) was used for the detection and analysis using an To identify the components, a liquid chromatography mass spectrometer (Waters Quattro premier XE, USA) was used. MS detection parameters are as follows: electrospray ion source (ESI); detection method is negative ion detection; scanning range m / z 50 ~ 1400; target relative molecular mass 500; drying gas temperature 350 ℃; dry gas volume ow rate 9.0 L / min; The atomizing gas pressure is 0.

Statistical analysis
All experiments were repeated three times. Data were expressed as mean ± SD (standard deviation). All results were analyzed by SPSS (USA) 11.5 statistical analysis software. A P value of less than 0.05 was statistically signi cant.

Results
The overall process of the experiment is shown in Fig. 1. Figure 2 and Table 1 are the HPLC plots of the grasshopper and the main compounds identi ed by LC-MS. After oral administration of 9 times of B rossica extracts, the mice developed blinking, unresponsiveness, lethargy, black stools and secrets, and no acute symptoms such as exophthalmos, restlessness, convulsions, shortness of breath, etc. In response, some of the mice lost their luster, but returned to normal within 2-3 days, and no mice died after 7 days of continuous observation. As shown in Table 2, the maximum tolerated dose and the half lethal dose of the mice could not be measured due to the limitation of the administration volume and the concentration of the administration, and it was considered that oral administration of the herbicide was safe. There was no difference in body weight between the groups before modeling (P > 0.05).
As shown in Fig. 3A, after 15 days of intraperitoneal injection of large dose of hydrocortisone, the body weight of the other groups was signi cantly lighter than the blank group (P > 0.05). As shown in Fig. 3B, after 15 days of administration the positive group, the BBR-L group and Acte-H group were heavier than the model group. As can be seen from Fig. 3C, the weight change and the rate of weight change in the other groups were signi cantly slower than that in the blank group (P > 0.05). As shown in Fig. 3D, the weight and weight change of BBR-L and Acte-H mice before and after treatment were signi cantly higher than that of the model group (P > 0.05). The results showed that the BBR group and the Acte-H group had signi cant differences in improving the body weight of the mice.
As shown in Fig. 4A, the SOD activity in the serum of the BBR-H,-M groups and the Acte-H group was signi cantly increased compared with the model group, and the difference was statistically signi cant (P < 0.05). Compared with the blank group, serum SOD activity was statistically signi cant in the BBR-M group and Acte-H group, and the difference was statistically signi cant (P < 0.05).
As shown in Fig. 4B, compared with the model group, the content of MDA in the serum of the BBR group and the Acte group decreased, and the difference was statistically signi cant (P < 0.05). There was no signi cant difference in MDA between the BBR group and the Acte group compared with the blank group.
The results showed that the model group mice exhibited KYDS status after treatment, while the positive group, BBR group and Acte group improved hydrocortisone-induced KYDS.
As shown in Fig. 5A, the T content in the serum of the BBR groups and the Acte groups were signi cantly increased as compared with the model group, and the difference was statistically signi cant. (P < 0.05) Compared with the blank control group, the serum T activity of the BBR-M group and the Acte-H group was signi cantly increased, and the difference was statistically signi cant.
As shown in Fig. 5B, the FSH content in the serum of the BBR group and the Acte group was signi cantly decreased as compared with the model group, and the difference was statistically signi cant. (P < 0.05) Compared with the blank control group, the serum FSH activity of the BBR-M, L groups and the Acte-L group decreased signi cantly, and the difference was statistically signi cant.
As shown in Fig. 5C, the cAMP activity level in the serum of the BBR groups and the Acte groups were signi cantly increased as compared with the model group, and the difference was statistically signi cant.
(P < 0.05) Compared with the blank control group, serum cAMP activity was signi cantly increased in the BBR-H group and the Acte-H group, and the difference was statistically signi cant.
As shown in Fig. 5D, the content of cGMP in BBR group and the Acte group was signi cantly lower than that in the model group, and the difference was statistically signi cant (P < 0.05). Compared with the blank control group, the serum cGMP level of the BBR-H group and Acte-H group decreased signi cantly, and the difference was statistically signi cant. There was no signi cant change in serum cGMP content in the BBR-M, -L and the Acte-L groups compared with the blank group.
The results showed that the mice in the model group showed KYDS status after treatment, while the positive group, BBR group and Acte group signi cantly improved T, FSH, cAMP and cGMP in the serum of KYDS mice.
As shown in Fig. 6A, the ActR2A content in the serum of the BBR group and the Acte group was signi cantly increased as compared with the model group, and the difference was statistically signi cant (P < 0.05). The activity of serum ActR2A in the BBR-H group and the Positive group was signi cantly higher than that in the blank control group, and the difference was statistically signi cant.
As shown in Fig. 6B, serum BMP-5 levels in the BBR-M, -L Acte-H and -L groups were signi cantly higher than those in the model group, and the difference was statistically signi cant (P < 0.05). Compared with the blank control group, the serum BMP-5 activity of the Acte-H group was signi cantly increased, and the difference was statistically signi cant.
The results showed that the mice in the model group showed KYDS status after treatment, while the positive group, BBR group and Acte group signi cantly improved the serum levels of ActR2A and BMP-5 in KYDS mice.

