Based On PI3K / AKT Signaling Pathway Study On The Molecular Mechanism Of Xinjia Guishen Pills Regulating The Proliferation And Apoptosis Of Ovarian Granulosa Cells In Rats

Part one Study on the GTW induced rat ovary granulosa cell injury modelOBJECTIVE:Developing the rat ovary granulosa cell injury model induced by rat medicated serum containing glucoside tripterygium wilfordine(GTW) to study the medical effect of Xinjiaguishenwan on granulosa cell in vitro.METHODS:Rats were intragastrically administrated with GTW with the concentrations of 160mg/kg·d、80mg/kg·d、40mg/kg.d twice a day respectively. Four days later, the rat medicated serum and the rat granulosa cell were prepared and divided into four groups, with cisplatin of 4.9mg/L as controlled group. Three GTW groups were cultivated with rat medicated serum. The OD values were measured in 48 hours with MTT methods. ELISA was used to test the E2 value.RESULTS:Both the inhibition effect and E2 value of 80mg/kg.d GTW serum were without statistic differences (P>0.05).CONCLUSION:The in vitro rat granulosa cells cultivated with 80mg/kg · d GTW serum presented the same inhibition as cisplatin goup, so 80mg/kg · d GTW concentration could be used in the following experiment.Part two Effect of Tonifying Kidney and activating blood method on the GTW serum cultivated rat ovary granulosa cell in vitro based on PI3K/AKT signaling pathwayOBJECTIVE:Based on the experiment of part one, we prepared the GTW serum induced rat granulosa cell model to study the cell proliferation effect of Tonifying Kidney and activating blood compound on the basis of PI3K/AKT signaling pathway. The molecular mechanism of the effect was also researched from the aspects of cell proliferation and apoptosis.METHODS:The granulosa cells were prepared as part one experiment and divided into 5 groups:normal controlled group, GTW group, FSH group, GTW+Chinese medicine low dosage group and GTW+Chinese medicine high dosage group, which were administered intragastrically with plain rat serum,80mg/kg.d GTW rat serum, FSH rat serum, high and low dosage Xinjiaguishenwan rat serums. In 48 hours after cultivation, RT-PCR was used to test p-AKTmRNA, p-mTORmRNA, p-p70S6KmRNA and PTENmRNA Western-blot was used to test cyclinD2, PNCA, caspase-3 and survivin. ELISA was performed to test E2 and P. The relevant analysis was used on the granulosa cell expression of cyclinD2, caspase-3 and p-AKT;of cyclinD2 and PCNA;of caspase-3and survivin.RESULTS: ① Comparing to the normal controlled group, the model group decreased on p-AKTmRNA, p-mTORmRNA, p-p70S6KmRNA, cyclinD2, PCNAand survivin proteins with significant differences (P< 0.05 or P< 0.01); The caspase-3 protein expressions increased significantly (P< 0.05 or P< 0.01);The PTENmRNA were not significant(P>0.05); The granulosa cell E2 and P decreased with significant differences (P< 0.05 or P< 0.01). ② Comparing to the model group, the high dosage group increased on the p-AKTmRNA, p-mTORmRNA, cyclinD2, PCNAand survivin expressions with significant differences (P< 0.05 or P< 0.01);The caspase-3 protein expressions decreased significantly (P< 0.05 or P< 0.01);The PTENmRNA were not significant(P>0.05); The granulosa cell E2 and P increased significantly (P< 0.05 or P< 0.01). Comparing to the model group, the low dosage group increased significantly on p-AKTmRNA (P< 0.05 or P< 0.01); the differences on other indicators were not significan(P>0.05). ③ The relevant analysis results showed positively between cyclinD2 and p-AKT (r=0.770,P<0.01), between caspase-3 and p-AKT (r=-0.779,P<0.01), between cyclinD2 and PCNA(r=0.547 P<0.05),between caspase-3 and survivin (r=-0.732, P<0.01)CONCLUSION:Via inhibiting the cascading activation reaction of granulosa cell AKT of PI3K/AKT signaling pathway, GTW may inhibit the activation of downstream signaling factor p-mTOR and p-p70S6K, promote the PTEN expression block the transmission of mitosis signals so as to decrease the cyclinD2 expression, block the process of cell cycle and inhibit the granulosa cell proliferation. On the other hand, GTW may up-regulate caspase-3, down-regulate survivin to speed granulosa cell over apoptosis, further lead to the decreasion of E2 and P secretion. Xinjiaguishenwan could release the GTW inhibition by activating p-mTOR and p-p70S6K, passing down the PTEN expression passing down the mitosis signal to promote the cyclinD2、PCNA expression and speed the cell cycle. On the other hand, Xinjiaguishenwan could down-regulate caspase-3, up-regulate survivin to inhibit the over apoptosis of granulosa cell so as to speed the recovery of E2 and P secretion.