Long Non-coding RNA-AK046375 Regulating Microrna To Reduce Oxidative Stress After Traumatic Brain Injury In Mice And Its Mechanism

ObjectiveTraumatic brain injury（TBI）is a disease with high morbidity and mortality which leads to severe neurological dysfunction,there is still no effective treatment for the secondary brain injury.Studies have shown that long non-codingRNA（LncRNA）plays an important role in the development regulation and the pathophysiological process of central nervous system（CNS）.Previous study showed that the expression of LncRNA-AK046375 increased significantly in the cortical tissue around trauma foci at 24 hours after TBI in mice,indicating that it involved in the pathophysiological process after TBI.MicroRNA（miRNA）can bind to the3’-untranslated region（3’-UTR）of the mRNA and inhibit the mRNA translation,thereby result in the downregulation the expression of its target gene.Studies have shown that LncRNA can inhibit the effect of miRNA on the regulation of its target genes by sponging the miRNA,and then increasing the expression of genes,and ultimately exerting its biological functions.Metallothionein 2（MT2）is a small molecular weight protein with abundant cysteine,which endows it a strong antioxidant ability.In this study,a mice TBI model in vivo and a H₂O₂induced oxidative stress model in vitro were established to explore the neuroprotection effect of LncRNA-AK046375 and its possible mechanism.Methods1.The LncRNA-AK046375 overexpression,knockdown adenovirus and their negative control vector adenovirus were constructed.Adenovirus were injected into the right lateral ventricle of C57BL/6J mice（n=237,8-12 weeks-old）,a controlled cortical impact（CCI）model on mice were used to simulate the TBI state at 7 days after injection.The mice were randomly divided into sham group（n=39）,TBI group（n=36）,LncRNA-AK046375 overexpression+TBI group（n=39）,overexpression vector group+TBI group（n=39）,LncRNA-AK046375 knockdown+TBI group（n=39）and knockdown vector+TBI group（n=39）,except the sham group only received craniotomy treatment,other groups received craniotomy and CCI treatment,Adenovirus was injected into lateral ventricle of 3 mice to observe the transfection of adenovirus in cerebral cortex.The mice Neurological severity scores（NSS）,grabbing test,rotating rod test and water maze test were used to detect the effect of LncRNA-AK046375 on the motor,learning and memory function of the mice after TBI.Quantity polymerase chain reaction（q-PCR）was used to detect the virus interference efficiency at 7 days after TBI.Immunofluorescence staining was used to detect the number of Neu N（+）stained cells.Western blot was used to detect the expression of tight junction proteins（occluding,claudin5 and ZO1）,apoptosis-related proteins（BCL-2,Bax,cleaved-caspase3）and the distribution of cytochrome C inside and outside the mitochondria respectively.Evans blue penetration test and the dry and wet weight test were used to detect the integrity of the blood-brain barrier（BBB）indirectly.One-step TUNEL kit was used to detect the apoptosis at or around the injury site.2.The LncRNA-AK046375 overexpression model in mice hippocampal neuron cell line（HT22）were constructed by transfecting the LncRNA-AK046375 overexpression adenovirus.TotalRNAs were extracted forRNA-sequencing to predict the biological function of LncRNA-AK046375 and its possible intermediate mediators.Fluorescence in situ hybridization（FISH）was used to detect the subcellular localization of LncRNA-AK046375 in mice primary cortical neurons and astrocytes.LncRNA-AK046375 overexpression or knockdown adenovirus and its negative control adenovirus were transfected into the primary cortical neurons and astrocytes respectively,the expression of Lnc-RNA-AK046375 and metallothionein 2（MT2）were detected by q-PCR,western blot and immunofluorescence.3.H₂O₂gradient concentrations method was used to construct the mice primary cortical neurons and astrocytes oxidative stress models.The condition of H₂O₂was determined based on the result of the cell viability by CCK-8 assay and the intracellular reactive oxygen species（ROS）levels by ROS assay kit.To explore the effect of LncRNA-AK046375 on primary cortical neurons and astrocytes after received H₂O₂treatment,western blot was used to detect oxidative stress related proteins（Malondialdehyde,（MDA）and Superoxide dismutase 2（SOD2）,Catalase（CAT））,apoptosis-related proteins（Bcl-2,Bax,cleaved-caspase3）,and the distribution of cytochrome C inside and outside the mitochondria.The GSH kit was used to detected the expression of GSH,GSSG and GSH/GSSG.Mito SOX^redstaining was used to detect the oxidative stress level in cells.One-step TUNEL kit was used to detect cell apoptosis.4.Previous studies