The Mechanism Of Nrf2-mediated Antioxidant Stress And Its Effect On P62 Protein In The Therapeutic Effect Of DEX On TBI

Objective:（1）Screening targets and signaling pathways related to the pathological changes of TBI secondary brain injury and the therapeutic effect of DEX.（2）To explore the role of Nrf2/P62 mediated interaction of antioxidant stress and autophagy in TBI.（3）To clarify whether Nrf2-mediated antioxidant stress and its effect on P62 protein are involved in the neuroprotective effect of DEX on TBI.Methods:（1）Preliminary exploration of the target and signal pathway related to the DEX’s neuroprotection on TBI.Free-falling Feeney method is used to build a rat TBI model,the rats were randomly divided into three groups:normal Control group（Control group）,model group（TBI）,treatment group（DEX）,the transcriptome sequencing and bioinformatics analysis were performed 24 h after modeling,and mRN A expression of the key molecules were validated by qRT-PCR.（2）To explore the molecular mechanism of the neuroprotective effect of DEX on TBI in vivo.Feeney free-fall method was used to construct the rat TBI model,combined with Nrf2 inhibition and P62 overexpression,the following tests were performed:（2.1）Behavioral tests:Modified neurological severity scores（mNSS）and the morris water maze（MWM）test were used to evaluate the therapeutic effect of DEX on TBI rats.（2.2）Detection of markers of oxidative stress injury in peripheral blood.Tailor venous blood was collected at 3 h,1 d,2 d,3 d,7 d and 10 d after injury or treatment,respectively.Plasma was separated to detect the levels of malondialdehyde（MDA）and lactic dehydrogenase（LDH）,markers of oxidative stress injury,to determine the optimal treatment course of DEX for TBI rats.（2.3）Transmission electron microscopy（TEM）was used to observe the changes of autophagic lysosomes in rat brain tissue.（2.4）The levels of neurotrophic factors（BDNF,IGF-1）and proteins such as Nrf2,HO-1,LC3II and P62 in brain tissues of rats in each group were detected by Western Blot.（3）To explore the molecular mechanism of the neuroprotective effect of DEX on TBI in vitro:In this section,PC 12 cell line was used,combined with Nrf2 inhibitor and P62 overexpression,the following experiments were conducted to evaluate the protective effect of DEX on TBI cell model and possible regulatory targets:（3.1）CCK8 assay was used to determine the optimal intervention dose and treatment time of H₂O₂ and DEX for PC12 cells.（3.2）Cell apoptosis level and intracellular ROS level were detected by flow cytometry to determine the optimal intervention dose and treatment time of H₂O₂ and DEX for PC 12 cells,and to evaluate the protective effect of DEX on PC 12 cell injury model.（3.3）Cell oxygen consumption rate（OCR）was measured to evaluate the effect of DEX on respiratory function of PC 12 cells damaged by oxidative stress.（3.4）The mRNA expression levels of Nrf2 and P62 were detected by qRT-PCR,and the changes of Nrf2 and P62 mRNA in cells under different treatment conditions were evaluated.（3.5）The protein levels of Nrf2 pathway related factors and autophagy related markers were detected by Western Blot,and the protein expressions of Nrf2 pathway related factors and autophagy related markers were compared under different treatment conditions.（3.6）Immunofluorescence assay further confirmed the protein expression of Nrf2 and LC3II under different treatment conditions.Results:（1）Transcriptome sequencing and bioinformatics analysis.（1.1）Compared with the Control group,174 mRNAs were upregulated and 335 mRNAs were down-regulated in the TBI group.The functions of mRNAs up-regulated are mainly enriched in PI3K signaling pathway,calcium ion signaling pathway and MAPK pathway;while in down-regulated mRNAs,Dusp1 may be involved in the regulation of the pathophysiological process of TBI secondary brain injury through phosphorylation of MAPK.（1.2）Compared with the TBI group,244 mRNAs were upregulated and 105 mRNAs were down-regulated in the DEX group.The upregulated mRNAs were mainly concentrated in cell division and cysteine sulfur transfer pathway,involving endogenous antioxidant stress-related Nrf2 signaling pathway.The functions of down-regulated mRNAs were mainly involved in the MAPK signaling pathway,which related to cell survival and cell fate regulation,and the HIPPO signaling pathway,which regulates cell proliferation and apoptosis.（1.3）There were 112 differentially expressed genes among the three groups,and their functions were mainly concentrated in the MAPK signaling pathway and the Nrf2 signaling pathway.（2）In vivo study.（2.1）Compared with the Control group,mNSS score increased and spatial learning and memory ability was impaired in the TBI group,which was marked by prolonged escape latency and shortened target quadrant time.DEX treatment decreased mNSS score and improved spatial learning and memory ability compared to TBI group.