Effects Of Bushen Tiaochong Recipe On Rats Ovarian Granulosa Cell Proliferation, Steroidogenesis And Associated Gene Expression

Objective: Bushen Tiaochong Recipy (BSTCR) is established by my tutor, professor Hanbing according to Chinese traditional medical theory and his clinical practice, which has the effect of tonifying and nourishing the liver and kidney, balancing Yin and Yang, dispersing the depressed liver-Qi as well as regulating Qi and blood in the Chong and Ren channels. It is applicable to a series of ovarian dysfunctional diseases caused by renal deficiency and the Chong and Ren channels disorder. The present study was undertaken to observe the effect of BSTCR on rats ovarian granulosa cell proliferation and steroidogenesis with both methods of direct regimen and serum pharmacology. Furthermore we examined the molecular mechanism at the level of ovary, by which BSTCR regulated the expression of follicle stimulating hormone receptor (FSHR), steroidogenic acute regulator protein (StAR) and Insulin-like growth factor-1 (IGF-1) mRNA in order to provide the theoretical basis for the application of BSTCR in clinic.Methods: Granulosa cells was isolated from rats ovaries and cultured in medium containing 20% calf serum for 24 hours preincubation, followed by culture in medium containing FSH (50ng/ml) and/or varying dosage of BSTCR (100,10,1, 0.1 u g/ml) for a further 48 hours. The serum contained FSH or varying dosage of BSTCR were prepared with 6-weeks-old Sprague Dawley rats using the method of serum pharmacology, with which granulosa cells were incubated for 48 hours. 3H-Thymidine incorporation assay were performed using a scintillation counter. Cell cycle analysis was carried out by flow cytometer(FCM). Quantification of Estrodial, progestogen and cAMP were examined by radio-immunity. FSHR StAR IGF-1 mRNA expression were measured by semiquantitative and real-time RT-PCR(SYBR GreenI).Results: 1. Effect of BSTCR on granulosa cell proliferation and steroidogenesis: (1) BSTCR increased [³H]thymidine incorporation into DNA of granulosa cells significantly; (2)BSTCR promoted cell conversion from G₀ phase to S and G2M phase and increased proliferation index (PI); (3)BSTCR improved the content of cAMP;(4)The result indicated a dose-dependent increase in granulosa cell proliferation and steroidogenesis at a certain extent. And the effect was maximum in the highest dosage(100 u g/ml) group, which was similar to FSH;(5)BSTCR reinforced FSH-induced increase of proliferation, steroidogenesis and cAMP.2. Effect of the serum contained BSTCR on granulosa cells function:(1)The serum contained BSTCR increased significantly granulosa cells proliferation, steroidogenesis and the content of cAMP; (2)There were no marked difference among the three groups of granulosa cells incubated by the serum contained large, medium dosage BSTCR and FSH, while the effect of the serum contained small dosage BSTCR were lower than those three serum. (3)Excessive androgen-treated granulosa cells showed reduced proliferation, steroidogenesis and cAMP. While the serum contained BSTCR rivaled excessive androgen-induced depressant effect and improved granulosa cells function.3. Effect of BSTCR on granulosa cells FSHR mRNA expression: (1)FSH treatment stimulated the expression of FSHR mRNA in FSH-exposed granulosa cells, whereas BSTCR had no significant effect on the expression of FSHR mRNA; (2) BSTCR increased FSH-induced expression of FSHR mRNA; (3)The serum contained BSTCR had a certain dosage-dependent effect on FSHR mRNA.4.Effect of BSTCR on granulosa cells StAR mRNA expression: (1)Both of BSTCR and BSTCR-treated serum stimulated the expression of StAR mRNA with a certain dose-dependent manner; (2)BSTCR-treated Granulosa cells showed increased expression of StAR mRNA in response to forskolin stimulation.5. Effect of BSTCR on granulosa cells IGF-1 mRNA expression: (1)Both of BSTCR and BSTCR-treated serum stimulated the expression of IGF-1 mRNA with a certain dose-dependent manner; (2)BSTCR-treated Granulosa cells showed increased expression of IGF-1 mRNA in response to forskolin stimulation.Conclusion: The present study indicated defferent mechanism of BSTCR to regulate ovarian granulosa cells proliferation and steroidogenesis in defferent conditions: 1. In culture of absence of FSH, BSTCR regulates granulosa cells...