Mechanism of action of chronic low-grade inflammation in ovulation dis-orders of polycystic ovary syndrome
Objective To verify the inflammatory status of ovarian granulosa cells in polycystic ovary syndrome(PCOS)mice,and to investigate the mechanism of chronic low-grade inflammatory status on ovulation disorders of PCOS.Methods Ten SPF healthy female C57BL/6 mice,21-25 d,14-16 g were selected,and they were divided into CONTROL group and PCOS group according to random number table method,with five mice in each group.The PCOS group received daily subcutaneous injection of dehydroepiandrosterone(6 mg/100 g,solvent:a mixture of corn oil and DMSO),while CONTROL group received daily subcutaneous injection of an equal volume of solvent,the drug was administered continuously for 21 d.Hemotoxylin-eosin staining was used to observe the morphology of ovarian tissue in two groups of mice,and the expression level of tumor necrosis factor-α(TNF-α)in ovarian granulosa cells of mice in both groups was detected by immunohistochemical staining.Lipopolysaccharide(LPS)0.1 μg/ml(LPS 0.1 group),0.2 μg/ml(LPS 0.2 group),0.5 μg/ml(LPS 0.5 group),1.0 μg/ml(LPS 1.0 group),and 5.0 μg/ml(LPS 5.0 group)were added to KGN cells,granulosa cell inflammation model was constructed,and KGN cells were used as control group.The expression levels of TNF-αand SMAD4 in KGN cells were detected by Western blot;the expression levels of YTHDF2 in KGN cells in control group and LPS 0.2 group were detected by immunofluorescence.YTHDF2-overexpressed granulosa cell model was constructed by transfecting PC3.1-YTHDF2(PC3.1-YTHDF2 group),and transfected PC3.1 plasmid as control(PC3.1 group).The expression levels of SMAD4 in KGN cells in PC3.1 group and PC3.1-YthDF2 group were detected by Western blot.Results Follicles in the ovaries were present of CONTROL group mice at all stages.Compared with CON-TROL group,there were a large number of immature follicles in ovaries of PCOS mice.Compared with CONTROL group,the expression level of TNF-α in granulosa cells of mice in PCOS group increased(P＜0.05).Compared with control group,the expression levels of TNF-α of KGN cells in LPS 0.2 group,LPS 0.5 group,LPS 1.0 group,and LPS 5.0 group were increased(P＜0.05);the expression levels of SMAD4 of KGN cells in LPS 0.1 group,LPS 0.2 group,LPS 0.5 group,LPS 1.0 group,and LPS 5.0 group were reduced(P＜0.05).Compared with control group,the expression level of YTHDF2 of KGN cells in LPS 0.2 group was increased(P＜0.05).Compared with PC3.1 group,the expression level of SMAD4 of KGN cells in PC3.1-YTHDF2 group was reduced(P＜0.05).Conclusion There is an inflammatory state in granulosa cells of PCOS mice,the inflammatory state of granulosa cells in PCOS can affect ovulation function,and the inflammato-ry state of granulosa cells in PCOS may affect the expression of SMAD4 by regulating YTHDF2,leading to ovulation disorders in PCOS.
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