Discussion
KYDS is one of the basic syndromes of traditional Chinese medicine. It is characterized by weakness, fatigue, waist and knee pain, easy to suffer from loose bones, memory loss, and decreased sexual function. 4,5 B. rossica is a traditional Chinese medicine, attending kidney de ciency and impotence, constipation, and also has a certain effect on rheumatoid arthritis. Therefore, we focus on oxidative stress, hormone immunity, energy metabolism and bone metabolism to study the possible mechanism of BBR extract on KYDS.
Oxidative stress, which is de ned as the cell's antioxidant defense system, produces an imbalance between reactive oxygen species (ROS) and is related to the high content of polyunsaturated fatty acids in the sperm plasma membrane due to the decline in fertility. Removal of Seminal plasma during semen storage may reduce the ability to protect enzymes and make sperm more susceptible to oxidative stress [12][13]. Superoxide dismutase (SOD) plays a fundamental but indispensable role in the antioxidant protection of biological systems against free radical attack. The enzyme is essential for cell health and protects the body's cells from excess oxygen free radicals, free radicals and other harmful substances that promote aging or cell death [14]. Malondialdehyde (MDA) is a reactive end product of lipid peroxidation. Lipid peroxidation products, which readily form adducts with nucleophilic groups of most cells, including DNA, lipids, peptides and proteins, leading to cellular metabolic disorders [15]. SOD and MDA are in a dynamic equilibrium state under physiological conditions, so as to control the content of reactive oxygen species and maintain cell function [16]. Recent studies have shown that low testosterone levels in diabetic men are associated with impaired metabolic status and mitochondrial function, as well as increased in ammation and leukocyte-endothelial cell interactions [17].
cAMP and cGMP are second messengers required for long-term enhancement (LTP) and memory formation/consolidation [18]. Changes in cyclic nucleotide signaling may lead to neurodegenerative diseases Alzheimer's disease, Huntington's disease and Parkinson's disease [19]. Studies have shown that cGMP and cAMP signaling pathways play an active role in osteoblast proliferation and differentiation. It has been observed that elevated intracellular cAMP enhances BMP action and increases ALP activity of osteoblasts in experimental animals [20].
The content of testosterone in the organism is closely related to the Nrf2 signal pathway [21]. If the SOD activity decreases and the MDA content increases, the mice will enter the oxidative stress state, which will cause defects in testosterone synthesis [22]. And cAMP can promote synthetic secondary hormones such as adrenocorticotropic hormone in the pituitary. After adrenocorticotropic hormone binds to adrenal cortical cells, it activates AC, increases cAMP content, thus activates protein kinase (PKA), and then activates synthetase such as corticosteroids in the adrenal gland after phosphorylation, thereby increasing testosterone content [23].
Testosterone (T) and FSH are the major endocrine regulators of spermatogenesis, which occurs in the speci c microenvironment of the testes. FSH-R is a G-protein coupled receptor (GPCR) bound to FSH. After activation, membrane-bound adenylate cyclase (AC) catalyzed the production of intracellular gproteins (e.g., stimulated gα), followed by an mediated pathway that stimulated multiple signal cascades. For example, PKA, PKC, PI3K, Akt / PKB and ERK1 / ERK2, to induce stem cell factor (SCF), glial-derived neurotrophic factor (GDNF) and androgen transcriptional binding protein (ABP) and transduction (TF) et al. The synergy between FSH and T to maximize the spermatogenic output process, which plays a key role in the spermatogenesis process and male fertility [24]. In adult fertile men and women, FSH mediates spermatogenesis and follicular development through its G-protein coupled receptor (FSHR) [25]. Activation of the FSH-induced INHA promoter cyclic AMP (cAMP) response element can be partially enhanced by androgens [4].
Activin is a growth factor in the transforming growth factor beta (TGFβ) superfamily. They have both endocrine and paracrine effects, which are essential for male reproductive biology and health. Areas of activin dysfunction may be linked to male reproductive symptoms, including infertility, in ammation and testicular cancer [7]. TGF-β1 can promote the proliferation and differentiation of osteoblasts, induce osteoclast apoptosis and improve bone formation [26]. Moreover TGF-β1 and activin A block the induction of Srebp1c (an inducer of adipogenesis) and TGF-β1 and activin A can act as inhibitors of adipogenesis [27]. Bone morphogenetic protein (BMP) is a member of the TGF-β family. It plays an important role in biological processes, including the involvement of vascular precursor cells in angiogenesis and the regulation of vascular cell population growth, differentiation and renewal [28]. ATP stimulates the formation of ALP and the expression of bone morphogenetic protein (BMP)-2, -4 and − 5 genes [29]. The action mechanism of the hypothalamic-pituitary-target gland axis is nally formed (Fig. 7).
Our results showed that the serum level of cAMP in the high-dose BBR extraction group (BBR-H) was signi cantly increased, which improved the balance between cAMP and cGMP, and thus activated the hypothalamic-pituitary-target gland. The axis signaling pathway demonstrates that BBR had a good therapeutic effect on KYDS mice.

Conclusions
Oral BBR-H dose greater than 1000 mg/kg/d in mice with kidney yang de ciency could increase serum testosterone and cAMP levels, and decrease serum FSH and cGMP levels. BBR anti-kidney yang de ciency is similar to Jingui Shenqi Pill. Acteoside was a representative component of phenylpropanol glycosides, phenylpropanol glycosides was the main component of the extract of B. rossica. Acteoside seems to be the active ingredient in B. rossica which has the ability to regulate ActR2A and BMP-5, but its mechanism of action on KYDS requires further research.