The Efficacy And Mechanism Study Of Yiru Tiaojing Granule On Treating Hyperprolactinemia In Vitro

Prolactin (PRL) is a protein hormone secreted by anteriorpituitary gland. It is not only necessary for mammal reproduction lactation hormones, but also can adjust the other pituitary hormones, such as luteinizing hormone, follicle stimulating hormone secretion and have a significant impact on the growth, development and immune function of people. Hyperprolactinemia (HPRL) is a common disease of the hypothalamus-pituitary-gonad dysfunction, which refers to the pathological phenomena that serum PRL value of patient is more than 25 ng/mL. The pathogenesis of the disease is complex and theprolactinoma is the most common cause. Most of the patients are women. Clinical symptoms of HPRL clinical are mainly for amenorrhea, menstrual scarce, anovulation, lactation, etc. Patients with serious condition even show infertility. According to the investigation of a study, the incidence of hyperprolactinemia in our country is as high as 0.4%, moreover, the incidence of women in reproductive age in China is as high as 9%-17%. Hyperprolactinemia is a common cause of infertility, which is one of clinical gynecological complicated diseases.A growing number of researches show that the neurotransmitter dopamine is typical prolactin release factor, synthesised by the hypothalamus funnel nodules, released by median eminence. Prolactin is regulated by the dopamine receptor in the hypothalamus. The factors blocking dopamine receptor can stimulate PRL secretion. Dopamine and its agonists inhibit the secretion of PRL in the body, whereas antagonists promote the PRL secretion. At present, dopamine agonists (Bromocriptine or Ergot Alkaloids) are commonly used in clinical to treat HPRL. The medicine is effective, but easily to cause adverse reactions such as headaches nausea, dizziness, posture hypotension. The early experiments used to prepare HPRL rat model through intraperitoneal injection of dopamine receptor blockers (Metoclopramide) continuously. After treatment with Bromocriptine and Yiru Tiaojing Granule, the expression of DA receptor in the pituitary increased and the PRL secretion derceased when compared with control group. These results strongly suggest the DA receptor is closely related with the regulation of PRL and HPRL disease.Currently, there is no safe, effective and convenient traditional Chinese medicine to treat HPRL on the market. Yiru Tiaojing Granule (YRTJ) is a Chinese medicine for HPRL developed by nanfang hospital, southern medical university, which is composed of Curculigo Orchioides, Morinda Officinalis, Radix Paeoniae Alba, and Glycyrrhiza. It has the protective effects for liver function and nourishing function to liver and kidney, mainly for the symptoms caused by HPRL, such as irregular menstruation, pain of abdomen, abnormal lactation, infertility etc. The clinical research showed that the efficient of Yiru Tiaojing Granule in the treatment of HPRL was 94.7%, especially for the type of liver and kidney deficiency. The observation in the early experiment in vivo, Yiru Tiaojing Granule reduced PRL levels in HPRL animal model by acting on dopamine D2 receptor (D2DR). This experiment used the method of serum pharmacology to investigate the effect of Yiru Tiaojing Granule (YRTJ)-medicated serum on rat pituitary tumor cells in vitro, and observed the PRL secretion and PRL, D2DR protein expression to validate the result of the experiment in vivo and revealed the main biological molecular mechanism of Yiru Tiaojing Granule to treat HPRL. It can provide important basis for developing Chinese traditional medicine to treat HPRL.ObjectiveThe rat pituitary tumor MMQ cells and GH3 cells are treated with YRTJ by the method of serum pharmacology. The method of real-time PCR and Western blotting were used to detect the expression of PRL, D2DR protein and PRL mRNA. The method of Elisa was used to detect the expression of cAMP and PKA. After dopamine receptors blocked by dopamine receptor blockers haloperidol, the expression of PRL and D2DR protein of the MMQ cells were observed to study specific efficacy of YRTJ-medicated on the model of HPRL in vivo. The observation of the cell proliferation, the expression of apoptosis protein Bax/Bcl-2 aimed to study YRTJ-medicated serum in promoting apoptosis of the pituitary tumor cells.Method1. The YRTJ affected on the growth of MMQ rat pituitary tumor cells.1.1 The preparation of low-, medium-and high dose YRTJ-medicated serum and bromocriptine-medicated serum of rats and blank serum.1.2 Make the MMQ cells as the research object, the cells were treated with different concentrations of high dose YRTJ-medicated serum. We choosed the most effective concentration and time by CCK-8 method after 24 h,48 h,72 h,96 h, respectively.1.3 