ABSTRACT
Objective To investigate the effects and mechanisms of glutamine (Gln) supplementation on oxidative stress,autophagy response and neurobehavioral outcome after traumatic brain injury (TBI) in rats.Methods TBI animal models were established using Feeney's method.Eighty SD rats were randomly divided into 4 groups:sham operation group (group Sham),Sham + glutamine supplementation group (group Sham+ GLN),traumatic brain injury group (group TBI),and TBI + glutamine supplementation group (group TBI+ GLN).We measured rat behavioral outcomes by modified neurologic severity score (mNSS) tests at day 1,3,7 and 14 after TBI.The apoptosis neurons in TBI cerebral cortex were determined by TUNEL staining.The expression of reactive oxygen species (ROS) was tested by ROS kits.Oxidative stress and autophagy related cytokines (HO-1,NQO1,Nrf2,LC3-Ⅱ and Beclin-1) were tested with Western blotting.Results Compared with the TBI group,the neurological function was improved [(9.79±0.43) vs.(8.43±0.30),F =6.775,P =0.010] and the apoptosis rate decreased (19.88％ ± 1.60％ vs.15.35％ ± 1.28％,P =0.013) in the TBI+ GLN group after 7-day treatment.Compared with the Sham group,the protein expression of ROS increased (P=0.000),and the expression of anti-oxidative stress factors (HO-1,NQO1) and Nrf2 pathway significantly decreased in the TBI group.After glutamine supplementation was given,the expression of ROS decreased and the expressions of HO-1 and NQO1 increased.The Nrf2 pathway and autophagy response also were activated with the expressions of Nrf2,LC3-Ⅱ and Beclin-1 increasing.Conclusion Glutamine supplementation can markedly reduce neuron apoptosis and improve neurological outcomes after TBI,thus has the protective effect on nerves by inhibiting TBI-induced oxidative stress response,activating Nrf2 pathway and autophagy response.
ABSTRACT
Objective To investigate the effects of glutamine (Gln) supplementation on neurologica severity score,brain edema,neuron apoptosis,and endoplasmic reticulum stress (ERS) response after traumatic brain injury (TBI) in rats.Methods TBI rat models were established using modified Feeney's method.Eighty Sprague-Dawley rats were divided into 4 groups with a random number table:sham operation group (Sham group),TBI group,Gln supplementation group (TBI + Gln group) and ERS inducer 2-deoxy-D-glucose group (TBI +Gln + 2-DG group).We measured the rats' neurobehavioral outcomes by modified neurologic severity score (mNSS) on day 1,3,7 and 14 after TBI.Neuron apoptosis was detected using TUNEL staining.Brain water content was measured with wet-dry weight method.The apoptosis-related protein (caspase-12,caspase3,and Bcl-2) and ERS-related cytokines [inositol-requiring enzyme 1 (IRE-1),C/EBP homologous protein (CHOP)] expressions in TBI cerebral cortex were determined by immunohistochemistry staining and Western blot.Results Compared with the Sham group,the levels of brain edema,mNSS,apoptosis-related protein (caspase-12,caspase-3,Bcl-2) and ERS-related proteins (IRE-1,CHOP) were significantly increased in the other three groups (all P =0.00).Compared with the TB1 group,the TBI +Gln group showed significant lower brain water content [3 d:(81.39±0.59)％ vs.(83.54±0.52)％,P=0.04;7 d:(74.86±0.38)％ vs.(77.32±0.66)％,P=0.03],improved mNSS (8.63 ±0.22 vs.10.37±0.29,P=0.03),suppressed expressions of apoptosis-and ERS-related proteins (caspase-12,caspase-3,IRE-1,and CHOP)(P =0.01,P ＜ 0.01),and increased expression of anti-apoptotic protein Bcl-2 (P =0.02).Compared withthe TBI + Gln group,the expression of ERS-related factors (IRE-1 and CHOP),brain edema level,and neurological severity were increased in the TBI + Glu + 2-DG group.Conclusion Glutamine supplementation may have neuroprotection function,demonstrated as reducing brain edema and neuron apoptosis,and improving neurobehaviroal outcomes after TBI,possibly mediated by inhibiting TBI-induced ERS response.
