The Role Of Aldose Reductase In Neurons After Spinal Cord Injury

Background:Aldose reductase（AR）is one of the key enzymes in the glucose metabolism pathway,and its physiological role is mainly to catalyze aldehydes or ketones into corresponding alcohols.As a member of the aldo-keto reductase superfamily,AR is widely expressed in a variety of cells.Research suggests that AR is not only involved in diabetes and other glucose metabolism-related diseases,but also played an important role in cardiomyopathy,asthma,ocular diseases and even nervous system damage.After spinal cord injury（SCI）,AR expression is up-regulated in microglias/macrophages,while AR inhibition,microglia/macrophage tend to polarize towards M2 type to promote SCI recovery;AR expression is also up-regulated in neurons,but its role and mechanism in neurons after SCI have not been reported.Studying the role and mechanism of AR in neurons after SCI will provide potential targets for the treatment of spinal cord injury.Objectives:1.Culture primary neurons of cerebral cortex in vitro,observe the expression of AR with various stimulatons,and analyze the sensitivity of AR expression with various stimulations.2.Infect primary neurons with AR overexpression lentivirus,observe and compare the responses of neurons with different levels of AR expression to inflammation stimulation.Establish spinal cord injury models after infecting neurons in T10 spinal cord anterior horn with AR overexpression lentivirus,and observe the effect of AR overexpression on the recovery of motor function in mice.Methods:1.The primary cerebral neurons were cultured,and the purity was identified by immunofluorescence staining;The primary cerebral neurons were stimulated by mechanical injury（scratch）,and then collected them for q RT-PCR and Western blot detection of AR expression at different time points after 12 h,24 h,48 h and 96 h;Culture primary cerebral neurons were stimulated with oxygen glucose deprivation/reoxygenation（OGD/R）for 0.5 h,1 h,2 h and 4 h,and then collected them for CCK-8 detection of neurons activity and q RT-PCR and Western blot detection of AR expression at different time points;The primary cerebral microglia/macrophages were stimulated with lipopolysaccharide（LPS）and interleukin-4（IL-4）for 24 h,respectively,and then collected the medium as condition medium（LPS-CM and IL-4-CM）.Culture primary cerebral neurons were stimulated with LPS-CM and IL-4-CM,respectively,and then collected them for q RT-PCR and Western blot detection of AR expression;The primary cerebral neurons were stimulated with 25 ng/m L tumor necrosis factor-α（TNF-α）and 20 ng/m L interferon-γ（IFN-γ）,and then collected them for q RT-PCR and Western blot detection of AR expression at different time points after12 h,24 h,48 h and 96 h.2.The primary cerebral neurons were infected with AR overexpression(AR^OE)lentivirus to determine lentivirus MOI and infection rate,and detects neuronal activity,and q RT-PCR and Western blot determine AR overexpression;WT,AR knockout（AR ko）and AR^OE primary neurons were cultured in groups at the same time,and processes of neurons were observed and counted by immunofluorescence staining;Neurons were infected with AR overexpression lentivirus in T10 spinal cord anterior horn,a week later,established WT,AR ko and AR^OE mice SCI models,evaluate mice motor function by BMS（Basso Mouse Scale,BMS）score.Results:1.qRT-PCR and Western blot results showed that the AR expression was up-regulated in primary neurons with stimulations of mechanical injury,LPS-CM/IL4-CM and TNF-α+IFN-γ.The up-regulation of AR expression in neurons with inflammation stimulations was more obvious,suggested that AR expression was more sensitive to inflammation stimulation compared with stimulations of mechanical injury;The primary neurons were stimulated with OGD/R,AR m RNA expression was up-regulated and AR protein expression was down-regulated,suggested that OGD/R stimulation induced AR expression at the transcriptional level in primary neurons.2.The primary neurons were infected with AR^OE lentivirus,and the neurons activity was no significant changes,but the expression of AR was up-regulated,indicating that the AR^OE primary neuron model was successfully established;The primary neurons were stimulated with Inflammation,The number of processes of AR ko control group were decreased while the number of processes of AR^OE group were increased,suggesting that AR is necessary for neuron survival,and the up-regulation of AR expression in neurons has a protection effect on neurons;Neurons in T10 spinal cord were infected AR^OE lentivirus,immunofluorescence staining indicated that the AR^OEmice model were successfully established;After SCI,the motor function recovery of AR ko mice group was better than that WT mice group,and the motor function recovery of AR^OE mice was better than that of WT mice group.Conclusions:1.Compared with mechanical injury,the expression level of AR in primary neurons is more closely related to inflammation stimulation.2.The up-regulation of AR expression in neurons has a protective effect on neurons;After SCI,the motor function recovery of AR ko mice group is significantly better than that WT mice group;The motor function recovery of AR^OE mice group better than WT mice group.