The Neuroprotective Effects And Mechanisms Of Hydrogen Sulfide On Mice Traumatic Brain Injury Model

Objective: Traumatic brain injury (TBI) is the most common cause of neurologicaldysfunction in young people. TBI contribute to a substantial number of deaths and casesof permanent disability, which is caused by secondary brain injury. Protecting neurons isan important therapeutic target after brain injury. Hydrogen sulfide (H2S) is likely tojoin nitric oxide (NO) and carbon monoxide (CO) as an endogenous gaseous signalingmolecule. H2S plays an important physiological and pathological role in the centralnervous system. In this study, we will explore the role of H2S on neurological functionafter traumatic brain injury in mice and whether the protective effect of H2S is related toneuronal cell death (apoptosis and autophagy).Methods: TBI model was established by weight drop device in adult mice. Theexpression of endogenous H2S synthesis enzyme cystathionine-β-synthase (CBS) inbrain tissue was detected using RT-PCR, Western blot at different times (1h,6h,12h,1d,2d,3d,7d), and the location of CBS in the brain was detected byimmunohistochemistry and the concentration of H2S was detected by methylene blueassay. In order to investigate the effect of H2S on neurological function after traumaticbrain injury, water content of brain tissue was detected at different concentrations(0.1-45μmol/kg) of H2S donor (NaHS) at different time points (pre-injury30min,15min,30min,1h,2h,4h after injury). The cumulative loss of brain tissue was detectedand behavior tests (motor function and Morris water maze test) were performed fordetecting whether H2S had a protective effect in the treatment of brain injury. To furtherexplore the neuroprotective mechanism of H2S after brain injury, loss of plasmalemmaintegrity was evaluated by intraperitoneal injection of propidium iodide (PI)1h beforesacrificing the animal. The number of PI-positive cells in injured cortex, dentate gyrus, CA1and CA3regions was counted. In the present study, we established a model of TBIaiming to determine whether supplementation with H2S would impart any tissueprotective effects against brain injury and explore its potential neuroprotectivemechanism through apoptotic and autophagic pathways.Results:(1) The levels of H2S in the serum, cortex and hippocampus showed the samechange tendency, declining first and then increasing. Double immunofluorescenceresults confirmed that CBS is widely expressed in brain tissue. There were entirelycolocalizations between TUJ1and CBS in the mice brain. There was no colocalizationof CBS with GFAP (astrocyte marker), CD11b (microglia marker), and Cnpase(oligodendrocyte marker). In the cortex and hippocampus surrounding the wound, CBSprotein decreased from postinjury, and reached a valley on3days and2days, thenincreased. The activity of CBS enzymes showed the same change tendency in the miceafter injury. TBI may attenuate the expression of CBS and the endogenous production ofH2S in the central nervous system.(2) Compared with sham group and contralateral hemisphere, the amount of watercontent of the injured hemisphere increased6h, peaked1d to2d, and lasted to3dafter TBI. Pretreatment30min before TBI by NaHS with dosage of1μmol/kg,5μmol/kg,10μmol/kg,25μmol/kg or45μmol/kg can reduced the percentage of watercontent in the injured ipsilateral cortex, but0.1μmol/kg dosage did not have the samefunction. Pretreatment30min before TBI by NaHS with dosage of1μmol/kg showedthe most obvious improvement in cerebral edema. Compared with TBI group,pretreatment with NaHS can significantly reduce lesion volume, attenuate the neuralinjury in the hippocampal CA1region, ameliorate motor deficits and improve spatialmemory acquisition.(3) Comparison with TBI group, pretreatment with NaHS (1μmol/kg) cansignificantly reduce the number of PI-positive cells in the cortex and hippocampus(CA1, CA3and DG) at6h after TBI. There was no PI-positive cells in the contralateralhemisphere and normal group. H2S reverses TBI-induced caspase-3cleavage and Bcl-2decline in the cortex and hippocampus. H2S also inhibits TBI-induced autophagic activation, which suppressed TBI-induced the increase of P62and reversed TBI-inducedVps34, LC3II and Beclin-1increase.Conclusion:(1) The levels of endogenous H2S, CBS expression and CBS enzyme activitygradually decreased, reached its lowest level and then increased, and TBI may reducethe expression of CBS and the levels of of endogenous H2S in the survival neurons afterTBI;(2) H2S pretreatment had reduced brain edema, improved motor performance andameliorated performance in Morris water maze test after TBI, and H2S may have atherapeutic potential against neuron damage;(3) The protective effect and therapeutic potential of H2S in the treatment of braininjury and the protective effect against TBI may be associated with suppressing cellularplasmalemma integrality disruption and regulating apoptosis and autophagy.