RMP Inhibits Irradiation-induced Apoptosis Of HCC Cells Through Reactive Oxygen Species

Objective:In this study,we explored the effect of RMP on radiation-induced apoptosis and intracellular reactive oxygen species level in hepatocellular carcinoma cells.Methods:1?The xenografts of hepatocellular carcinoma(HCC)cells in nude mice were applied to determine the effects of RMP on xenografts formation.The growth curves of xenografts were drown and HE staining was performed.2?Immunohistochemical staining(IHC)was applied to detect the expression of RMP,proliferating factor Ki67,DNA damage factor ?-H2AX,and p-p65 in the xenografts of nude mice.3?IHC was applied to detect the expression of apoptosis-related factor Bcl-xl in the xenografts of nude mice.4?The reactive oxygen assay kit was applied to detect the effect of RMP on the intracellular ROS level after irradiation in HepG2 and 7701.5?The reactive oxygen assay kit was applied to detect the effect of different doses RMP on HepG2 and 7701 intracellular ROS level after irradiation.6?After the treatment of irradiation,cell apoptosis of HepG2 was measured by flow cytometry,meanwhile the intracellular ROS level was measure7?Western blot was applied to detect the effect of RMP on oxidative stress d.related factor GSS,Catalase,TrxRl,Nrf2 and TrXl protein expression in irradiation treated HepG2.8?Western blot was applied to detect the effect of RMP doses on TrX1 protein expression in irradiation induced HepG2.Results:1?The results of xenografts in nude mice showed that,compared with the control group,the tumor volume of RMP overexpression group increased significantly and the RMP interference group decreased significantly.The HE staining showed that,compared to the control group,the cell density of RMP overexpression group was much higher and that of the RMP interference group was lower.2?IHC results showed that the expression of proliferation factor Ki67 was significantly increased in the RMP overexpression group,and decreased in the RMP interference group either with or without irradiation.Before irradiation,the expression of DNA damage factors y-H2AX and p-p65 in the RMP overexpression and interference gyroups showed no significant change.After irradiation,the expression of DNA damage factors y-H2AX was significantly decreased,and p-p65 was increased.3?IHC results showed that,compared with the RMP control group,the expression of cell apoptosis gene Bcl-xl in the RMP overexpression group was significantly higher either with or without irradiation,and the expression of Bcll-xl in the RMP interference group was significantly lower.4?The Reactive Oxygen Species assay showed that,compared with the control group,the ROS level in HepG2 in the RMP overexpression group decreased significantly,and the ROS level in HepG2 in the RMP interference group increased significantly after irradiation.The HepG2 intracellular ROS level decreased with the increased concentration of RMP overexpression plasmid,and the HepG2 intracellular ROS level increased with the increased concentration of RMP interfering plasmid.5?The Reactive Oxygen Species sssay showed that,compared with the control group,the ROS level of 7701 in the RMP overexpression group and RMP interference group has no significant change after irradiation.When increasing the concentration of RMP overexpression plasmid or RMP interfering plasmid,the 7701 intracellular ROS level has no significant change.6?Flow Cytometer showed that,compared with the RMP control group,the apoptosis of HepG2 in the Nac group was significantly decreased before irradiation.After 24 hour of irradiation,the apoptosis of the RMP overexpression group and Nac group was significantly reduced,RMP interference group was significantly increased.The ROS assay kit results showed that,after 24 hour irradiation,the ROS level of the RMP overexpression group and the Nac group was significantly decreased,the RMP interference group was significantly increased.7?Western blot results showed that,compared with the RMP control group,there was no significant difference in the expression of GSS,Catalase or TrxRl between RMP overexpression group and RMP interference group.After irradiation,the expression of Nrf2 and TrX1 in the RMP overexpression group was much higher.In the RMP interference group,the expression of Nrf2 was not significantly changed,and the expression of TrXl was significantly decreased.8?Western blot results showed that,after irradiation,the expression of TrXl increased with the increased concentration of RMP overexpression plasmid,and the expression of TrXl decreased with the increased concentration of RMP interfering plasmid.Conclusion:1?RMP promoted DNA damage repair and inhibited irradiation-induced apoptosis in hepatocellular carcinoma cells.2?RMP inhibited irradiation-induced reactive oxygen species in HepG2 cells through promoting the expression of oxidative stress factors Nrf2 and TrX1.