Ginseng polysaccharides on HCG-induced luteal cell function of the particle
Author: Sun Yan, Feng Li, Hong-Jun Wang, the
spring-chi, Chu Zheng, Qiang Wang, Zhang Zhiqiang, Wu Yimin
【Abstract】 Objective To study the ginseng polysaccharides
on the role of ovarian function in rats. Methods Cell culture in vitro observation of ginseng polysaccharides on HCG-induced
luteal progesterone secretion in granulosa cells and the effects with cAMP and examined the survival rate of oocytes.
Isolated and cultured luteal, particles and oocytes, as control group with different concentrations of ginseng polysaccharide experimental group were measured using radioimmunoassay of progesterone and cAMP generated content, cell viability detected by MTT method. Results Compared with control group, progesterone content: luteal cells (6 573.46 ?
185.14) and (4509.55 ? 126.82) pmol, granule cells (197.16 ?
42.87) and (320.42 ? 12.46) pmol. cAMP: luteal cells (31.20 ? 17.13) and (65.26 ? 15.93) fmol, granule cells (121.15 ?
19.96) and (296.42 ? 27.28) fmol. Conclusion ginseng
polysaccharides inhibit HCG-induced progesterone secretion of luteal cells to promote the HCG-induced progesterone secretion in granulosa cells. The luteal cells together with the
granular cell cAMP generation, ginseng polysaccharides so that the rate of oocyte growth inhibition lower interval was dose-
Key words Panax ginseng polysaccharide; progesterone; cyclic adenosine monophosphate; human chorionic gonadotropin
The Effect of Ginseng Polysugar on Secretion Function of Luteal and Granulose Cell of Rat in vitro
Abstract: ObjectiveTo study using of ginseng polysugar on secretion function of female rat ovary. The experiment had observed progesterone and cAMP by HCG inducing luteal and
granulose cell as well as living cell rate of oocyte in vitro.MethodsRat luteal and granulose cell and oocyte was cultivated , cell counting was worked by microscope, progesterone and cAMP content was determined by
radioimmunoassay kit (RIA kit). Cell exist rate of oocyte was determined by MTT way. ResultsProgesterone: the luteal cell test groups compare with control group, 6573.46 ? 185.14 and
( 4509.55 ? 126.82) pmol.The granulose cell test groups compare with control group, 197.16 ? 42.87 and (320.42 ?
12.46) pmol.cAMP: the luteal cell test groups compare with control group, 31.20 ? 17.13and (65.26 ? 15.93) fmol, the
granulose cell test groups compare with control group, 121.15 ? 19.96 and (296.42 ? 27.28) fmol.ConclusionThis treatise
had demonstrated that ginseng polysugar can inhibit HCG inducing progesterone secretion and enhancing cAMP content of luteal and granulose cell of rat, ginseng polysugar has lower oocytes cell grow up inhibitory rate. It is evident that ginseng polysugar possesses characteristic of dose dependent.
Key words: Ginseng polysugar; Progesterone; cAMP; HCG
Many studies have shown that ginseng polysaccharides have a nourishing vitality, enhance the role of gonadal function , its adoption under the influence of the hypothalamus pituitary gonadal axis secretion system, the target organs and tissues through the cAMP intracellular information transmission system to start a series of physiological and biochemical reaction, ovary, as the hypothalamus - pituitary -
gonadal axis role in the reproductive system, its subject the role of ginseng polysaccharides to accelerate the process of sexual maturation and reproductive cycle also occurs in different functional changes. Ginseng polysaccharides on
reproductive cells more complicated, yet lack of a comprehensive system, detailed reports, in order to explore the ginseng polysaccharides on human chorionic gonadotropin (HCG)-induced rat luteal and granulosa cells function, this study used in vitro culture methods observed ginseng
polysaccharides on HCG-induced rat corpus luteum and granulosa cells secreted progesterone and cAMP content changes and cell
viability were detected. The content of this study no relevant reports, hereby reports the following results.
1.1 Animals and cells in body weight of about 250 ~ 300 g adult female rats were purchased from the Experimental Animal Center, China Medical University, in this laboratory sub-cage
farming, grain feed, automatic water, to adapt to 1 week after the experiment. Luteal cells, granule cells and oocytes from rat ovary.
1.2 Reagent and instrument MoCoy's 5a medium dry, collagenase, progesterone, human chorionic gonadotropin and heparin were purchased from Sigma Company; calf serum were
purchased from Shanghai Biological Engineering Company; pregnant mare serum were purchased from Experimental Animal Center of Tianjin ; ginseng polysaccharide Chemistry, Jilin University teaching and research benefit from the gift (2 g /
L); 3H-progesterone were purchased from Shanghai Institute of Biological Products; 125I-cAMP RIA kit were purchased from
Shanghai Traditional Chinese Medicine, xylene, PPO, POPOP, HCI , NaOH, etc. purchased from Shenyang Chemical Reagent Company, were analytically pure; CO2 Incubator: The United States Shel-Lab Inc., model 1815TC; Clean Bench: Jiangsu Suzhou Antai Air Technology Co., Ltd., model SW-CJ-2FD;
inverted phase contrast Microscope: Japan Olympus Corporation, model CX21FS1. BECKMAN: American Beckman Company, model J2-21;
liquid scintillation counter: Sweden LKB Company (machine efficiency 50%), model LKB1211; Kontro γ - Counter: Swiss
company, model Kontro-28D.
