| Objective: Effect of YIGU Capsule drug-containing serum on cell proliferation and on release of IL-1β ,TGF- β1 from osteoblasts in vitro are studied in order to further explore the mechanism of YIGU Capsule on preventing and treating osteoporosis. Methods: ①Preparation of YIGU Capsule drug-containing liquid: Mixed eight types of herbal pellets, included Epimedium and Angelica, soaked and melted fully with distilled water, then deal with percolated, isolated and 95% ethanol etc., preserved for the experiment after condensed. ②Preparation of YIGU Capsule drug-containing serum: Forty female Spragne-Dawley(SD) rats at the age of 12 months were randomly divided into 4 groups (high dose, medium dose, low dose and the blank control group) for orally administering YIGU Capsule liquid or physiological saline daily. Different concentrations of liquid were also given. They were administered for 12 days. At 1.5 hours after the last administration, under sterile condition, blood was collected from the rat's right ventricle, the collected blood was isolated and preserved under 56℃, 30min inactivation, and preserved under -70 ℃ for the experiment. ③Groups: Osteoblasts were obtained from neonatal SD rat's skull using the method of twice enzyme digestion, subculture, took the tertiary cells as experimental model. Built up the culture model of neonatal rat osteoblasts with drug-containing serum in YIGU Capsule in vitro, and prepared two groups: drug-containing serum and blank control group (both final concentrations were 25%). Each group took 16 holes, eight were cultivated 24 hours, and the other eight were cultivated 36 hours, the content of IL- 1β and TGF- β1 were assayed. Observed cell cycle of osteoblasts, took the fifth generation cells as experimental model, with the same groupings (as above), each group took four flasks, two were cultivated 24 hours, and the other two were cultivated 36 hours, then the cell cycle was determined. ④Assay method: ELISA method was used to observe the content of IL-1 β and TGF- β1 secretion and Flow Cytometry was used to observe cell cycle of osteoblasts. ⑤Statistic method: Data was shown in x ±s, which was analyzed by the statistics software SPSS (Statistical Package for the Social Science) 10.0, comparison with control groups were analyzed byt-Test.Results: ①Effect of YIGU Capsule drug-containing serum on the cell cycle of osteoblasts: At 24 hours and 36 hours, the G0/G1 phase rate (61.61 ± 1.27% and 61.54 ± 3.97 % respectively) in groups with drug-containing serum were obviously lower than in the blank control group (P<0.01); at 24 hours and 36 hours, the S phase rate (19.71 ± 1.48% and 23.27±0.50 % respectively), Proliferation Index (PI) (31.66 ± 1.46% and 32.57 ±2.97 % respectively) and G2/M phase rate (8.85 ± 0.15% and 6.41±1.60 % respectively) in groups with drug-containing serum were obviously higher than in the blank control group (P<0.05). ②Effect of YIGU Capsule drug-containing serum on the IL-1β secretion of osteoblasts: at 24 hours and 36 hours, the content of IL-1β (14.83 ± 1.96 pg/ml and 11.31 ±2.06 pg/ml respectively) in groups with drug-containing serum were obviously lower than in the blank control group (P<0.0 1);③ Effect of YIGU Capsule drug-containing serum on the TGF- β1 secretion of osteoblasts: at 24 hours and 36 hours, the content of TGF-β , (367.88±34.41 pg/ml and 469.50±26.30 pg/ml respectively) in groups with drug-containing serum were obviously higher than in the blank control group (P<0.01); The content of TGF- β1 with drug-containing serum at 36 hours was higher than at 24 hours (P<0.01).Conclusion: YIGU capsul drug-containing,serum,can,effectively,promote,the proliferation of osteoblasts,increases the secretion of TGF-β1 and decreases the secretion of IL-1β from osteoblasts. It is suggested that these results could be parts of the mechanism for YIGU capsule preventing and treating osteoporosis.
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