Astragalus Polysaccharide Inhibits Ionizing Radiation-Induced Bystander Effects by Regulating MAPK/NF-kB Signaling Pathway in Bone Mesenchymal Stem Cells (BMSCs)

Abstract

BACKGROUND This study investigated the effect of Astragalus polysaccharides (APS) on radiation-induced bystander effects (RIBE) in human bone mesenchymal stem cells (BMSCs) induced by irradiated A549 cells. MATERIAL AND METHODS A549 cells were irradiated with 2 Gy X-rays to obtain conditioned medium. BMSCs were incubated with the conditioned medium or APS. The levels of reactive oxygen species (ROS) and TGF-β were detected by ELISA. Cell survival, genomic instability, and DNA damages were detected by CCK-8 assay, colony formation assay, the micronucleus test and immunofluorescence assay, respectively. The protein and phosphorylation protein expression of p38, c-Jun N-terminal kinase (JNK), extracellular regulated protein kinase (ERK1/2), P65, and cyclooxygenase-2 (COX-2) in bystander effect cells were detected by Western blot. RESULTS The expression of COX-2 and ROS increased following stimulation with conditioned medium; this effect was inhibited by pre-exposing the cells to APS. BMSCs growth and colony formation rate decreased following stimulation with conditioned medium; this effect was suppressed by pre-exposing the cells to APS. In addition, the micronucleus rate and 53BP1 foci number increased after treatment with conditioned medium; this increase in BMSCs was inhibited by APS. The levels of phosphorylated p38, JNK, ERK1/2, NF-κB P65, and COX-2 proteins were increased by conditioned medium but were decreased by pre-treatment with APS. CONCLUSIONS RIBE in BMSCs induced by the irradiated A549 was mediated by the ROS in the conditioned medium and might be related to MAPK/NF-κB signal pathways in BMSCs. APS may block RIBE through regulating the MAPK/NF-κB pathway.

Figure 1

Analysis of the growth of the bystander BMSCs. BMSCs were divided into the control group, APS group, CM group, and APS+CM group. (A) Cell proliferation was detected with CCK-8 assay. (B) Colony formation by cells of each group. Compared with control group or APS group, * P<0.05, ** P<0.01. Compared with CM group, ^# P<0.05, ^## P<0.01.

Figure 2

Genomic instability analysis of the bystander BMSCs. BMSCs were divided into the control group, APS group, CM group, and APS+CM group. (A) Frequency of micronuclei (MNF) in bystander cells at 24 h were calculated and compared. (B) The number of 53BP1 foci per 100 cells at indicated time points was calculated and compared. (C) Representative images of the 53BP1 foci in each group at 2 h. Compared with control group or APS group, * P<0.05, ** P<0.01. Compared with CM group, ^# P<0.05, ^## P<0.01.

Figure 3

Analysis of protein levels in the bystander BMSCs. BMSCs were divided into the control group, APS group, CM group, and APS+CM group. Western blot was used to detect levels of JNK, p-JNK (A), ERK, p-ERK (B), P38, p-P38 (C) and, NF-κB, and p- NF-κB (D). Representative and quantitative Western blot results are shown, respectively. Compared with control group or APS group, * P<0.05, ** P<0.01. Compared with CM group, ^# P<0.05, ^## P<0.01.

Figure 4

The COX-2 protein level in the bystander BMSCs. BMSCs were divided into the control group, APS group, CM group, and APS+CM group. Western blot was used to detect COX-2 level in bystander cells at 6 h and 12 h after co-culturing with conditioned medium and APS. Representative (A) and quantitative Western blot results (B) were shown, respectively. Compared with control group or APS group, * P<0.05, ** P<0.01. Compared with CM group, ^# P<0.05, ^## P<0.01.

Figure 5

The levels of ROS and TGF-β in conditioned medium and in supernatant of the bystander BMSCs. The levels of ROS and TGF-β were detected by ELISA. The conditioned medium was harvested at 0 h, 0.5 h, 2 h, 3 h, 6 h, 8 h, and 12 h after exposure to X-rays. The supernatant of each experiment group was collected at 0 h, 3 h, 6 h, 9 h, 12 h, 15 h, and 18 h after co-culturing with conditioned medium. (A) The relative concentrations of TGF-β in conditioned medium. (B) The relative concentrations of ROS in conditioned medium. (C) The relative concentrations of ROS in supernatant of each experiment group. Compared with control group or APS group, * P<0.05, ** P<0.01. Compared with CM group, ^# P<0.05, ^## P<0.01.

Figure 6

A model of RIBE in this study. In summary, the ROS delivered from irradiated cells and bystander cells can inhibit cell growth, induce DNA DSB, and cause genomic instability. The activated MAPK/NF-κB pathways synergistically increase cellular injury. APS might block RIBE through regulating the MAPK/NF-κB pathway.