have confirmed that LncRNA can modulate the target mRNA by sponging the microRNAs（miRNAs）,therefore the miRNAs that may be involved in this study were screened out by searching the"microrna.org"database and integrating with threeRNA interaction sequences analyze software"mi Randa","PITA"and"RNAhybrid".The dual luciferase test was used to verify whether the predicted miRNAs could directly bind to MT2 mRNA.q-PCR and western blot were used to detect the expression of miRNA-491-5p and MT2 in primary astrocytes after transfected with miRNA-491-5p mimics or inhibitor.CCK-8 and lactate dehydrogenase（LDH）release tests were used to detect the effect of miRNA-491-5p on the cell viability and cytotoxicity of astrocytes after received the H₂O₂treatment.Mito SOX^redstaining was used to detect the oxidative stress level in the primary astrocytes after received the H₂O₂treatment.Ago2-RNA binding protein immunoprecipitation（Ago2-RIP）test and dual luciferase test were used to verify whether LncRNA-AK046375 and miRNA-491-5p could bind with each other directly.Primary cortical astrocytes were divided into non-treated group,LncRNA-AK046375 overexpression group,LncRNA-AK046375overexpression+miRNA-491-5p mimics group,LncRNA-AK046375knockdown group and LncRNA-AK046375 knockdown+miRNA-491-5p inhibitor group to detect whether the MT2 expression was regulated by LncRNA-AK046375 and miRNA-491-5p.Results1.The LncRNA-AK046375 overexpression and knockdown models in mice cerebral cortex were constructed successfully.Compared with the overexpression vector group,the overexpression of LncRNA-AK046375could significantly improve the motor,memory and learning functions in mice after TBI.Compared with the knockdown vector group,LncRNA-AK046375 knockdown could significantly aggravate the motor,memory and learning dysfunction in mice after TBI.Compared with the overexpression vector group,the overexpression of LncRNA-AK046375could significantly reduce the apoptosis,the loss of cortical neurons,the exudation of Evans blue,the brain water content and the loss of the tight junction proteins at 7 days in mice after TBI.Compared with the knockdown vector group,knockdown of LncRNA-AK046375 could significantly increase the cell apoptosis,the loss of cortical neurons,the exudation of Evans blue,the brain water content and the loss of tight junction proteins at 7 days in mice after TBI.Compared with the knockdown vector group,knockdown of LncRNA-AK046375 could significantly increase the cell apoptosis,the loss of cortical neurons,the exudation of Evans blue,the brain water content and the loss of tight junction proteins at 7 days in mice after TBI.2.TheRNA-sequencing showed that a total of 1342 mRNAs were significantly changed due to the overexpression of LncRNA-AK046375 in HT22 cells.Of which 717 mRNA were significantly up-regulated,while the other 625 mRNAs were significantly down-regulated.Metallothionein（MT2）was the most significantly changed mRNA.We also confirmed that LncRNA-AK046375 could increase the expression of MT2 in vitro experiments.3.FISH probe test confirmed that LncRNA-AK046375 existed in the cytoplasm and nucleus of mouse primary cortical neurons and astrocytes,but mainly located in the cytoplasm.Determined conditions of H₂O₂to construct the oxidative stress model on mouse primary cortical neurons and astrocytes were as followed 200μmol/L for 12 hours for neurons,400μmol/L for 3 hours for astrocytes.It was confirmed that the overexpression of LncRNA-AK046375 could alleviate the oxidative stress level and the cell apoptosis in primary cortical neurons and astrocytes after received the H₂O₂treatment compared to the overexpression vector group.Knockdown of LncRNA-AK046375 could aggravate the oxidative stress and the cell apoptosis in the primary cortical neurons and astrocytes after received the H₂O₂treatment compared to the overexpression vector group.4.Bioinformatics analysis showed that miRNA-491-5p and miRNA-505-3p were able to bind to the MT2 mRNA.Dual luciferase test confirmed that only mi R-491-5p could bind to the 3’-UTR of MT2 mRNA,downregulation of mi R-491-5p could reduce the oxidative stress level in primary astrocytes after received the H₂O₂treatment.In addition,Ago2-RIP test and dual luciferase test confirmed that LncRNA-AK046375 could bind to miRNA-491-5p directly.Conclusion1.LncRNA-AK046375 could decrease the cell apoptosis,reduce the loss of neurons,maintain the integrity of the BBB,and improve the neurological function of mice after TBI.2.LncRNA-AK046375 exerts its anti-oxidative stress effect by increasing the expression of MT2.3.LncRNA-AK046375 reduces the inhibitory effect of miRNA-491-5p on MT2 by sponging miRNA-491-5p,thereby increasing the expression of MT2.