| Objective:To observe the secretion function and apoptosis of human ovarian granulosa cells in vitro by cisplatin,and explore the protection mechanism of Zuo gui Pill-contained serum in order to reduce the damage of ovarian function by regulating the PI3K/AKT signaling pathway,and this also further provide experimental evidence for the study of ovarian premature senescence after the treatment of chemotherapy.Methods:To select optimum concentration in each drug group after preculturing human ovarian granulosa cell.Group A was the control group in Pure culture medium.Group B was the model group choose Cisplatin with 10 mg/L for 24 h.After modeling,there were divided in to 3 groups: Group C,Group D and Group E.Group C was the inhibitor group,with LY294002(PI3K inhibitors)early intervention 30 min.Group D was the tonifing Kidney-yin drug serum low dose group,with with 5% medicated serum concentration.Group E was the tonifing Kidney-yin drug serum high dose group,with with 10% medicated serum concentration.Conventional collection of extracellular fluid training after 48 h,enzyme linked immunosorbent method the expression level of E2,FSH and LH.Finally collect granular cell,use the number of flow cytometry analysis of GC apoptosis,Q-PCR and Western Blot methods on PI3 K pathway between groups of GC was detected in related gene and protein phosphorylation and protein expression.Results: 1.Cell ApoptosisEach cell shows different degree apoptosis.But group B cell apoptosis rate is significantly higher than the other groups,especially compared with group A(P< 0.01).That cisplatin had apoptosis effect of ovarian granulosa cells.Compared with group B,group D and group E had a decrease in the apoptosis rate(P< 0.05),and the results showed that each dose of tonifing Kidney-yin drug serum group can reduce the isolated GC apoptosis rate.2.ELISA MethodCompared with group A,the level of FSH,LH and E2 in GC were decreased largely(P<0.01).After treatment,and compared with group B,the expression of E2 enhanced largely(P< 0.01),while the LH level reduced(P< 0.01).FSH in group C had been continued to decrease(P< 0.05),especially in group D and group E(P < 0.01).3.Real-time Q-PCR Detection ResultFOXO3a: compared with group A,the expression of FOXO3 a quantity increased significantly in group B(P < 0.05);Compared with group B,the values of group D decreased(P < 0.05),while that of group C and E reduced largely(P< 0.01).Bax: compared with group A,the expression of Bax increased significantly in group B(P< 0.01),while that of group E decreased(P< 0.05);Compared with group B,each amount of Bax protein expression significantly reduced,while that of group C and E reduced largely(P < 0.01).Bcl-2: compared with group A,the expression of Bcl-2 quantity decrease significantly in group B(P< 0.05),while that of group C and D increased(P< 0.05),and the content of Bcl-2 protein in group E was lager than other groups(P< 0.01);Compared with group B,the values of group C increased(p < 0.05),while that of group D and E increased largely(P < 0.01).4.Western Blot Detection ResultFoxo3a: compared with group A,the expression of FOXO3 a quantity increase significantly in group B(P< 0.05),but there was no statistically significant difference in the other groups(p > 0.05);Compared with group B,the expression of FOXO3 a were decreased in the other groups,especially in group E(P < 0.05).AKT: compared with group A,the expression of AKT marked increase in group B(P< 0.01);Compared with group B,the expression were decreased in the other grups,but there was no statistically significant difference in group C(P > 0.05),while the level in group D was difference(P< 0.05),and in group E was significant difference(P < 0.01).P-AKT: compared with group A,after using CDDP,the expression of p-AKT in group B and group C were decreased(P < 0.01),obviously,a marked drop was in group C(P< 0.01);Compared with group B,the expression in group C and group E were increased,especially in group E(P< 0.05).Conclusions:1.The prescription of reinforcing kidney-Yin could stimulate the production of E2 in granulosa cells,which like the kind of estrogen effects of kidney in TCM,and make sure the endocrine system consists of a certain adjustment.2.The effect of tonifying the kidney-Yin on the function of ovarain reserve can be reached by delaying the apoptosis of ovarian granulosa cells.3.The prescription of reinforcing kidney-Yin could up-regulation the related cytokin e levels of p-AKT,and influence the ratio of p-AKT/AKT,then drop the expression of FOXO3 a by the PI3K/AKT signaling pathway,so as to impact a certain degree of inhibit ion the damage of granulosa cells,for preventing ovarian follicles depletion effectively a nd achieving the aim of protecting ovarian function. |
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