18-?Glycyrrhetinic Acid Suppress Radiation-induced Inflammatory Responses In Macrophages Through NADPH Oxidase-ROS/P38MAPK Signaling Pathway

Objective:Observing the effect of 18-?Glycyrrhetinic acid on radiation-induced inflammatory reaction in Macrophages and explore its potential molecular mechanism.Methods:1.Raw 264.7 macrophage cells were cultured in vitro and divided into five groups to observe whether 18-?Glycyrrhetinic acid have inhibitory effect on radiation-induced inflammatory reaction,including control group(none irradiated)as group A,solvent control group(1%DMSO+irradiation)as group B,GA5(GA5ug/ml+ irradiation)as group C,GA10(GA10ug/ml+ irradiation)as group D and GA20(GA20ug/ml+irradiation)as group E.Using MTT method to determine the cytotoxicity of 18-PGlycyrrhetinic acid firstly,and then treating cells with drug or 4Gy X-ray irradiation.Reactive Oxygen Species(ROS)production in each group was detected by Luciferase ELIAS A and the concentration of IL-1?,IL-6 and TNF-a in cellular mediums were detected by ELISA.2.Raw264.7 cells were cultured in vitro and divided into six groups.control group(none irradiated)as group A,solvent control group(1%DMSO+irradiation)as group B,GA10(GA10ug/ml+ irradiation)as group C,only drug(GAlOug/ml)as group D,NADPH oxidase inhibitor(diphenyl iodide DPI+ irradiation)as group E and p38MAPK inhibitor(SB203580+ irradiation)as group F.At first,using MTT method to determine the dual cytotoxicity of 18-?Glycyrrhetinic and X ray on cells,and then and then treating cells with drug or 4Gy X-ray irradiation.Using Luciferase ELIASA to detect the production of Reactive Oxygen Species(ROS)in group A,B,C,D and E.Nicotinamide Adenine Dinucleotide Phosphate(NADPH)was detected by cytochrome C reduction method in group A,B,C,D and E.Using cell-based ELISA to detect levels of Phosphorylated p38MAPK and Phosphorylated NF-kB in group A,B,C,D and E.DNA-binding activity of NF-kB and AP-1 were detected by electrophoretic mobility shift assays(EMSA).mRNA expression of IL-1?,IL-6 were measured by RT-PCR in group A,B,C,D and E.The concentration of IL-1? and IL-6 in the cell culture medium of A,B,C,D and F were detected by ELISA.Results:1.After 4Gy X-ray irradiation exposure to mouse macrophage RAW264.7 cells,generation of ROS increased(P<0.01),secretion levels of IL-1? and IL-6 increased(P<0.01),but no increase in TNF-a secretion(P>0.05).Compared with the irradiated group,GA can inhibit the secretion of IL-1? and IL-6 in cells treated with irradiation(P<0.01)and reduce the production of ROS(P<0.01).2.After mouse macrophage RAW264.7 cells exposure to 4Gy X-ray irradiation,the production of ROS increased in cells(P<0.01),the activition NADPH oxidase increased(P<0.01),p38MAPK and NF-kB phosphorylation levels increased(P<0.01,P<0.01),DNA-binding activity of NF-kB and AP-1 increased(P<0.01,P<0.01),the mRNA expression and secretion of IL-1? and IL-6 increased(for expression and secretion,P<0.01).GA and DPI can inhibit the generation of ROS after irradiation(P<0.01,P<0.01)and can also inhibit the NADPH oxidasic activity.GA and SB203580 can reduce phosphorylation level of NF-kB(P<0.01)and DNA-binding activity of NF-kB and AP-1(P<0.01,P<0.01).Only GA reduced phosphorylation level of p38MAPK,whereas SB203580 can not reduce the phosphorylation level(P<0.01,P>0.05).Conclusions:1 18-?Glycyrrhetinic acid can significantly reduced the irradiation-induced inflammatory responses in Raw 264.7 macrophage cells;2 18-pGlycyrrhetinic acid inhibits radiation-induced inflammation in Raw 264.7 macrophage cells may through NADPH oxidase-ROS/P3 8MAPK signaling pathway.