2.1 Cell Culture
2.1.1 Preparation of luteal cells and cultivation of the
female rats were injected subcutaneously neck, pregnant mare serum 50U / only, 48 h after the subcutaneous injection of human chorionic gonadotropin 45U / only, 1 week later, animals were decapitated, sterile take ovaries, the corpus luteum of
ovary Shredded, 0.2% collagenase 5 ml digestion at 37 ?
incubator every 15 min straw dogs new tricks, 1 h, add 5 ml MoCoy's 5a medium, blending, 200 r / min , centrifugation 2 min. Abandoned precipitation, 1300 r / min, centrifuge 10 min,
discard supernatant, add culture medium 10 ml, 1 300 r / min, centrifuge 10 min, washed 2 times. Add a new medium, blending, take 50 μl, together with 50 μl 0.4% trypan blue, cell
counting, calculating the percentage of cells in the total number of living cells. With 5% fetal calf serum MoCoy's 5a medium raised to 300,000 cells / 0.5 ml, by adding ginseng polysaccharide (30 ng / ml), inoculated 24-well plate, 5% CO2,
95% ventilation, 37 ? cultured 24 h, 0.5 ml serum-free
MoCoy's 5a medium wash one time, plus ginseng polysaccharides
containing 0.9 ~ 1 ml serum-free culture medium, 24 h later,
for fluid, washed 1 time. Serum-free culture medium plus 1 ml,
as control group and experimental group ginseng
polysaccharide, ginseng polysaccharides containing cell
culture medium 48 h, collected culture medium, boil 5 min, -
20 ? storage, for progesterone and cAMP radioimmunoassay.
2.1.2 Preparation of granulosa cells and cultivation of the female rat abdominal subcutaneous injection of dehydroepiandrosterone diethylstilbestrol oil 0.5 mg/0.1 ml / only, continuous injection of 4 d, 5 days, animals were decapitated, sterile taken ovary, will be used dehydrogenase diethylstilbestrol oil deal with the trumpet of the rat ovarian needle (41 / 2) punctured follicles and release
granule cells. Centrifugal, closed cell, plus an appropriate culture medium, take 50 μl, add 50 μl 0.4% trypan blue, and
counting cell number and percentage of living cells. Living cells is generally between 40% ~ 60%. According to number of viable cells with 5% fetal calf serum MoCoy's 5a medium raised to 300,000 cells / 0.5 ml, by adding ginseng polysaccharide, inoculated 24-well plate 24 h, washed with serum-free culture
medium 1, the control group is located 37 ? the experimental
group and ginseng polysaccharide, ginseng polysaccharides containing cell culture medium 24 h, collected culture medium, boil 5 min, -20 ? storage, for progesterone, cAMP of the RIA.
2.1.3 Preparation of oocytes and cultivation of the neck and subcutaneous injection of pregnant female rats horse
serum, 25U / only to stimulate follicular development, 48 h later, the animals were decapitated, the abdomen with 75% alcohol disinfection, in the ultra-clean Inside the remove the
ovaries, will deal with pregnant mare serum 48 h of the
ovaries, as far as possible in the dissection microscope, containing a few follicles were divided into small pieces,
with the needle punctured follicles, released into the containing 100 μl / ml of culture Aminophylline solution, centrifugation can be collected oocytes, the oocytes collected in 24-well plate by adding ginseng polysaccharide culture 8 ~ 10 h. Containing 0.2% after hyaluronidase, 1% sodium citrate and 10 mmol / L EDTA culture medium, located 37 ? control
group and experimental group ginseng polysaccharide, in the difference between the mirror count the survival rate of oocytes.
2.2 Steroid hormones and second messengers Determination
2.2.1 Progesterone RIA kit reference to the specific operation manual, sample preparation reference .
2.2.2 cAMP radioimmunoassay for the specific operation carried out according to kit instructions.
