| Objective: To use the method of Chinese herbal seropharmacology, to investigate the effect of the rats sera containing Benefiting-bone capsule (BBC) on the cell proliferation, cell differentiation and cell mineralization of rat osteoblast in vitro skulls of neonatal SD rats and to research the relationship between the best time-effectiveness and dosage-effectiveness, and at the same time, to study the significant changes of both alpha and beta estrogen receptors in the osteoblast and the production of Interleukin-6(IL-6) by adding the rats sera containing BBC by studying the expression level of both the mRNA and the protein. This is performed to explore the mechanism of BBC on the prevention and treatment osteoporosis by means of estrogenic modulation and control.Methods: Forty female SD rats, aged 12 months, were used and randomly divided into 4 groups including N.S., low dosage, middle dosage and high dosage with BBC, the sera were extracted at different times. The MTT method was used to determine the best time-efficiency and dosage-efficiency. To obtain osteoblasts from neonatal SD rat's skull using the method of twice enzyme digestion, conducted primary culture and subcultures.There are three groups including Group I (containing deactivating sera with BBC), Group II (containing deactivating sera without BBC) and Group III (simple DMEM medium group). Also, spectrophoto- meter colorimetry assays were used to examine the activity of the alkaline phospha tase. Alizarin at 0.1% was used to count the mineralized nodes in discussing the influence involving the differentiation and the mineralization of the osteoblast of the rats sera containing BBC. RT-PCR technology was used to study the estrogenic receptor α, βmRNA, IL-6 mRNA and p-actin mRNA. The RBA method was used to study the affinity of estrogen receptors and the number of receptors. The RIA method was used to study IL-6 in the culture (including the sera) of the osteoblasts.Results: The MTT value reached its highest point 1.5 hours after oral administration, which was considered to be statistically significant when compared with others (P<0.05). MTT of high dosage met the maximum value with the concen tration of 25%, and was considered to be statistically significant (P<0.05). Thereby increasing the number of ALP, mineralized nodes and up-modulating the expression of ERp mRNA ( P<0.05), without boosting the affinity of ER compared to the control groups (P>0.05) but increasing the receptors number statistically significantly(P<0.01).Both can down-modulate the expression of IL-6 mRNA and protein ( P<0.05). Conclusions: The rats sera containing BBC can promote proliferation, differentiation and mineralization of osteoblasts, there is an up-modulating of the osteoblastular ERβ mRNA and an increase in the number of their receptors' protein, at the same time, a down-modulating of the osteoblastular IL-6 mRNA and a decrease in the amount of IL-6 secreted by osteoblasts. These indicate an estrogenic action on bones through ERβ modulation. BBC, a Chinese herbal complex prescription, can effectively treat Osteoporosis through the modulating mechanism of estrogenic pathway. This is worth further research. |
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