Allylmethylsulfide Down-Regulates X-Ray Irradiation-Induced Nuclear Factor-kappaB Signaling in C57/BL6 Mouse Kidney

Abstract

Allylmethylsulfide (AMS), a volatile organosulfur derivative from garlic, has been shown to have radioprotective effects in radiation-challenged cell and animal models, but the mechanism of radioprotection is not well understood. To determine the mechanism of radioprotection in an in vivo model, we first verified the antioxidant capacity of AMS using 2,2'-azobis(2-amidinopropane) dihydrochloride-induced human embryonic kidney 293T cells by measuring reactive oxygen species generation, reduced glutathione, protein tyrosine kinase/protein tyrosine phosphatase balance, and nuclear factor-kappaB (NF-kappaB) protein levels. We then investigated the protective effects of AMS (55 and 275 micromol/kg, intraperitoneal treatment) on 15 Gy X-ray-irradiated mouse kidney. The results showed that AMS decreased the free radical-induced lipid peroxidation in mice exposed to X-rays. Moreover, the antioxidative AMS suppressed the activation of NF-kappaB and its dependent genes such as vascular cell adhesion molecule-1, inducible nitric oxide synthase, and cyclooxygenase-2 through inhibition of IkappaBalpha phosphorylation and activation of IkappaB kinase alpha/beta and mitogen-activated protein kinases (MAPKs). Based on these results, AMS may be a useful radioprotective agent by down-regulating the MAPKs and NF-kappaB signaling pathway that can be induced via X-ray irradiation.

FIG. 1.

Effect of AMS on AAPH-induced (A) ROS generation and (B) GSH depletion in HEK293T cells. Cells were preincubated with or without AMS (1 μM or 5 μM) or N-acetylcysteine (NAC) (200 μM) for 6 hours prior to AAPH treatment. ROS and GSH levels were measured 1 hour after AAPH treatment. Three independent experiments were performed with quintuple assays. Results of one-factor ANOVA followed by Fisher's protected LSD post hoc test were used: **P < .01, ***P < .001 versus untreated control; ^##P < .01, ^###P < .001 versus AAPH-treated cells, respectively.

FIG. 2.

Effect of AMS on AAPH-induced PTK/PTP imbalance in HEK293T cells: (A) PTK and PTP activities and (B) PTK/PTP ratio. Cells were preincubated with or without AMS (1 μM or 5 μM) or N-acetylcysteine (NAC) (200 μM) for 6 hours prior to AAPH treatment. PTK and PTP activities were measured 1 hour after AAPH treatment and applied to measure the PTK/PTP ratio in cell lysate. The balance point is set to the value that PTK/PTP ratio is 1.0 for the control group. Data are mean ± SE values of triplicate assays in five separate experiments. Results of one-factor ANOVA followed by Fisher's protected LSD post hoc test were used: *P < .05, ***P < .001 versus untreated control; ^#P < .05, ^##P < .01, ^###P < .001 versus AAPH-treated cells, respectively.

FIG. 3.

Effect of AMS on NF-κB activation in HEK293T cells. HEK293T cells were grown to 80% confluence in 100-mm-diameter dishes in Dulbecco's Modified Eagle's Medium and then stimulated with AAPH with or without AMS (1 μM or 5 μM) or N-acetylcysteine (NAC) (200 μM) for 6 hours. (Top panel) Western blot was performed to detect p-p65, p65, and p50 protein levels in nuclei (30 μg of protein) from HEK293T cells. Levels were normalized to transcription factor ΠB (TFΠB). One representative blot of each protein is shown from three experiments that yielded similar results. (Bottom panel) Values are the relative optical intensity of each band normalized as a percentage of the untreated control. Results of one-factor ANOVA followed by Fisher's protected LSD post hoc test were used: *P < .05, **P < .01 versus untreated control; ^#P < .05, ^##P < .01, ^###P < .001 versus AAPH-treated cells, respectively.

FIG. 4.

Influence of AMS on lipid peroxidation in X-ray-exposed mouse kidney. MDA+HAE assays were performed as described in Materials and Methods. Data are shown for kidneys of mice 6 hours after 15 Gy irradiation. Data are mean ± SE values representing triplicate assays of five mice from each group (n = 5). Results of one-factor ANOVA followed by Fisher's protected LSD post hoc test were used: ***P < .001 versus nonexposed mouse kidney; ^##P < .01 versus X-ray-exposed mouse kidney, respectively.

FIG. 5.

Effect of AMS on X-ray-exposed phosphorylation of MAPKs in mouse kidney. Mice were treated as described in Materials and Methods. Kidney homogenates were prepared to determine the phosphorylated levels of JNK, ERK, and p38 using western blot. A representative blot is shown from three independent experiments with identical observations (n = 5), and equivalent protein loading was confirmed by probing stripped blots for total JNK, EKR1/2, and p38. Blot density was detected with FluorChem and standardized to a percentage of the untreated group. Results of one-factor ANOVA followed by Fisher's protected LSD post hoc test were used: *P < .05, **P < .01, ***P < .001 versus nonexposed mouse kidney; ^#P < .05, ^###P < .001 versus X-ray-exposed mouse kidney, respectively.

FIG. 6.

Effect of AMS on X-ray-induced activation of NF-κB, phospho-IκBα, and phospho-IKKα/β in mouse kidney. Mice were treated as described in Materials and Methods. The activations of (A) NF-κB and (B) p-IκBα and p-IKKα/β were determined using western blot. One representative blot of each protein is shown from three experiments that yielded similar results (n = 5). Equivalent protein loading was confirmed by probing stripped blots for transcription factor ΠB (TFΠB) and β-actin. Blot density was detected with FluorChem and standardized as a percentage of the untreated group. Results of one-factor ANOVA followed by Fisher's protected LSD post hoc test were used: *P < .05, **P < .01, ***P < .001 versus nonexposed mouse kidney; ^##P < .01, ^###P < .001 versus X-ray-exposed mouse kidney, respectively.

FIG. 7.

Effect of AMS on X-ray-induced activation of NF-κB-dependent genes in mouse kidney. Mice were treated as described in Materials and Methods. Kidney homogenates were prepared to determine the levels of VCAM-1, iNOS, and COX-2 using western blot. A representative blot is shown from three independent experiments with identical observations (n = 5), and equivalent protein loading was confirmed by probing for β-actin. Blot density was detected with FluorChem and standardized as a percentage of the untreated group. Results of one-factor ANOVA followed by Fisher's protected LSD post hoc test were used: **P < 0.01, ***P < .001 versus nonexposed mouse kidney; ^##P < .01, ^###P < .001 versus X-ray-exposed mouse kidney, respectively.