| BackgroundMammalian follicle is the basic functional unit of the ovary,consisting of an oocyte and surrounding granulosa cells.Primordial follicles develop from the fetal period,and the total amount does not increase after birth.The number of surviving primordial follicles in the ovary determines the reproductive reserve level of the mother.Activation of primordial follicles occurs during puberty and goes through each menstrual cycle from primary,secondary,preantral,and antral follicles to subsequent recruitment and ovulation of the dominant follicle.In the process of follicle development and egg maturation,the granulosa cells around the oocyte are the most important somatic cells in the follicle.They participate in the regulation of the growth of theca cells and oocytes by synthesizing and secreting a variety of sex hormones and growth factors.Differentiation and maturation are critical for primordial follicle activation and subsequent follicular development.Abnormal follicle development can lead to diseases such as polycystic ovary syndrome(PCOS),premature ovarian failure(POF),empty follicle syndrome(EFS),and ultimately infertility.The parietal granulosa cells that develop to the antral follicle stage regulate oocyte meiotic maturation by secreting EGF-like factor.Urs et al.found that granulosa cells are an important supporting factor for follicular development,and their mitochondrial function directly affects oocyte quality.Kuntai Capsules are mainly used to assist estrogen and progesterone in hormone replacement therapy in the field of reproductive medicine,and are often used clinically for the treatment of perimenopausal syndrome.Animal studies have shown that Kuntai Capsules can alleviate the clinical symptoms of premature ovarian failure through the Phosphatidylinositol 3 kinase/Protein kinase B(PI3K/AKT)signaling pathway.Berberine is one of the important active ingredients of Kuntai Capsules,which was widely used in fertility clinic.In view of its good anti-inflammatory and antibacterial effects,it is commonly used in clinical treatment of dysentery and diarrhea in adults and children.In addition,Berberine also has the effects of anti-tumor,lowering blood lipids,and improving metabolic syndrome.Zhang et al.reported that Berberine can resist oxidative stress injury through the Pl3K/AKT/Bcl-2 pathway,reduce inflammatory response,and protect nerve cells.In the field of reproductive medicine,studies of Berberine have found it can assist metformin in improving insulin resistance in PCOS patients.Nevertheless,the molecular mechanism of Berberine’s effect is still unclear,especially the role and molecular mechanism of berberine in regulating ovarian follicle development.In view of this,this study used in vitro cultured granulosa cells and neonatal mice as models to elucidate the regulatory effect of Berberine on granulosa cell function and the molecular mechanism of its regulatory effect on ovarian follicle development,and further explored its potential role in follicular development.Objective1.To determine the dose range of Berberine in human ovarian granulosa cells.2.To explore the molecular mechanism of Berberine regulating ovarian granulosa cell proliferation and hormone secretion.3.To explore the molecular mechanism of Berberine regulating ovarian follicle development.4.To explore the clinical application value of Berberine on women’s clinical reserve and ovarian function.MethodHuman ovarian primary granulosa cells were extracted from follicular fluid during oocyte retrieval from patients undergoing in vitro fertilization and embryo transfer(IVF-ET).The level of estradiol in the in vitro culture supernatant of human primary granulosa cells was determined by electrochemiluminescence detection system.Realtime fluorescence quantitative polymerase chain reaction technology and Western blot analysis technology were used to determine the expression levels of granulosa cell apoptosis indicators Caspase-3,Bax,Bcl-2,steroid synthesis key genes StAR and P450arom.The growth curves of granulosa cells and KGN cell lines were determined by cell proliferation analysis CCK-8 method,and the expression levels of related proliferation target Ki-67 and apoptosis related target Bcl-2 were detected by immunofluorescence staining and western blot analysis.The fluorescence level of oxidative stress damage indicator DCF was quantitatively detected by fluorescence microplate reader,and the fluorescence level of mitochondrial membrane potentialrelated indicator JC-1 polymer/monomer was quantitatively detected by JC-1 fluorescent probe method under fluorescence microplate reader.In human primary granulosa cells and KGN cell lines,hydrogen peroxide was used to induce oxidative stress injury in granulosa cells to create a ROS model,and the cell proliferation curve was analyzed by CCK-8 method.model,and the early follicular development status was detected by HE staining.Results1.The effect of Berberine on ovarian granulosa cells is dose-dependent.Berberine at low concentration can maintain the growth state of granulosa cells,while Berberine at concentrations of 50 μM and above has inhibitory effect on cell growth.In the control group,the primary granulosa cells gradually deformed,the number of adherent cells gradually decreased,and the cell colonies gradually dissipated,while the low-concentration Berberine treatment group(5μM,10μM)in vitro cultured for 72h was better than the control group.2.Berberine treatment significantly increased the viability of primary granulosa cells cultured for 72h and 120h in vitro(p<0.001).The activity of KGN cells in the Berberine-treated group was significantly higher than that in the control group(p<0.001).In addition,Berberine treatment up-regulated the expression level of the anti-apoptotic protein Bcl-2 in primary granulosa cells,and increased the expression of immunofluorescence proliferation index Ki-67 under the condition that the cell density was basically the same.3.Berberine treatment significantly reduced the secretion level of estradiol in granulosa cells in vitro(p<0.001),down-regulated the expression levels of key steroid synthesis proteins P450arom and StAR in granulosa cells,and significantly downregulated the mRNA expression levels of P450arom and StAR genes.(p<0.01).4.Berberine treatment significantly recovered the decrease of granulosa cell viability(p<0.001),the increase of reactive oxygen species,the decrease of mitochondrial membrane potential and the deterioration of cell morphology caused by hydrogen peroxide stress.5.Berberine treatment significantly upregulated the expression level of p-AKT protein,while treatment with PI3K inhibitor LY294002 reversed the protective effect of Berberine on reactive oxygen species levels and mitochondrial membrane potential in granulosa cells.Berberine treatment significantly increased the activation rate of primary ovarian follicles in D4 neonatal mice(p<0.01),inhibited the follicular atresia caused by the PI3K/AKT pathway inhibitor LY294002,and maintained the survival of primordial follicles.6.Berberine treatment increased the expression level of p-AKT,an immunofluorescence indicator in mouse ovary,and increased the mRNA expression level of PI3K/AKT pathway genes(p<0.05),and decreased the mRNA expression levels of apoptosis genes Bax and Caspase-3(p<0.05).ConclusionBerberine treatment increases the viability of granulosa cells in vitro by activating the PI3K/AKT signaling pathway,promotes the proliferation of granulosa cells,resists cellular oxidative stress damage,and promotes the activation of primary follicles,inhibits follicular atresia,and is conducive to maintaining primordial follicles survival in the follicle pool. |