ABSTRACT
Objective To investigate the effects of omega-3 polyunsaturated fatty acids (ω-3 PUFA)supplementation on brain edema,autophagy response and neurobehavioral outcome after traumatic brain injury (TBI) in rats and the related mechanisms.Methods TBI rat models were established using Feeney's method.Seventy-two SD rats were divided into 4 groups using random number table:sham operation group,TBI group,ω-3 PUFA supplementation group (TBI + ω-3 group) and autophagy inhibitor 3-methyladenine group (TBI + 3-MA group) (all n =18),each group was further divided into 3 sub-groups (n =6) corresponding to 3 time points (days 1,3,and 7 after TBI).On each of the 3 time points,we measured rat behavioral outcomes with modified neurologic severity score (mNSS) tests;brain water content was measured with wet-dry weight method.The mRNA and protein expressions of autophagy-related factors (LC3-Ⅱ and Beclin-1) in TBI cerebral cortex were determined by immunohistochemistry staining,reverse transcription-polymerase chain reaction and Western blot on day 3 after TBI.Results Compared with the sham group,on days 1,3,and 7 after injuary,the TBI group,the TBI + ω-3 group,and the TBI + 3-MA group had significantly higher mNSS scores (TBI group:12.42±0.27vs.1.34±0.32,12.07±0.27vs.1.16±0.29,10.22±0.39vs.1.22±0.30;TBI+ω-3 group:12.05 ±0.23 vs.1.34 ±0.32,11.38 ±0.21 vs.1.16±0.29,8.20 ±0.21 vs.1.22±0.30;TBI +3-MA group:11.93 ±0.20 vs.1.34 ±0.32,11.09 ±0.19 vs.1.16 ±0.29,7.93 ±0.17 vs.1.22 ± 0.30;all P =0.00) and brain water content [TBI group:(79.82 ± 0.61) ％ vs.(71.87 ± 0.43) ％,(83.04±0.42)％ vs.(72.13 ±0.53)％,(75.12 ±0.72)％ vs.(71.78 ±0.38)％;TBI+ω-3 group:(76.81 ±0.63)％ vs.(71.87 ±0.43)％,(79.39 ±0.59)％ vs.(72.13 ±0.53)％,(73.86 ±0.38)％ vs.(71.78 ±0.38)％;TBI+3-MAgroup:(75.98 ±0.49)％ vs.(71.87 ±0.43)％,(77.14 ±0.46)％ vs.(72.13 ±0.53)％,(72.24 ±0.37)％ vs.(71.78 ±0.38)％;all P =0.00].The mRNA and protein expressions of LC3-Ⅱ and Beclin-1 in the brain were also significantly higher on day 3 in the TBI group,the TBI + ω-3 group,and the TBI + 3-MA group (all P =0.00).Compared with the TB1 group,on day 3 and day 7 after injury,the TBI + ω-3 group and the TBI + 3-MA group had significantly lower mNSS scores (TBI + ω-3 group:11.38±0.21 vs.12.07±0.27,P=0.04,8.20±0.21 vs.10.22±0.39,P=0.01;TBI+3-MA group:11.09±0.19vs.12.07 ± 0.27,P=0.01,7.93 ± 0.17 vs.10.22±0.39,P=0.00).Ondays1,3,and 7,compared with the TBI group,the TBI + ω-3 group and the TBI + 3-MA group had significantly lower brain water content [TBI + ω-3 group:(76.81 ± 0.63) ％ vs.(79.82 ± 0.61) ％,P =0.04,(79.39 ±0.59)％ vs.(83.04±0.42)％,P=0.01,(73.86±0.38)％ vs.(75.12±0.72)％,P=0.03;TBI+3-MAgroup:(75.98 ±0.49)％ vs.(79.82 ±0.61)％,P=0.01,(77.14 ±0.46)％ vs.(83.04 ±0.42)％,P =0.00,(72.24 ± 0.37) ％ vs.(75.12 ± 0.72) ％,P =0.02].On day 3,the TBI + ω-3 group and the TBI + 3-MA group had significantly reduced LC3-Ⅱ and Beclin-1 mRNA expression compared with the TBI group (TBI +ω-3 group:P=0.04,P =0.01;TBI +3-MA group:P =0.01,P =0.00) and protein expression (TBI+ω-3 group:P=0.01,P=0.03;TBI +3-MA group:both P=0.00).Conclusion ω-3 PUFA supplementation could markedly reduce brain edema and improve neurological functions after TBI,showing a neuroprotective effect,possibly through inhibiting TBI-induced autophagy responses.