Neuroprotective effect of hydrogen sulfide against glutamate-induced oxidative stress is mediated via the p53/glutaminase 2 pathway after traumatic brain injury

Abstract

Several reports suggest that hydrogen sulfide (H₂S) exerts multiple biological and physiological effects on the pathogenesis of traumatic brain injury (TBI). However, the exact molecular mechanism involved in this effect is not yet fully known. In this study, we found that H₂S alleviated TBI-induced motor and spatial memory deficits, brain pathology, and brain edema. Moreover, sodium hydrosulfide (NaHS), an H₂S donor, treatment markedly increased the expression of Bcl-2, while inhibited the expression of Bax and Cleaved caspase-3 in TBI-challenged rats. Tunnel staining also demonstrated these results. Treatment with NaHS significantly reduced the glutamate and glutaminase 2 (GLS-2) protein levels, and glutamate-mediated oxidative stress in TBI-challenged rats. Furthermore, we demonstrated that H₂S treatment inhibited glutamate-mediated oxidative stress through the p53/GLS-2 pathway. Therefore, our results suggested that H₂S protects brain injury induced by TBI through modulation of the glutamate-mediated oxidative stress in the p53/GLS-2 pathway-dependent manner.

Keywords: glutamate; hydrogen sulfide; oxidative stress; p53; traumatic brain injury.

Figure 1

H₂S alleviated TBI-induced motor and spatial memory deficits and brain edema. (A) A wire-grip test was performed to analyze the motor function at 0 to 7d after TBI (n = 5). (B) A Rota-rod test was performed to analyze the motor function at 0 to 7d and 14d after TBI (n = 5). (C) A Morris water maze test was performed to test spatial memory ability on days 8 to 15 (n = 5). (D) A spatial probe test was performed to test spatial memory ability on days 16 (n = 5). (E) The pathological changes was analyzed by H&E staining. (F) The brain water content was measured at 24 h after TBI (n = 5). #P<0.05 vs. sham group.* P<0.05, vs. TBI and TBI+ vehicle groups. #P<0.05 vs. sham group.

Figure 2

Treatment with H₂S inhibited TBI-induced apoptosis. (A) Western blot was performed to analyze the protein level of Bcl-2, Bax, and Cleaved caspase-3 in TBI-challenged rats after treatment with NaHS. (B–D) The band density of Bcl-2 (B), Bax (C), and Cleaved caspase-3 (D) was analyzed using Image J. (E) The apoptosis rate of cerebral cortex in TBI rats was analyzed by Tunnel staining. * P<0.05, vs. TBI and TBI+ vehicle groups. #P<0.05 vs. sham group.

Figure 3

Treatment with H2S inhibited TBI-induced glutamate. (A) The level of glutamate in TBI-challenged rats after treatment with NaHS. (B) Western blot was performed to analyze the protein level of GLS-2 in TBI-challenged rats after treatment with NaHS. (C) The band density of GLS-2 was analyzed using Image J. * P<0.05, vs. TBI and TBI+ vehicle groups. #P<0.05, ###P<0.001 vs. sham group.

Figure 4

Treatment with H2S inhibited TBI-induced oxidative stress and HO-1 expression. (A) The level of MDA in TBI-challenged rats after treatment with NaHS. (B, C) The activities of SOD (B) and GPx (C) in TBI-challenged rats after treatment with NaHS. (D) The mRNA level of HO-1 in TBI-challenged rats after treatment with NaHS. (E) The protein level of HO-1 in TBI-challenged rats after treatment with NaHS. (F) The band density of HO-1 was analyzed using Image J. * P<0.05, vs. TBI and TBI+ vehicle groups. #P<0.05, ##P<0.01 vs. sham group.

Figure 5

p53 inhibition reversed the effect of H2S on glutamate and glutamate-mediated oxidative stress after TBI. (A) The protein level of GLS-2 and p53 in TBI-challenged rats after co-treatment with NaHS and pifithrin-α. (B, C) The band density of GLS-2 (B) and p53 (C) was analyzed using Image J. (D) The level of glutamate in TBI-challenged rats after co-treatment with NaHS and pifithrin-α. (E) The level of MDA in TBI-challenged rats after co-treatment with NaHS and pifithrin-α. (F, G) The activities of SOD (F) and GPx (G) in TBI-challenged rats after co-treatment with NaHS and pifithrin-α. * P<0.05, vs. TBI+NaHS+ pifithrin-α group. #P<0.05, vs. TBI+ vehicle group. @P<0.05, @@<0.01, vs. sham group.