（2.2）From 3 h after injury,the levels of MDA and LDH in peripheral blood of rats in TBI group were higher than those in Control group,and reached the highest level at 3 d,then decreased slowly.DEX treatment decreased the levels of oxidative stress markers in peripheral blood of rats at each time point.（2.3）The expression of neurotrophic factor BDNF was down-regulated and IGF-1 was slightly up-regulated in TBI group.DEX treatment significantly promoted the expression of BDNF and IGF-1.（2.4）Transmission electron microscope scanning showed that the number of autophagy lysosomes increased in TBI group,DEX treatment further increased the number of autophagy lysosomes.（2.5）After Nrf2 inhibition,the expression of neurotrophic factor BDNF and IGF-1 in brain tissue of TBI rats was significantly inhibited,and the effect of DEX on the up-regulation of neurotrophic factor expression in brain tissue of TBI rats was also inhibited.（2.6）After Nrf2 inhibition,autophagy levels in brain tissues of TBI rats were decreased,indicating as the expression of autophagy-related markers LC3Ⅱ and P62 were down-regulated,and the effect of DEX on promoting autophagy was also weakened.（2.7）The overexpression of P62 had a neuroprotective effect on TBI rats,which indicated as the expression of neurotrophic factors BDNF and IGF-1 were significantly up-regulated.（2.8）In addition,P62 overexpression promoted the expression of Nrf2 and its downstream HO-1.（3）In vitro study.（3.1）H₂O₂ could decrease the survival rate and increase the apoptosis of PC 12 cells in a concentration-dependent manner,and the effect of reducing cell viability and promoting cell apoptosis was most significant at the concentration of 200 μM.（3.2）Different concentrations of DEX（0.1 μM,10 μM,50μM,100 μM,150 μM and 200 μM）had no significant effect on the viability of PC 12 cells,100 μM,150 μM and 200 μM of DEX could significantly reduce the apoptosis induced by H₂O₂.（3.3）The ROS level and apoptosis ratio in H₂O₂ group were significantly higher than those in the Control group,and DEX treatment could effectively reduce the ROS and apoptosis in cells.（3.4）Compared with the Control group,OCR in the H₂O₂ group was significantly decreased,and was up-regulated by DEX treatment.（3.5）After H₂O₂ treatment,the expression of Nrf2 pathway related proteins（Nrf2 and HO-1）and autophagy related proteins（P62,LC3Ⅱ）in PC12 cells were up-regulated,DEX treatment further promoted the expression of these proteins.（3.6）Compared with the Control group,the mRNA level of Nrf2 was not significantly changed in the H₂O₂ group,while the mRNA expression of P62 was slightly up-regulated.The mRNA expressions of Nrf2 and P62 in the DEX treatment group were significantly higher than those in the H₂O₂ group.（3.7）Immunofluorescence results showed that the expression of Nrf2 protein increased and the translocation of Nrf2 to the nucleus was enhanced after treatment with H₂O₂.DEX treatment further promoted the expression and translocation of Nrf2 to the nucleus.（3.8）The proportion of LC3Ⅱ positive cells increased in the H₂O₂ treatment group,and DEX treatment further promoted the expression of LC3Ⅱ.（3.9）After Nrf2 inhibition,H₂O₂-induced PC 12 cell injury was further aggravated,which was characterized by decreased cell viability,increased intracellular ROS accumulation and increased cell apoptosis ratio,and the protective effect of DEX against oxidative stress injury of PC 12 cells was also weakened by Nrf2 inhibition.（3.10）Western Blot and immunofluorescence results showed that the autophagy level of PC 12 cells subjected to oxidative stress injury decreased after Nrf2 inhibition,which was reflected by the decreased expression of autophagy-related markers LC3Ⅱ and P62.Meanwhile,the effect of DEX on promoting autophagy was also weakened.（3.11）P62 overexpression can promote the repair of PC 12 cell model of oxidative stress,which is manifested by increased cell viability,decreased intracellular ROS accumulation and apoptosis ratio.Meanwhile,the aggravation of oxidative stress injury caused by Nrf2 inhibition can be partially corrected by P62 overexpression.（3.12）Western Blot and immunofluorescence results showed that P62 overexpression promoted the expression of Nrf2 and its downstream HO-1.Conclusions:（1）The mRNA expression profile of rat brain tissue changed after TBI injury and DEX treatment,the MAPK signaling pathway and Nrf2 signaling pathway may be closely related to the pathophysiological process of TBI and the neuroprotective effect of DEX.（2）DEX exerted neuroprotective effect on TBI injury rat and oxidative stress injury PC 12 cells.（3）DEX may play a neuroprotective role by promoting the elimination of oxidative stress products and enhancing the level of autophagy.（4）The mechanism of neuroprotective effect of DEX may be closely related to Nrf2-mediated antioxidant stress and its effect on P62 protein.