Peroxidase 6 Knockout Alleviates Neuronal Damage In The Hippocampus Of Epilepsy Rats

Epilepsy is a kind of nervous system disease characterized by abnormal firing of neurons caused by the excitatory and inhibitory imbalance of the central nervous system.Its pathogenesis is very complex and is closely related to neurotransmitter imbalance,oxidative stress,and immune inflammation.Oxidative stress produces a large number of oxygen free radicals.Under normal physiological conditions,the body will utilize and eliminate the excess free radicals.However,Excessive harmful stimulation,such as epilepsy,can lead to hypoxia,damage and even necrosis of tissues and cells.Oxidative stress damage was reported to be found in Kainic acid(KA)-induced epilepsy in rats and was distributed across all areas of the brain.Peroxiredoxin6(PRDX6)is a member of the mercaptospecific peroxidase-reductase family and contains two structural functional domains,both antioxidant peroxidase-reductase activity and non-calcium-dependent phospholipase A2 activity that promotes inflammation.Therefore,on the one hand,PRDX6 can play an important role in anti-oxidative stress by eliminating reactive oxygen species(ROS)and participating in the repair of peroxidation cell membrane and the transport of cytophospholipids.On the other hand,it can promote inflammation by activity of phospholipase A2.In 2008,Myung-Jeom Ryu et al.Published an article on neurochem neuroproteomic analysis of stargazer mutant mice with seizures.Stargazer(stg)mutant mice had mutations in the calcium channel 2 subgroup stargazin,showing several neurological disorders,including spontaneous seizures,cerebellar ataxia,and head twitches.The results showed that the expression levels of phosphoprotein 1 and PRDX6,two proteins related to oxidative stress in the hypothalamus of mice,were decreased,but whether PRDX6 was involved in the pathogenesis of epilepsy or not has not been reported.Therefore,this experiment is intended to observe the expression level,cell location and role of PRDX6 in KA induced epileptic rats and provide theoretical basis and experimental basis for studying the disease mechanism of epilepsy and screening therapeutic drug targets.Objective: 1.To observe the expression of PRDX6 in epileptic rats;2.To observe the localization of PRDX6 in epileptic rat brain;3.To investigate the role of PRDX6 in epileptic rats and its possible mechanism.Methods:Wild type and PRDX6 knockout SD rats were injected with KA to induce epilepsy in the lateral ventricle.Those with epilepsy scores greater than 4 were included as successful models,and the rats were sacrificed and brain tissue samples were collected on 1,3,and 7 days after KA injection.Fluoro-jade B staining was used to detect neuron damage.Nissl staining is used to detect neuron morphology.Immunohistochemistry(IHC)was used to observe the expression level and encephalic region localization of PRDX6 in epileptic rats.Immunofluorescence technique(IF)was used to observe the localization of PRDX6 cells.Western blot(WB)was used to detect the expression level of PRDX6 in hippocampus of epileptic rats.Enzyme linked immunosorbent assay(ELISA)was used to detect PRDX6 in serum of epileptic rats.Quantitative real-time reverse transcription polymerase chain reaction(qRT-PCR)was used to detect the m RNA levels of PRDX6 and other peroxidase reductase family members in the hippocampus of epileptic rats,and m RNA levels of oxidative stress related factors,inflammation-related factors.RNA sequencing(RNA-seq)was used to detect gene expression changes in the hippocampus of PRDX6 knockout rats epilepsy models.Results: 1.PRDX6 is upregulated in hippocampus of KA-induced seizure ratsAccording to the seizure score of the rats,epileptic rats with more than 4 points or above were selected to be included in the group,and were sacrificed on day 1,3,and 7 after KA injection and brain tissue samples were collected.FJB staining showed that severe neuron damage in KA induced epileptic rats,and neurons with degraded functions were mainly distributed in the hippocampus CA3 and CA1 regions.Nissl staining showed significant loss of neurons in hippocampus CA3 and CA1 of epileptic rats.IHC results showed that the expression of PRDX6 increased significantly in the brain of epileptic rats,and it was mainly located in the hippocampus and cortex,and also expressed around the lateral ventricle.WB showed that the expression of PRDX6 in hippocampus of epileptic rats was up-regulated,which began to increase at KA1 d,was significantly up-regulated at KA3 d,and remained high at KA7 d.Because PRDX6 is a secretory protein,we used ELISA to detect the serum level of PRDX6,and the results showed that the serum level of PRDX6 in epileptic rats was significantly increased in KA3 d.The results of qRT-PCR also showed that the m RNA level of PRDX6 in hippocampus of epileptic rats increased significantly.2.PRDX6 is expressed in astrocytes in KA-induced seizure ratsIn order to explore the cellular expression localization of PRDX6,we used glial fibrillary acidic protein(GFAP)as a marker for astrocytes.The results of