The Role And Mechanism Of Astragaloside IV In The Induction Of Cell Bystander Effects By Radiation Stimulating Solution

Purpose: The aim of the study is to observe the role and its mechanism of Astragalobystander?on LO2 cells exposed to radiation stimulation solution to provide theoretical basis for the development of new radiation-protective drugs.Methods: Constructing a radiation-induced cell bystander effect model using the transfer radiation condition stimulation mode;(1)Using reactive oxygen fluorescent probe to detect the change of intracellular ROS content after ?-ray 0,1,2,4,6,8 Gy irradiation for 1h and transfer conditioned medium for 1h,select the appropriate dose of radiation to construct the bystander effect model.According to the dose,the pretreatment with different concentrations of Astragaloside IV(0,20,40,60,80,100?g/mL)for 3h and then the radiation condition of the stimulating solution for 24 h and then detect the content change of cell ROS to screen the appropriate concentration of Astragaloside IV;(2)The effect of Astragaloside IV on cell viability after irradiation with stimulating solution was detected by CCK-8 method,and the concentration of Astragaloside IV was further screened.There are four groups in this study including NC group,IR group,ICM group and ASIV+ICM group.(3)Growth curve and clone formation experiments were carried out to determine the effects of Astragaloside IV on cell growth rate and colony formation rate after irradiation with stimulating solution;(4)The damage of cell DNA was detected by Micronucleus test;(5)The effect of Astragaloside IV on apoptosis after irradiation with stimulating solution was detected by Annexin V-PI double staining combined with flow cytometry;(6)The effect of Astragaloside IV on the cell cycle after irradiation with stimulating solution was detected by Flow cytometry;(7)Microplate method was used to detect the effects of Astragaloside IV on the activity of total superoxide dismutase SOD,reduced glutathione GSH and malondialdehyde MDA in the cells after irradiation with stimulating solution;(8)Rhodamine 123 fluorescent probe was used to detect the change of cell mitochondrial membrane potential after the action of Astragaloside IV on radiation condition stimulating solution;(9)Western blot was used to detect the effects of Astragaloside IV on Nrf2/Keap1 signal-related proteins Nrf2,Keap1,HO-1 and NQO-1 after irradiation with stimulating solution.Results are as follows:1.Compared with the control group,the intracellular ROS content increased with the increase of radiation dose(P<0.05).Compared with the control group,the 8Gy corresponding conditional stimulating solution had the highest ROS content(P<0.05).Compared with the ICM group,intracellular ROS decreased significantly after treatment with a concentration of 80?g/mL and 100?g/mL of Astragaloside IV(P<0.05).2.The survival rate of the cells was decreased after 24 hours of transfer of the conditioned medium(P<0.05 VS.control group).Compared with the ICM group,the cells were treated with the concentration of 80?g/mL and 100?g/mL.Survival rate increased(P<0.05).3.Compared with the control group,the growth rate of the cells in the ICM group decreased and the colony formation rate decreased(P<0.05).Compared with the ICM group,the growth rate of the cells in the ASIV+ICM group increased and the colony formation rate increased(P<0.05).4.Compared with the control group,the micronucleus formation rate of the ICM group increased after 24 h and 48 h of the conditional stimulating solution(P<0.05).Compared with the ICM group,the micronucleus of the ASIV+ICM group after 48 h The rate was reduced(P<0.05).5.Compared with the control group,the apoptotic rate of ICM group increased after 48 h of the conditional stimulating solution(P<0.05).Compared with the ICM group,the apoptosis rate of ASIV+ICM group decreased at 48h(P<0.05).6.Compared with the control group,the ratio of G2/M phase in IR group and ICM group increased from 4h,peaked at 51.04% and 41.01% at 12 h,and G2/M phase in ASIV+ICM group also started from 4h,increased to reach a peak of 34.86% at 12 h.7.Compared with the control group,the activity of SOD and GSH in the ICM group decreased and the MDA content increased in the ICM group after 48 hours of treatment with the stimulating solution(P<0.05).Compared with the ICM group,the activity of SOD and GSH in the ASIV+ICM group and MDA content decreased(P<0.05).8.Compared with the control group,the membrane potential of mitochondria in the ICM group decreased after 48 h of the conditional stimulating solution(P<0.05).Compared with the ICM group,the membrane potential of the mitochondria in the ASIV+ICM group recovered(P<0.05).9.Compared with the control group,the expression of Nrf2/Keap1 signal-related proteins Nrf2,HO-1 and NQO-1 in ICM group increased after 48 hours of treatment with stimulating solution,and Keap1 decreased(P<0.05).Compared with the ASIV+ICM group,the expressions of Nrf2,HO-1 and NQO-1 decreased,and Keap1 increased(P<0.05).Conclusion:1.The content of ROS and MDA increased after the cells stimulated by the stimulating solution,the cell growth rate decreased,the cell survival rate and the clone formation rate decreased,the micronucleus and apoptosis rate increased,and the cell cycle showed G2/M phase arrest,mitochondria membrane potential decreased;2.Astragaloside IV can increase the production of ROS induced by stimulating solution by reducing the content of GSH and SOD,reduce the formation of micronucleus,reduce the apoptosis rate,reduce the cell cycle G2/M phase arrest,and partially restore the membrane potential of mitochondria.This may be related to the activation of the Nrf2/keap1 signaling pathway by Astragaloside IV.