Research Of Resveratrol On The Regulation Of Autophagy And Apoptosis Induced By Ox-LDL In Granulosa Cells Of Ovarian

Polycystic ovary syndrome(PCOS)is a common reproductive endocrine disease that causes female infertility.Follicular development process of patients with PCOS is chaotic and multiple antral follicles are arrested before the selection of follicles.Studies have shown that the development of follicles is closely related to the functional status of granulosa cells.The occurrence of autophagy and apoptosis can regulate the balance between proliferation and death of granulosa cells.Therefore,studying the autophagy and apoptosis of granulosa cells is very important for the exploring of pathophysiological mechanism of PCOS.The production of oxidized low density lipoprotein(ox-LDL)is an important part of lipid metabolism,which is closely related to the occurrence and development of various metabolic diseases such as cardiovascular diseases and diabetes.Ox-LDL levels in serum and follicular fluid were found significantly higher in PCOS patients.Previous studies have found that ox-LDL can induce restorative autophagy or autophagic death in granulosa cells by affecting expression of the corresponding receptors for granulosa cells,including LOX-1 and TLR4.Resveratrol(RES),as a common antioxidant,can reduce the number of antral follicles,increase secondary follicles,reduce the death of granule cell,promote its proliferation and reduce oxidative stress levels.In addition,RES can reduce androgen levels and increase sensitivity of insulin.Many studies have confirmed that RES has certain clinical value in the treatment of PCOS.Whether or not RES has effect on autophagy and apoptosis of granulosa cells has not been reported at present.This study was aimed to explore the regulatory effect of RES on ox-LDL-induced granulocyte autophagy and apoptosis,and to lay a foundation for further investigation of the pathophysiological mechanism of ovulation dysfunction in patients with PCOS.ObjectiveTo explore the regulation and the underlying molecular mechanism of RES on the autophagy induced by ox-LDL in ovarian granulosa cells.To investigate the regulation of RES on apoptosis induced by ox-LDL in ovarian granulosa cells.Material and MethodsThirty patients who received IVF treatment at the Reproductive Center of The First Affiliated Hospital of Zhengzhou University were enrolled into the study.The follicular fluid was collected on the oocyte retrieval day and the granulosa cells were extracted and primary cultured in vitro for 48 hours.The granulosa cells were divided into 4 groups: 100 μg/ml ox-LDL was added to the experimental group 1,30 μM RES was added to the experimental group 2,and 100 μg/ml ox-LDL+30 μM RES was added to the experimental group 3.The control group was not treated.The expression of LOX-1 and autophagy markers including Beclin1,LC3-II/LC3-I and apoptosis marker cleaved caspase3,and other protein including ROS,p-AKT,AKT,p-PI3 K,PI3K,p-Ser2448-mTOR,mTOR was detected 36 hours after the addition.The protein expression of LOX-1,Beclin1,LC3-II/LC3-I,p-AKT,AKT,p-PI3 K,PI3K,p-Ser2448-mTOR and mTOR was detected by Western blot and real-time fluorescence quantification was performed.Polymerase chain reaction(RT-qPCR)was used to detect the mRNA expression of LOX-1,and ROS kit was used to detect intracellular ROS content.To explore RES on the regulation of autophagy and apoptosis induced by ox-LDL in ovarian granulosa cells.Results1.The expression of Beclin1,LC3-II/LC3-I and cleaved caspase3 were higher in ox-LDL group than in the control group(Beclin1: P<0.001,LC3-II/LC3-I: P<0.001,cleaved caspase3: P<0.001).The expression of Beclin1,LC3-II/LC3-I and cleaved caspase3 in ox-LDL+RES treatment group was lower than in ox-LDL treatment group(Beclin1: P<0.01,LC3-II/LC3-I: P<0.001,cleaved caspase3: P<0.001).2.The expression of LOX-1 mRNA in ox-LDL treatment group was higher than in control group(P<0.001).The LOX-1 mRNA in ox-LDL+RES treatment group was lower than in ox-LDL treatment group(P<0.001).The expression of LOX-1 in ox-LDL treatment group was higher than in control group(P<0.001),and LOX-1 in ox-LDL+RES treatment group was lower than in ox-LDL treatment group(P<0.01).3.The ROS in the ox-LDL treated group was higher than in the control group(P<0.001).The ROS content in the ox-LDL+RES treated group was lower than in the ox-LDL treated group(P<0.001);ox-LDL the phosphorylation levels of AKT,PI3 K,and mTOR in the treatment group were lower than those in the control group(PI3K: P<0.001,AKT: P<0.001,mTOR: P<0.001).The phosphorylation levels of AKT,PI3 K,and mTOR in the ox-LDL+ RES group were high.In the ox-LDL treatment group(PI3K: P<0.001,AKT: P<0.05,mTOR: P <0.01).Conclusions1.RES can inhibit autophagy and apoptosis of granulose cells induced by ox-LDL.2.RES can inhibit the autophagy interfered with PI3K/AKT/mTOR signaling pathway,thereby inhibiting autophagy induced by ox-LDL through down-regulating expression of LOX-1 expression and reducing intracellular content of ROS in granular cell.