Study On The Mechanism Of Resveratrol Relieving Granulosa Cell Injury Induced By Aflatoxin B1 In Porcine Ovary

In this study,porcine ovarian granulocyte cells(PGCs)cultured in vitro were used as the research object to understand whether resveratrol could resist the toxic effect of AFB1 on PGCs and whether resveratrol could regulate cell proliferation and apoptosis through Akt/PI3 K pathway.This experiment was divided into four groups,namely blank group control group,AFB1 group,RES group,RES+ AFB1 group,and a series of detection experiments were conducted,including MTT detection of cell viability,fluorescence microscope observation of cell morphological changes and changes in DAPI nuclear staining,and enzyme plate instrument detection of glucose concentration in cell supernatant.Cell reactive oxygen species(ROS)and membrane potential changes were detected by the kit.Cell apoptosis rate was detected by flow cytometry.Expression levels of apoptotic genes(Bad /Bax/Bcl-2/XIAP/Aif),Akt/PI3 K pathway and key upstream and downstream genes(PI3K/Akt/PTEN/Fox O1/m TOR)were detected by qPCR.Western Blot was used to detect the protein expression levels of Akt/PI3 K signaling pathway and related downstream molecules.The results are as follows:1.MTT assay showed that the concentration of RES(8 μM)had the best protective effect on cells,and AFB1(0.7 μM)was the optimal challenge concentration.2.Microscope observation of cell morphology showed that after AFB1 treatment for 24 hours,cell morphology significantly changed compared with blank group and RES group,cell gap increased,and a large number of cells became round,died and fell off.However,compared with the AFB1 intervention group,the phenomenon of cell shrinkage and round was alleviated.And the morphology is relatively regular,it can be seen that RES has a relieving effect on the toxicity of AFB1.DAPI nuclear staining also showed that the number of nuclear staining was significantly reduced in the AFB1 group,and the nuclear concentration became small and dense,while in the RES+AFB1 group,the phenomenon of nuclear concentration became small and dense and dense was significantly alleviated compared with the AFB1 group.3.The kit was used to detect the glucose concentration in the cell superdefinition and calculate the glucose consumption rate.The results showed that compared with the blank group and RES group,the glucose consumption rate of AFB1 was the lowest,while the glucose consumption rate of RES+AFB1 group was significantly increased compared with the AFB1 group.4.The detection results of reactive oxygen species(ROS)showed that compared with the blank group and RES group,the level of reactive oxygen species in the AFB1 group was significantly increased,while the level of reactive oxygen species in the RES+AFB1 group was significantly decreased compared with the AFB1 group.The cell membrane potential test results showed that compared with blank group and RES group,the cell membrane potential of AFB1 group was significantly decreased,while the cell membrane potential of RES+AFB1 group was significantly increased compared with AFB1 group.5.Annexin V-FITC/PI flow analysis showed that the apoptosis rate of blank group,AFB1 group,RES group and RES+ AFB1 group were 6.72%,38.78%,5.35% and 15.82%,respectively.The apoptosis rate of AFB1 group was the highest.The apoptosis rate of RES+AFB1 group was significantly lower than that of AFB1 group.6.The results of qPCR and Western blot analysis showed that RES could affect related genes in the Akt/PI3 K signaling pathway to inhibit apoptosis induced by AFB1.According to the above results,aflatoxin B1 can induce the apoptosis of porcine ovarian granulosa cells with concentration-dependent changes,while resveratrol can effectively alleviate the toxic effects of AFB1 on PGCS.And low concentration of RES can proliferate PGCs.Meanwhile,qPCR and Western blot analysis showed that RES could affect related genes in the Akt/PI3 K signaling pathway to inhibit apoptosis induced by AFB1.