Effects Of Icariin On Mouse Ovarian Granulosa Cells Proliferation,Secretion And Associated Genes Expression

Objective: To explore the molecular mechanism of Icariin promoting the proliferation and secretion of mouse ovarian granulosa cells through the effect of Icariin on the proliferation and secretion of mouse ovarian granulosa cells in vitro.Methods: Mouse ovarian granulosa cells were isolated and cultured in vitro for 24 hours.The adherent ovarian granulosa cells were divided into 6groups: blank group,solvent control group,Icariin 1μg/L group,Icariin 10μg/L group,Icariin 50μg/L group and Icariin 100μg/L group.the proliferation rate and apoptosis rate of ovarian granulosa cells in each group were detected by CCK-8 method and flow cytometry apoptosis,and the growth curve of ovarian granulosa cells was drawn.The concentration of progesterone in the culture medium of granulosa cells in each group was detected by enzyme-linked immunosorbent assay(ELISA),and the expression of HGF,c-Met,IGF,TGF-β,LIF,GDF-9 and Caspase-3 protein in ovarian granulosa cells of mice in each group was detected by Western blotting.The m RNA expressions of HGF,c-Met,IGF,TGF-β,LIF,GDF-9 and Caspase-3 in ovarian granulosa cells were detected by reverse transcriptase polymerase chain reaction(q-PCR).Results: Compared with the blank group,Icariin 1μg/L group and Icariin100 μg/L group decreased the survival rate of ovarian granulosa cells,and there was no significant difference between the two groups(P>0.05).The results of CCK-8 showed that Icariin 1μg/L group and Icariin 100μg/L group decreased the survival rate of ovarian granulosa cells.Both Icariin 10μg/L group and Icariin 50μg/L group could promote the survival of ovarian granulosa cells,and there was no significant difference in Icariin 50μg/L group(P>0.05).There was significant difference in Icariin 10μg/L group(P<0.05).The results of flow cytometry apoptosis showed that the apoptosis rate of each experimental group was lower than that of the blank group,and there was no significant difference between Icariin 1μg/L group and Icariin100μg/L group(P>0.05).There was significant difference in the apoptosis rate between Icariin 10μg/L group and Icariin 50μg/L group(P<0.01),and the apoptosis rate was the lowest in Icariin 10μg/L group.The results of ELISA detection showed that the level of progesterone in the culture medium of ovarian granulosa cells in each group was significantly higher than that in the blank group(P<0.01 or P<0.05);The results of Western Blotting detection showed that the protein expression of HGF,LIF,GDF-9,TGF-β and IGF in ovarian granulosa cells in each group was higher than that in the blank group.There was significant difference in HGF protein between Icariin 1μg/L group and Icariin 10μg/L group,but there was no significant difference in Icariin50μg/L group and Icariin 100μg/L group;LIF protein between Icariin 10μg/L group and Icariin 50μg/L group.There was significant difference in GDF-9,TGF-β and IGF protein in Icariin 10μg/L group,but there was no significant difference in other groups(P>0.05).The expression of c-Met protein in ovarian granulosa cells increased at first and then decreased in each group,and there was significant difference in Icariin 10μg/L group,but there was no significant difference in other groups(P>0.05).The expression of Caspase-3protein decreased in all groups(P<0.01 or P<0.05).The results of q-PCR detection showed that compared with the blank group,the expression levels of HGF and LIFm RNA in ovarian granulosa cells of all groups were firstly increased and then decreased,and the expression levels of HGF and LIFm RNA were only significantly different in Icariin 10μg/L group(P<0.01).The expression of c-Metm RNA in each group was firstly decreased and then increased,and the difference was statistically significant only in Icariin 10μg/L group(P<0.01).The m RNA expression of GDF-9,TGF-β,IGF in ovarian granulosa cells increased in all groups,the m RNA expression of Caspase-3 was increased at first and then decreased in each group.The m RNA expression of GDF-9 and IGF in ovarian granulosa cells decreased at first and then increased in each group,but there was no significant difference among the other groups except Icariin 10μg/L group(P<0.01 or P<0.05).There was no significant difference in the expression of TGF-β between Icariin 10μg/L group and Icariin 50μg/L group,but there was no significant difference in Caspase-3m RNA expression except in Icariin 10μg/L group and Icariin50μg/L group(P<0.0l).There was no significant difference in the expression of Icariin between Icariin 10μg/L group and Icariin 50μg/L group(P>0.05),except in Icariin 10μg/L group and Icariin 50μg/L group,there was no significant difference among the other groups(P>0.05).Conclusion(s): Icariin can promote the proliferation and inhibit the apoptosis of mouse ovarian granulosa cells in vitro,and increase the secretion of progesterone in mouse ovarian granulosa cells.The molecular mechanism is to promote the expression of related proliferation factors in ovarian granulosa cells.