Axonal Injury Induced By Cytoskeletal Protein Carbonylation After Traumatic Brain Injury

Objective: Oxidative stress contributes to diffuse axonal injury in several neuropathologies,however the underlying mechanism remains unclear.We have recently shown that traumatic brain injury induces reactive oxygen species(ROS)generation and cytoskeleton protein carbonylation in injured brain tissue.Objective to study the changes of oxidative stress and the level of cytoskeletal protein carbonylation in brain tissue after traumatic brain injury(TBI)and its significance.Given the important role of actin/tublin filament dynamics in maintaining axon integrity,here we further investigated the impact of carbonyl modification on actin/tublin filament dynamics and axonal injury in an in vitro model of oxidative stress.Materials and methods: 15 adult SD rats were randomly divided into mild TBI group(n=5)and severe TBI group(n=5)and sham operation group(n=5),the application of hydraulic head injury instrument according to the corresponding parameters of preparation of mild and severe animal model and sham operation group.Glutathione 24 h rat brain tissue was detected by ELISA and Western blotting after TBI(GSH),malondialdehyde(MDA)and cytoskeletal proteins [beta actin,beta tubulin and glial fibrillary acidic protein(GFAP)] carbonyl level,and the application of Western blotting detection of axonal injury marker phosphorylated tau protein level(p-tau).Rat pheochromocytoma(PC-12)cells were exposed to various concentrations of H2O2 for 24 or 48 hrs before assessing intracellular redox status,actin/tublin carbonylation,proteasome activity and actin/tublin filament polymerization.Results 1.Mild and severe MDA level of brain tissue in rats of TBI were(389.62+29.95)mol/g,(642.50+37.56)umol/g,compared with the sham group(233.94+25.08)mol/g increased significantly,the differences were statistically significant(P<0.05);2.The levels of GSH were(352.10+37.75)umol/g,(153.27+43.49)mu mol/g,which were significantly lower than those of the sham operated group(492.48 + 41.43)umol/g,respectively,and the differences were statistically significant(P<0.05);3.?-actin,?-tubulin and GFAP in mild TBI group carbonylation ratio was 0.099 ±0.104,0.194±0.114,0.643±0.037,severe TBI group were 0.142±0.017,0.290±0.029,0.902±0.021,compared with the sham group,0.068±0.017,0.108±0.016,0.673±0.032)significantly increased,the differences were statistically significant(P<0.05);4.The proportion of p-tau in mild and severe TBI group was 0.289±0.014 and 0.373±0.012,respectively,compared with the sham operation group(0.185±0.009),the difference was statistically significant(P<0.05);5.Our data showed that incubation of PC-12 cells with H2O2 increased malondialdehyde(MDA)and 8-hydroxydeoxyguanosine(8-OHd G)contents and decreased glutathione(GSH)levels in a dose-and time-dependent manner.6.Along with these changes,carbonyl modification to total proteins as well as ?-actin/?-tubulin were increased in H2O2-treated cells,which was accompanied by increased phosphorylation of neurofilament heavy chain(p-NFH)indicative of axonal injury.7.?-actin/?-tubulin carbonylation led to increased depolymerization of their filaments.Lastly,H2O2 treatment also suppressed the activity of the proteasome,and over expression of proteasome 5 subunit significantly reversed the above changes in PC-12 cells.Conclusion:In conclusion,these results demonstrate that under prooxidant conditions,carbonylated actin/tublin proteins accumulate due to increased carbonyl modification and suppressed proteasome-mediated degradation,which leads to decreased polymerization of their filaments and eventually axonal injury.