2.3 Cell survival MTT test taking subculture luteal and granulosa cells, trypsin digestion and counted using trypan blue exclusion staining of living cells measured by the number of 90%, and inoculated in sterile 96-well plate, each hole to
1 × 104. Experimental group, with different doses of ginseng polysaccharide, the control group received PBS solution, each complex is located five holes. The hole filled with culture medium to a final volume of 200 μl. Purchase 37 ?, 5% CO2
incubator box at 24 h after incubation, add PBS solution of MTT (5 g / L), each well 20 μl, 37 ? reaction 4 h after the
culture medium and the suction abandoned unreacted thiazole blue, DMSO solution of each hole by adding 100 μl, after the
room temperature oscillation static 10 min, with the
microplate reader at 570 nm was measured absorption of light (A value), calculate the rate of cell growth inhibition. Reposted elsewhere in the paper for free download http://
2.4 Experimental measurement of information on statistical methods are mean ? standard deviation of that
difference between the groups using t test.
3.1 ginseng polysaccharides on HCG-induced luteal
progesterone production and particle effects of cell-based
ginseng polysaccharides with 30 ng / ml in good condition
acting on the growth of luteal cells 48 h, and granulosa cells 24 h, changed with HCG 0.15 U / ml The new medium to continue at 37 ? CO2 incubator. Luteal cell culture 8h, granule cell cultures 6 h after the collection of culture medium to measure
the content of progesterone production. The results showed that ginseng polysaccharides inhibit HCG luteal cells to stimulate progesterone production and strengthening of granular cell progesterone production, specifically manifested in the ginseng polysaccharides so that the basis of luteal cells, progesterone production and decreased with the increase in granulosa cells, ginseng experimental group compared with control group P "0.05. Table 1.
Table 1 ginseng polysaccharides on HCG-induced luteal
cell progesterone and particle content (abbreviated)
Compared with control group, * P <0.05
3.2 ginseng polysaccharides on HCG-induced luteal cell-
based cAMP generation and particle effects of ginseng polysaccharide in experimental group by 30 ng / ml were pre-
luteal and granulosa cells 48 h and 24 h, new ones culture medium by adding 0.15 U / ml HCG, continues at 37 ? in CO2
incubator 4 h. Determination of cAMP content (Table 2). Ginseng polysaccharides on the basis of luteal and granulosa
cells cAMP generated content regulation, reflected in the ginseng polysaccharides so that the basis of luteal and granulosa cells cAMP generated content increased, ginseng experimental group compared with control group P <0.05. That ginseng polysaccharide and HCG has a synergistic effect.
3.3 ginseng polysaccharides on oocyte survival rate of low-temperature stress effects of different doses of ginseng polysaccharide medium acting on the oocyte cell, compared with the control group, plus ginseng polysaccharide group starting from 20 μl, ie the cell has obvious effect on the
proliferation, and along with the increase in the concentration of ginseng polysaccharide on the proliferation and promoting cell division also increased in a dose-dependent
positive relationship, indicating that ginseng polysaccharide on the regulation of cell growth curve was the amount - effect
relationship, but in 35 ~ 60 μl between the plateau,
indicating an additional dose of ginseng polysaccharides can no longer be unlimited to promote cell division. Cell growth inhibition rate of (1 - mean value of experimental group A / A value of the control group mean) × 100% (see Figure 1).
Description ginseng polysaccharides can promote cell growth, mortality.
Table 2 Panax ginseng polysaccharides on HCG-induced
luteal and granulosa cell-based cAMP content (abbreviated)
Compared with control group, * P <0.05
Figure 1 Effect of different concentrations of ginseng polysaccharides on the inhibition rate of oocytes effects
Ginseng polysaccharide on the corpus luteum and granulosa cells of the role of the more complex of different species of cell types, cells at different developmental stages, or the existence of factors to observe the role of different
indicators results to differ. Observed in this experiment of ginseng polysaccharides on HCG stimulation of luteal and granulosa cells produce progesterone and the relationship between cAMP showed that, HCG can luteal and granulosa cells
produce progesterone and cAMP increase, ginseng
polysaccharides inhibit HCG-stimulated progesterone luteal
cells build and enhance progesterone secretion in granulosa cells, while the corpus luteum and granulosa cells together to increase cAMP. With regard to the principle of the role of ginseng polysaccharide may be with the body's reaction to the process, especially for nerve - pituitary - adrenal cortex
system of feedback effects on . Ginseng has obvious strong role, but also may be due to feedback inhibition of FSH and LH secretion . Therefore, ginseng polysaccharides on HCG stimulation of luteal progesterone production and the granular cells showed different biological effects. Ginseng polysaccharides of this experiment in collaborative mechanism
for the increase in cAMP generation is complex, according to inspection of the literature and the results of this experiment is also difficult to make accurate conclusions, but
one thing is clear, steroid hormones is regulated by the
second messenger cAMP, Intracellular information transmission system of mutual inhibition between both, have a mutually reinforcing effect , should we look at ginseng polysaccharide in intracellular signaling mechanism between the conduction process. Ginseng polysaccharides to reduce the rate of cell growth inhibition showed a "mitogen-like" effect
on the body's reproductive cells, the role of health care and rehabilitation have some physiological significance.
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