The Role Of Proanthocyanidin In The Proliferation Of Female Germline Stem Cells And Ovarian Aging

Objective:Female follicles decreases with increasing age,and ovarian aging caused by follicle pool’s depletion is an unavoidable reproductive health problem for women.With ability of differentiating into oocytes and constantly self-renewing,female germline stem cells（FGSCs）can delay ovarian aging by replenishing primordial follicle pool.Proanthocyanidin is recognized as natural antioxidants that have anti-aging and anti-inflammatory effects.This study aims to explore whether proanthocyanidins can promote the proliferation of female germline stem cells and its relationship with SIRT1 in vitro.Furthermore,we confirmed that proanthocyanidin could delay ovarian aging in vivo.Methods:In the cell experiment,female germline stem cells isolated from mice ovaries were identified by RT-PCR,immunofluorescence staining and alkaline phosphatase staining.After selecting the best administration time and concentration of proanthocyanidins according to CCK-8 results,FGSCs are divided into normal culture group（Control）and proanthocyanidin administration group（PAC,10mg/L）.After 48hours,the mRNA and protein expression levels of FGSCs markers MVH,Oct4 and SIRT1、p53、p21 were determined to explore the effect of proanthocyanidin on FGSCs.In animal experiments,after constructing a pathological ovarian aging model,mice administrated with proanthocyanidin were divided into low-dose group（Treatment-low,100 mg/kg/d）,medium-dose group（Treatment-mid,200 mg/kg/d）,and high-dose group（Treatment-high,400mg/kg/d）.To analyzethe effect of proanthocyanidins on ovarian aging,the change of ovarian function was determined by measuring the ovarian index,the number of follicles,the concentration of serum E₂ and AMH.Results:（1）The results of RT-PCR,immunofluorescence staining and alkaline phosphatase staining showed that the extracted cells conform to the standards of female germline stem cells.（2）The results of CCK-8 showed that proanthocyanidin promotes the proliferation of FGSCs（P<0.05）,while SIRT1 inhibitor EX527 inhibite the proliferation of FGSCs（P<0.001）.q-PCR and WB results showed that proanthocyanidins（10 mg/L）increased the mRNA and protein expression of female germline stem cell markers（MVH,Oct4）as well as SIRT1（P<0.05）,meanwhile,the mRNA and protein of p53 and p21 decreased（P<0.05）.（3）After proanthocyanidins treatment,the ovarian index,follicle number,E₂ and AMH concentration of the mice ovaries were increased（P<0.05）.Conclusions:（1）Proanthocyanidin can promote the proliferation of female germline stem cells,which may be related to the SIRT1-p53-p21 signaling pathway;（2）Proanthocyanidin has a salvage effect on pathological aging of female mice.