A Preliminary Study Of Quercetin Alleviating Endoplasmic Reticulum Stress-induced Apoptosis In Buffalo Follicle Granulosa Cells

There are thousands of follicles on the ovary of mammals,but most of the follicles will be atresia during the development of follicles,which will affect the fertility of female animals and cause a waste of excellent female resources.Apoptosis of granulosa cells is one of the main causes of follicular atresia,so exploring the apoptosis of granulosa cells is very important to improve the fecundity of female animals.Endoplasmic Reticulum Stress can induce apoptosis and affect the normal function of cells.However,the effect of endoplasmic reticulum stress on apoptosis and function of buffalo(Bubalus bubalis)granulosa cells is unclear.As a plant-derived flavonoid,Quercetin QC can protect cells through various biological pathways such as antioxidant and anti-inflammatory.Whether QC can alleviate the effect of endoplasmic reticulum stress on buffalo granulosa cells remains to be explored.Therefore,in this study,buffalo granulosa cells were used as the research object,and tunicamycin was used to construct an endoplasmic reticulum stress model of buffalo granulosa cells to explore the effect of endoplasmic reticulum stress on buffalo granulosa cells.Alleviating the effect of endoplasmic reticulum stress on buffalo granulosa cells.The main research contents of this paper are as follows:1.Isolation and identification of buffalo granulosa cells and construction of endoplasmic reticulum stress model.Buffalo granulosa cells were obtained by isolating and purifying buffalo follicular fluid and identified by cell immunofluorescence.The results showed that FSHR protein fluorescence signal was detected in all the obtained cells,indicating that the isolated cells were all buffalo granulosa cells.The endoplasmic reticulum stress model of buffalo granulosa cells was constructed by adding tunicamycin,and the apoptosis rate was detected by flow cytometry,and the changes of apoptosis and endoplasmic reticulum stress-related genes and proteins were detected by q RT-PCR and Western Blot.The results showed that treatment of granulosa cells with 2.5μg/m L tunicamycin for 48 h significantly increased the apoptosis rate of granulosa cells;q RT-PCR analysis showed that the expression of pro-apoptosis-related genes was up-regulated,and the expression of anti-apoptotic-related genes was down-regulated,the expression of endoplasmic reticulum stress-related genes increased significantly;Western blot analysis confirmed that the changes in protein expression were consistent with the changes in m RNA.It indicated that the model of endoplasmic reticulum stress in buffalo granulosa cells was successfully constructed.2.QC alleviates the apoptosis of buffalo granulosa cells caused by endoplasmic reticulum stress.Granular cells were pretreated with 2 μg/m L QC for 8 h,and then an endoplasmic reticulum stress model was constructed.Changes in stimulator-related genes and proteins.The results showed that QC pretreatment significantly reduced the apoptotic rate and intracellular ROS(Reactive Oxygen Species)activity of granulosa cells,and at the same time could significantly reduce the expression of pro-apoptotic and endoplasmic reticulum stress-related genes and proteins in granulosa cells,and increased anti-apoptosis.expression of genes and proteins.In order to further explore the mechanism of QC alleviating endoplasmic reticulum stress,the pc DNA3.1-CHOP overexpression plasmid was transfected to explore the mechanism of QC on endoplasmic reticulum stress.The results showed that the apoptosis rate and ROS activity were significantly enhanced after overexpression of Chop gene,while the ROS activity and apoptosis rate of granulosa cells were significantly decreased after QC pretreatment.This indicated that QC pretreatment could alleviate the effects on granulosa cells by regulating the endoplasmic reticulum stress pathway.3.QC alleviates the effect of endoplasmic reticulum stress on the function of granulosa cells.The contents of estradiol(E2)and progesterone(PG)in granulosa cells were determined by ELISA,and it was found that endoplasmic reticulum stress could inhibit the secretion of E2 and PG in granulosa cells,and QC pretreatment could promote E2 and PG secretion.PG secretion.The expression levels of E2 and PG synthesis and secretion-related genes and proteins were detected by q RT-PCR and western blot.The results showed that after endoplasmic reticulum stress,the expression of CYP19A1 and HSD3B1 genes and proteins decreased.After QC pretreatment,CYP19A1 and HSD3B1 The expression levels of genes and proteins were significantly up-regulated.The above results indicated that endoplasmic reticulum stress in buffalo granulosa cells inhibited the synthesis and secretion of E2 and PG,while QC pretreatment significantly increased the synthesis and secretion of E2 and PG.In conclusion,this study successfully established an endoplasmic reticulum stress model in buffalo granulosa cells,which clarified that QC inhibited the apoptosis of buffalo granulosa cells and promoted steroid hormone secretion by alleviating endoplasmic reticulum stress.The mechanism of reticulum stress provides a new idea for using QC to relieve buffalo follicle atresia and improve the reproductive performance of female buffalo.