Protective Effect Of Aspartate Aminotransferase On Neurons In Rats With Traumatic Brain Injury

BackgroundTraumatic brain injury（TBI）is one of the major diseases currently threatening human health.Because of its high morbidity,high mortality and high disability rate,it is still one of the important topics of medical research in the world today.At present,the specific brain injury mechanism of TBI is not fully understood,and the main purpose of treatment is to reduce brain injury and improve prognosis.A number of studies have shown that neuronal apoptosis and autophagy are closely related to the mechanism of brain injury,and it is also one of the hotspots in current brain injury research.The neuronal cytoprotective effect of Glutamate oxaloacetate aminotransferase（GOT）on TBI has been confirmed in previous studies,but the protective mechanism of GOT on neurons after TBI and the relationship between neuronal apoptosis and autophagy need to be further explored.ObjectiveTo investigate the regulatory effect of aspartate aminotransferase（GOT）on neuronal apoptosis and autophagy after traumatic brain injury（TBI）in SD rats.Methods1.Forty healthy SD rats（male,8-9 weeks,280 ± 20 g）were purchased from the Experimental Animal Center of Shanxi Medical University.Rats were randomly divided into blank control group（control）,sham-operated group（sham）,model group（TBI）,and administration group（TBI+GOT）.GOT group was injected with GOT solution after TBI modeling,drug injection method（tail vein injection）,dose（35mg/kg）.2.Traumatic brain injury animal model establishment:SD rat TBI model was established according to the modified Feeney free fall method.3.Neurological Severity Score:The test is used to assess neurological deficits 24 after TBI in rats.4.24 hours after the successful modeling,the rat brain tissue was fixed,embedded,and sliced for Nissl staining,immunohistochemistry to detect the expression of cleaved caspase-3,and immunofluorescence to detect LC.The expression of-3 and Beclin-1,apoptosis was detected by TdT-mediated dUTP Nick-End Labeling（TUNEL）,and cleaved caspase-3 in the cortex around the lesion was detected by western blot.,LC-3,Beclin-1,cytoplasmic protein Bax,Cyt-c,mitochondrial protein Bax,Cyt-c expression.5.Statistical methods:GraphPad Prism 7.00 and SPSS21.0 were used for statistical analysis and graph drawing.Using ANOVA to compare means between groups,p<0.05 was considered significant and statistically significant.Results1.The nerve severity score test was performed at 24 hours after TBI to evaluate post-traumatic neurological dysfunction.The results showed that the scores of the GOT injection group were significantly lower than those of the TBI group,indicating that the nerve damage caused by the GOT injection group was alleviated.2.Nissl staining was performed 24 hours after TBI,and the damaged neuronal cells exhibited shrinkage,loss of nuclear structure,and intense cytoplasmic staining.The number of dead neurons in the GOT group was significantly lower than that in the TBI group,indicating that GOT had a significant protective effect on neurons.3.TUNEL staining was performed 24 hours after TBI:a large number of apoptotic cells in the TBI group were positive for TUNEL staining,and the number of apoptotic neurons in the cortex around the injury foci in the GOT group was significantly reduced.4.The expression of apoptotic protein cleaved caspase-3 was detected by immunohistochemistry（IHC）24 hours after TBI:IHC showed that the expression of cleaved caspase-3 was significantly up-regulated in the cortical neurons around the lesion in the TBI group.The GOT-treated group significantly reduced the number of cleaved caspase-3-positive cells in the cortex around the injury foci after TBI,indicating that GOT could inhibit the TBI-induced up-regulation of cleaved caspase-3 expression.5.Autophagy protein LC-3 was detected by immunofluorescence 24 h after TBI:the initiation molecule LC-3 for autophagosome formation was stained with red fluorescence.A large number of LC3-positive cells were found in the cortex around the lesion in the GOT-treated group,indicating that neuronal autophagy was highly active.The number of LC-3-positive cells in the GOT group was significantly higher than that in the TBI group.6.Western blot was performed to detect the expressions of cleaved caspase-3,Bax and Cyt-c proteins in the cortex around the lesion 24 hours after TBI:Western blot showed that the expression of Bax in the cytoplasm of the GOT group was higher than that of the TBI group,and the expression of Cyt-c was lower than that of the TBI group.The opposite findings were observed in mitochondria:Bax expression was lower in the GOT group,whereas Cyt-c expression was higher in the TBI group compared to the TBI group.The expression of cleaved caspase-3 in the GOT group was significantly decreased,indicating that GOT can inhibit the high expression of cleaved caspase-3 after TBI,and GOT can inhibit the binding of the apoptosis regulator Bax to the mitochondrial outer membrane,reduce cell membrane permeability and The pro-apoptotic protein cytochrome c（Cyt-c）is released into the cytoplasm,thereby inhibiting the occurrence of apoptosis.These results suggest that GOT may reduce neuronal apoptosis induced by TBI through a mitochondria-mediated pathway.7.Western blot was performed to detect the expressions of LC-3 and Beclin-1 in the cortex around the lesion 24 hours after TBI:the ratio of LC3-Ⅱ/LC3-IⅠand the expression of Beclin-1 in the autophagy-related protein GOT group were significantly higher than those in the TBI group.group,which indicated that the expression level of autophagy was significantly increased in the brain tissue of the GOT-treated group after TBI.Conclusions1.GOT reduces neuronal damage by inhibiting neuronal apoptosis after TBI.2.GOT activates neuronal autophagy after TBI and reduces neuronal damage.3.GOT exerts neuroprotective effects by inhibiting neuronal apoptosis and activating neuronal autophagy.