PRDX6 and GFAP fluorescence co-standardization showed that they exist in the cerebral cortex,hippocampus and lateral ventricle.The colocalization of PRDX6 and GFAP suggests that PRDX6 is expressed in astrocytes.3.PRDX6 is not expressed in activated microglia or neurons in KA-induced seizure ratsWe use Ionized calcium binding adapter molecule-1(Iba-1)to label microglia.The immunofluorescence double labeling results showed that PRDX6 was not co-located with Iba-1,indicating that Prdx6 was not expressed in microglia.Neural nuclei protein(Neu N)is a marker of neurons.The double labeling results of PRDX6 was not co-located with Neu N,which show that PRDX6 is not expressed in neurons.4.Oxidative stress markers i NOS and 4-HNE were both expressed in cerebral astrocytes of epileptic ratsIn order to understand whether there is oxidative stress in the brain of rats with KA-induced epilepsy.Immunofluorescence was used to detect the cell localization of inducible nitric oxide synthase(i NOS)and 4-hydroxynonenal(4-HNE).It was found that both i NOS and 4-HNE were expressed in GFAP positive cells.Co-localization with PRDX6 suggested that epilepsy could induce upregulation of oxidative stress in astrocytes.5.PRDX6 KO significantly reduced the number of recurrent seizures,mortality and neuron lossWe established an epileptic model of PRDX6 knockout rats,and found that there was no significant change in the seizure score of PRDX6 knockout rats within 3 hours after KA administration compared with wild-type rats.However,the number of recurrent seizures was significantly reduced(P <0.05)and the death rate was also significantly reduced(P <0.001).FJB staining and Nissl staining showed that the loss of neurons in hippocampus of PRDX6 knockout rats was significantly reduced.These results suggest that PRDX6 knockout can reduce hippocampal neuron damage caused by KA.6.Effect of PRDX6 knockout on proliferation of glial cells in brain of KA-induced epilepsy ratsReactive hyperplasia of glial cells and formation of glial scars are important pathological features of epilepsy.In order to understand the effect of PRDX6 knockout on the number or proliferation of glial cells,we first use GFAP and Iba-1 to label astrocytes and microglia cells.Compare the effects of PRDX6 on the number or proliferation of glial cells without modeling.The results showed that PRDX6 knockout alone had no significant effect on the number of glial cells.To further investigate the effects of PRDX6 knockout on the number or proliferation of glial cells in epileptic rats,Proliferating Cell Nuclear Antigen(PCNA)was co-labeled with GFAP or Iba-1.The number of astrocytes did not change significantly,while the number of microglia was significantly lower than that of wild rats,and it proliferation was also reduced.7.Effects of PRDX6 knockout on oxidative stress in the hippocampusPRDX6 is only a member of the peroxidase reductase family in mammals.Will PRDX6 gene knockout affect another peroxidase expression? To this end,first,we used qRT-PCR to detect the m RNA levels of other PRDXs and oxidative stress-related factors in hippocampal tissues of wild-type and PRDX6 knockout unmodeled modules.The results showed that after PRDX6 knockout,the m RNA expression of PRDX1 decreased,the m RNA expression of PRDX2 and PRDX4 was up-regulated,and there was no significant change of oxidative stress-related factors in the hippocampus.After that,we examined the m RNA levels of other PRDXs and oxidative stress-related factors in hippocampus 3 days after KA administration in WT and PRDX6 knockout rats.The results showed that after PRDX6 knockout,m RNA expression of PRDX1 gene decreased,m RNA expression of PRDX5 m RNA was up-regulated,and there was no significant change in levels of oxidative stress-related factors in hippocampus.8.Effect of PRDX6 knockout on hippocampal inflammation levelIn order to investigate whether PRDX6 knockout has an effect on inflammation levels in rats,we used qRT-PCR to detect m RNA levels of inflammation-related factors in hippocampus tissues of wild-type and PRDX6 knockout rats in the unmodeled group.The results showed that PRDX6 knockout had no significant effect on the inflammatory level of the hippocampus.In order to explore whether PRDX6 knockout had an effect on the inflammation level of epileptic rats,we used qRT-PCR to detect m RNA levels of inflammation-related factors in wild-type and PRDX6 knockout hippocampus tissue 3 days after KA administration.The results showed that PRDX6 knockout had no significant effect on the inflammatory level of hippocampus.Meanwhile,we used RNA-seq to screen genes with significant expression differences,and found that the expression of CCL2 and CCL7 were significantly down-regulated in knockout rats.Conclusion: 1.PRDX6 was significantly upregulated in hippocampus of KA-induced seizure rats 2.PRDX6 is specifically expressed in astrocytes in KA-induced seizure rats 3.PRDX 6 knockout alleviates neuronal damage in the hippocampus of epilepsy rats 4.PRDX6 may reduce neuron damage during epilepsy by reducing microglial cell proliferation and chemokine levels.