Improvement Of DHT-induced Ovarian Granulosa Cell Dysfunction And Abnormal Lipid Metabolism Via PPARγ-mediated Action Of Atractylenolide Ⅰ

With the popularization of health concepts and the acceleration of the aging process,people’s demand for functional foods is increasing,which has also become a hot topic in the food field.Atractylodes macrocephala Koidz is a food with both medicinal and edible properties,and it is listed in the“List of Items that Can Be Used for Health Foods”.AtractylenolideⅠ（AO-I）is the main active ingredient of Atractylodes macrocephala Koidz.At present,most of the research on Atractylodes Macrocephala Extract（AME）and atractylenolide focuses on digestion and anti-cancer aspects,while there are few studies on its effects on ovarian function.It has been reported that Atractylodes macrocephala Koidz has some effects on polycystic ovary syndrome（PCOS）rats,but its mechanism is not clear.Therefore,this study used dihydrotestosterone（DHT）to induce ovarian granulosa cell line cells（KGN）to construct a model of ovarian dysfunction caused by hyperandrogenemia.Based on this model,the main research includes:（1）DHT induces KGN cells to establish a model of ovarian dysfunction caused by hyperandrogenemia.The impact of DHT on cell viability was assessed using the CCK-8 assay kit.（2）The effects of Atractylodes macrocephala Extract（AME）,AO-I,and AO-III on the proliferation and apoptosis of KGN cells exposed to DHT were investigated.The CCK-8 assay and flow cytometry were used to assess the effects of AME on KGN cells.The results showed that high concentrations of DHT(10^-4 M)reduced the proliferation of KGN cells but had no significant effect on cell apoptosis.AME was found to reverse the decrease in KGN cell proliferation induced by DHT.AO-I and AO-III are the major active components of AMK,and their pharmacological effects were compared and screened using the CCK-8.The results showed that AO-I can reverse the decrease in KGN cell proliferation induced by DHT,while AO-III had no significant effect on the cell viability induced by DHT.Therefore,AO-I was selected as the main component for further study.（3）Effects of AO-I on ovarian function and lipid metabolism under the induction of hyperandrogenic.The secretion of estradiol was measured by enzyme-linked immunosorbent assay（ELISA）.DHT caused an abnormal increase in estradiol secretion in KGN cells.AO-Ⅰsignificantly reversed the estradiol secretion in KGN cells,with no significant difference compared with the normal group.The expression of CYP19 in KGN cells was detected by real-time fluorescence quantitative PCR（q PCR）and protein immunoblotting（Western Blot,WB）,and the results showed that compared with the normal group,DHT significantly increased the expression of CYP19,while AO-Ⅰsignificantly downregulated the expression of CYP19,with no significant difference compared with the normal group.In terms of lipid metabolism,the probe staining method was used to detect lipid droplet formation and fatty acid uptake in KGN cells.The results showed that under DHT induction,lipid droplet formation increased significantly,while fatty acid uptake had no significant difference.After AO-Ⅰreversal,lipid droplet formation decreased significantly,with no significant difference compared with normal cells.To further explore the mechanism of action,q PCR was used to detect the expression of FABP4 and C/EBPα.The results showed that DHT significantly upregulated the expression level of FABP4 and C/EBPαm RNA.Under AO-Ⅰintervention,FABP4 and C/EBPαexpression decreased significantly,with no significant difference compared with the normal group.（4）Based on PPARγ,AO-Ⅰregulates its downstream targets CYP19,FABP4 and C/EBPα,thereby affecting the DHT-induced ovarian function and lipid metabolism.The main contents include:1)The effect of AO-Ⅰas a PPARγagonist.q PCR,WB,and immunofluorescence（IF）were used to detect the expression of PPARγand histone deacetylase 3（HDAC3）genes in KGN cells.The results showed that AO-Ⅰsignificantly increased the expression of PPARγin KGN cells under DHT induction,but had no significant effect on the expression of HDAC3.2)AO-Ⅰreversed the abnormal estradiol secretion and lipid metabolism disorder caused by the PPARγinhibitor.The results showed that compared with the normal group and inhibitor group（GW9662）,AO-Ⅰsignificantly reduced the abnormal increase of estradiol secretion and CYP19protein expression caused by GW9662 in KGN cells,while reducing lipid generation,lowering FABP4 and C/EBPαprotein expression.In summary,AO-Ⅰactivates PPARγ,further regulates CYP19,and reverses the hormonal imbalance induced by DHT in KGN cells.It also affects its downstream genes FABP4 and C/EBPαby regulating PPARγ,and reverses the lipid metabolism disorder induced by DHT in KGN cells.This also provides a theoretical basis for Atractylodes macrocephala Koidz as a functional food.