The MMQ cells in logarithmic phase were divided into five groups:control group, bromocriptine-medicated serum group, low-, medium-, high-dose YRTJ-medicated serum group. After cultured for 24 h,48 h,72 h,96 h,10 u L CCK-8 solution were added, respectively. After continued to incubate for 2h, the optical density was determinated and calculated the cell proliferation inhibition rate.1.4 After treated with drugs, all the groups of MMQ cell apoptosis ratio was detected by flow cytometry; the Bax/Bcl-2 protein expression in cells was analyzed by western blot method.2. The pharmacological effects of YRTJ on MMQ and GH3 cells.2.1 The MMQ cells and GH3 cells were cultured with the blank serum, bromocriptine-medicated serum, low-, medium-, high-dose YRTJ-medicated serum for 48 h. Then cells and cell culture supernatant were collected, the content of PRL in the supernatant fluid were determined by ELISA method, the expression of PRL protein in MMQ cells and GH3 cells, and the expression of D2 receptor protein in the MMQ cells were determined by western blot method at the same time. The expression of PRL mRNA in MMQ cells and GH3 cells were detected by RT-PCR method.2.2 After pre-incubated with dopamine receptor blockers haloperidol for 4 h, then MMQ cells were added with high dose of YRTJ-medicated serum and blank serum and cultured for 48 h. The PRL protein expression and the expression of D2DR protein in the MMQ cells were determined by western blot. The expression of PRL mRNA in MMQ cells and GH3 cells were detected by RT-PCR.2.3 The MMQ cells and GH3 cells were treated with the blank serum, bromocriptine-medicated serum, low-, medium-, high-dose YRTJ-medicated serum for 48 h. Then cells were collected. The intracellular PRL of MMQ cells and GH3 cells were determined by ELISA method.3.1 Material basis of YRTJWe detected qualitative of peoniflorin, liquiritin, monotropein and orcinol glucoside in YRTJ by high performance liquid chromatography (HPLC). The mobile phase of Paeoniflorin was a mixture of acetonitrile-0.1% phosphoric acid solution (14:86).The UV detective wavelength was 231nm. The mobile phase of liquiritin was a mixture of acetonitrile-0.1% potassium dihydrogen phosphate (17:83). The UV detective wavelength was 231 nm;The mobile phase of monotropein was a mixture of acetonitrile-0.1% potassium dihydrogen phosphate (6:94). The UV detective wavelength was 231 nm;The mobile phase of monotropein was a mixture of mobile phase of acetonitrile-0.1% potassium dihydrogen phosphate (5:95). The UV detective wavelength was 216 nm. Chromatographic column was Diamonsil C18(250 mm×4.6 mm,5 μm). The column temperature was 30℃.The flow rate was 1.0 mL/min3.2 Material basis of YRTJ-medicated serumThe mobile phase consisted of 0.1% formic acid aqueous solution (A)and acetonitrile (B)with a gradient eluention program:80-20%(v/v),0.1-0.5 min;40-60%, 0.5-2.8 min; 10-90%,2.8-3.5 min; 10-90%,3.5-4.5min; 20-80%,4.5-5 min, at a flow rate of 300 μL/min. The liquid chromatography was performed on an Agilent 6460 Series liquid chromatography equipped with a ZORBAN SB-C18 column (3.0×100mm,1.8μm, Agilent Corporation, USA). The most suitable heated capillary temperature, spray voltage and collision energy for all target analysts were selected by manual optimization.Results1. The YRTJ affected on the growth of MMQ rat pituitary tumor cells.1.1 The MMQ cells were cultured with different concentrations of high dose YRTJ-medicated serum. We found the cell inhibition rate of 10% of high dose-medicated serum reached the maximum in 48 h. So we choosed the concentration of 10% of YRTJ-medicated serum for subsequent tests.1.2 The different concentration YRTJ-medicated serum made significant inhibitory effect on cell proliferation, and showed in a dose-response relationship. YRTJ-medicated serum increased cell inhibition rate with the increase of drug dose. At 48 h, cell inhibition rate of the high dose, medium dose group was significantly increased (P<0.001) when compared to that of low dose group. In 72 h, compared with that of low dose group, the cell inhibition rate of high dose group showed significant differences (P<0.05).1.3 The results of flow cytometry showed that the high, medium dose YRTJ-medicated serum and bromocriptine-medicated serum can induce the early apoptosis of the MMQ cell significantly (P<0.001) in dose-response manner.1.4 The MMQ cells were incubated with low-, medium- and high dose of YRTJ-medicated serum. The expression of Bcl-2 protein gradually decreased along with the increase of drug dose, whereas the expression of Bax protein expression gradually increased. The Bax protein expression in high-, medium- and bromocriptine group increased compared with the control group (P<0.01). And compared with the bromocriptine group, the Bax protein expression of