Protective Effects And Mechanism Of α-tocopherol Against Oxidative Stress In GCDH^-/- Rats

Background and ObjectiveGlutaric aciduria type I is a rare autosomal recessive neurodegenerative disease caused by mutations in the glutaryl-CoA dehydrogenase gene.Mutation of glutaryl-CoA dehydrogenase gene leads to deficiency of glutaryl-CoA dehydrogenase,resulting in lysine,hydroxylysine and tryptophan catabolism blocked,and intermediate metabolites glutaric acid,3-hydroxyglutaric acid,and glutaric acid accumulate in tissues and body fluids,causing a series of neurological damage.Children often suffer from acute encephalopathy crisis due to non-specific disease at 3-36 months after birth,leading to irreversible acute striatum degeneration and cortical damage.Clinical manifestations are macrocephaly,progressive dystonia,dyskinesias,ataxia and seizures.Imaging showed atrophy of the frontotemporal cortex,leukoencephalopathy,basal ganglia degeneration,extracerebral space,and widening of the lateral cleft cistern.Pathologically,the most prominent feature of Glutaric aciduria type Ⅰ is the reduction of striatum neurons.The research on the pathogenesis of glutaric aciduria type Ⅰ striatum degeneration mainly focuses on three aspects:excitotoxicity,depletion of energy metabolism,and oxidative stress.Glutaric acid and 3-hydroxyglutaric acid mediate excitotoxic injury through N-methyl-D-aspartate receptors,glutaric acid,3-hydroxyglutaric acid and glutaryl-CoA,which interfere with tricarboxylic acid cycle,lead to energy metabolism disorders,and lead to neuronal damage.The brain has extremely high oxygen consumption,high levels of oxidizable unsaturated fatty acids,low antioxidant defense system activity,and the nervous system is highly sensitive to oxidative stress.Vitamin E is an important antioxidant and membrane stabilizer in the cell membrane,which can effectively prevent the oxidation of fat to produce reactive oxygen species.α-tocopherol is the main endogenous lipophilic natural antioxidant.lt is the most biologically active form of vitamin E.As a peroxide free radical scavenger,it has neuroprotective effects.The research team used the early construction of GCDH-/-rats as a research model,and induced glutaric aciduria type I disease state rat model by high lysine diet.Model rats were intervened by intragastric administration of α-tocopherol.We observed the general condition(weight and viability)of each group of rats,and HE staining was used to observe the pathomorphological changes of frontotemporal cortex and striatum in each group.We used ELISA kits to detect oxidative stress indicators MDA,the antioxidant enzyme defense system GPx,GSH,CAT and SOD in the frontotemporal cortex and striatum.We detected the expression levels of extracellular regulated protein kinase(ERK),c-Jun-NH2-terminal kinase(JNK),and p38 MAPK proteins in mitogen-activated protein kinase(MAPK)pathway of the frontotemporal cortex and striatum tissues by western blotting.From the animal model level,we explored oxidative stress injury and possible pathways in glutaric aciduria type Ⅰ,and the protective mechanism of classic antioxidant α-tocopherol against oxidative stress injury induced by high lysine diet in GCDH-/-rats.This study provides a research model for studying glutaric aciduria type Ⅰ,which is similar to the disease state of the body,and also provides an experimental basis for studying the potential pathophysiology of glutaric aciduria typeⅠ,and exploring clinical treatment options.Methods1.This study was based on the GCDH-/-rat model constructed by the research group.Male and female 3:1 mating and breeding,and the pups were genotyped,and we obtained 36 homozygotes rats(GCDH-/-)and 21 wild type rats(GCDH+/+).2.We determined the genotype of the pups,and the pups were randomly divided into 6 groups:the wild-type standard diet group(Al),the homozygous standard diet group(B1),the wild type homolysine diet group(A2),Homozygous homolysine homolysine diet group(B2),wild type homolysine homolysine diet and α-tocopherol group(A3),homozygous homolysine diet and α-tocopherol Protective agent group(B3).3.At 4 weeks of age,A1 and B1 group were given standard feed,A2,B2,A3 and B3 group were given 4.7%lysine feed,freely fed with water.Then A3 and B3 group were intragastric administered α-tocopherol once a day at 10 am at a dose of 100 mg/kg.d.Specimens were retained after 28 days of treatment according to different groups.The body weight was measured every day,and the average of the three measurements is taken as the weight of the day.4.HE staining was used to observe the pathomorphological changes of the frontotemporal cortex and striatum.ELISA method was used to detect the changes of oxidative stress indexes in frontotemporal cortex and striatum.Western blot was used to detect the expression level of JNK,P38,ERK in MAPK pathwayResults1.Genotype identification:three genotypes of GCDH-/-,GCDH-/+ and wild-type SD rats can occur in the offspring,transcription product electrophoresis appeared a 725 bp band(WT primer amplification)was the wild type,appeared a 498 bp band(GT primer amplification)was the homozygote(GCDH-/-),and appeared two bands simultaneously was the heterozygote(GCDH-/+).2.General condition and survival rate of rats in each group:on the 7th day of the diet and drug intervention,two rats in the B2 group showed gait abnormality,which mainly included a tilted head to the left and foreleg paralysis.On the 15th day of the intervention,the B3 group one rat showed its head tilted to the left.There were 4 deaths in the B2 group,the survival rate was 69.23%(9/13).There was 1 death in the B3 group,and the survival rate was 91.67%(11/12).The rats in A1,B1,A2 and A3 group were generally in good condition with a survival rate of 100.00%.3.The weight change of rats in different groups in diet and drug intervention at different times:there was no significant difference in body weight between the 6 groups before diet and drug intervention(day 0).After 7 days of diet and drug intervention,with the extension of the intervention time,the weight of rats in the B2 and B3 groups was significantly lower than other groups,and the weight of rats in B2 group was significantly reduced.4.Pathological and morphological changes of rat brain tissue in each group:histopathology showed that frontotemporal cortex and striatum neuron cells of B2 group had deep constriction staining,the boundary between cytoplasm and nucleus was unclear,the shape of the cells was irregular,the interstitial was edema,the cells are arranged disorderly.In B3 group,a small number of neuron cells in the frontotemporal cortex were densely stained and the cells were regular in shape.the striatum neurons were arranged regularly with clear boundaries.the other groups were not significantly abnormal.5.Indexes of oxidative stress:compared with A1 group,MDA was increased significantly,GSH was significantly reduced,and the activity of GPx,CAT and SOD were weakened in frontotemporal cortex and striatum of B2 group.Compared with B2 group,MDA was significantly reduced,the antioxidant enzymes recovered part of activity in frontotemporal cortex and striatum of B3 group.6.Expression of JNK,P38,and ERK proteins in the MAPK pathway:western blot results showed that,compared with A1 group,the expression level of P38 protein were significantly up-regulated in frontotemporal cortex,the expression levels of JNK and P38 proteins were significantly up-regulated in striatum.Compared with B2 group,the expression levels of JNK and P38 proteins were significantly down-regulated in frontotemporal cortex and striatum of B3 group.Conclusions1.GCDH-/-rats with high lysine diet developed oxidative stress injury,causing damage to the nervous system.2.Oxidative stress in GCDH-/-rats with high lysine diet activated P38 and JNK pathways,which activated the MAPK signaling pathway,and this participated in GAI nerve damage.3.α-tocopherol relieved oxidative stress injury and had a neuroprotective effect on GCDH-/-rats.