high dose group showed no significant difference. The Bcl-2 protein expression of low and medium dose group increased in compared with that of the control group serum (P<0.01). The Bcl-2 protein expression of high dose, medium dose group showed no significant difference from the bromocriptine group.2. The pharmacological effects of YRTJ on MMQ and GH3 cells..2.1 The high-, medium dose YRTJ-medicated serum and bromocriptine-medicated serum can inhibit the secretion of PRL in MMQ cell when compared with the control group(P<0.001). And high dose of YRTJ-medicated serum and bromocriptine-medicated serum showed the most significant effect to inhibit the secretion of PRL level (P>0.05). But PRL level of GH3 cells culture supernatant in all the treatment group had no significant difference.2.2 Both the high dose of YRTJ-medicated serum (P<0.001) and bromocriptine-medicated serum (P<0.05) inhibits the PRL proteins expression of MMQ cells. The effect of all the YRTJ-medicated serum groups showed a dose-response relationship. Compared with the bromocriptine-medicated serum group, the inhibitory effect of high dose YRTJ-medicated serum on PRL protein expression of the MMQ cells more significantly (P<0.001). However, the PRL protein expression of GH3 cells in all the treatment group showed no significant difference.2.3 After pre-incubated with dopamine receptor blockers haloperidol, the inhibition of high dose YRTJ-medicated serum on the PRL protein expression of the MMQ cells significantly reduced (P<0.01), but showed no significant difference when compared with that of control group.2.4 In the same way, after the drug treatment, the trend of PRL mRNA expression in MMQ cells is similar to the trend of PRL protein expression. Both expression of PRL mRNA and PRL protein in high YRTJ-medicated serum group are significantly lower than that in cells with co-treatment (P< 0.01 or P<0.001).2.5 The D2DR protein expression in MMQ cells increased along with the concentration of YRTJ-medicated serum in a dose-dependent manner. Compared with the control group, the high-and medium-dose YRTJ-medicated serum significantly increased the expression of D2DR protein in MMQ cells (P<0.05 and P<0.001). The D2DR expression in the high-and low-dose YRTJ-medicated group showed statistically significant differences from the bromocriptine-medicated serum group (P< 0.05 and P<0.001).2.6 High YRTJ-medicated serum group stimulated D2DR protein significantly compared to in cells with co-treatment (P<0.001). There were significant differences among co-treatment group and control group compared with haloperidol group (P<0.01 or P<0.05).2.7 Compared with control group, the PKA and cAMP in MMQ cells were significantly attenuated dose-dependently by treatment with high-and medium-dose YRTJ-medicated serum (P<0.05). There was no significant difference among high-and medium-dose YRTJ-medicated group compared with bromocriptine-medicated group.3. The major components of YRTJ granule were analyzed by HPLC. By comparison with the standard reference compounds, four compounds were identified:peoniflorin, liquiritin, monotropein, orcinol glucoside, eluting at approximately 14.505min, 14.601mm,9.757min,15.298min, respectively. The LC-MS/MS analysis showed that the YRTJ-medicated serum contained complicated components. The components included prototype components in crude drugs. The retention times of paeoniflorin, orcinol glucoside and liquiritin were approximately 2.9 min,3.0 min, and 3.1min, respectively.Conclusion1. The YRTJ-medicated serum could significantly inhibited the proliferation of MMQ cells. The cell inhibition rate of 10% of high dose-medicated serum reached the maximum in 48 h.2. The YRTJ-medicated serum induced the cell early apoptosis and promoted the expression of Bax/Bcl-2 apoptosis protein.3. The YRTJ-medicated serum made effect reduced the PRL protein expression and secretion of PRL in the MMQ cells, a HPRL model in vitro.High dose YRTJ-medicated serum can produce similar pharmacological effects as bromocriptine-medicated serum.4. The YRTJ-medicated serum produced no suppression on the PRL protein expression and secretion of PRL in GH3 cells. With the treatment by the dopamine D2 receptor blocker haloperidol, the effect of reducing PRL expression by YRTJ-medicated serum is blocked. The main pharmacological effects of YRTJ may reduce the PRL expression by acting on dopamine D2 receptor, which was related to the second messenger cAMP and PKA.5. It can be seen from the results of HPRL that the YRTJ contains peoniflorin, liquiritin, monotropein, orcinol glucoside. As shown in the result of LC-MS/MS, YRTJ drug-medicated serum containing peoniflorin, liquiritin, monotropein, orcinol glucoside. However, monotropein had not been identified in serum and may transform into other products.