Study On The Changes And Neuroprotective Mechanisms Of Parkin After Traumatic Brain Injury

【Background】As one of the most common acute and severe cases in clinic,TBI is characterized with high mortality and disability.It can be classified into primary brain injury and secondary brain injury during the injury process.The type and severity of primary brain injury are determined by external force,which is controlled mainly by prevention.How to reduce secondary brain injury is the responsibility of doctors and the key to treatment.However,there is a lack of effective neuroprotective drugs in clinic.Therefore,it is of great significance to find potential molecular targets to reduce TBI.Parkin is an E3 ubiquitin ligase closely related to neurodegenerative diseases.Previous literature has confirmed that Parkin is widely involved in neuronal signal transduction,synaptic homeostasis regulation,and plays a protective role in ischemic brain diseases.But the role and mechanism of Parkin in TBI are still unclear.In addition,if Parkin can play a neuroprotective role in TBI,how to realize its protective role also needs to be further explored.【Purpose】The study aims to:（1）illustrate the change and role of Parkin in the occurrence and development of TBI;（2）explore the molecular mechanism of Parkin in TBI;（3）preliminarily find the means to realize the neuroprotective effect of Parkin.【Methods】1.Establish the mouse controlled cortical injury（CCI）model and the mouse primary cortical neuron mechanical injury（TNI）model as the experimental models of TBI in vivo and in vitro,respectively.2.Adeno-associated virus transfection was used to upregulate the expression level of Parkin in mouse cerebral cortex.3.Parkin knockout mice were introduced to observe the effect of Parkin knockout on CCI.4.The HE and Nissl staining were used to observe the damages of brain tissue after CCI in mice.5.The brain edema of mice after CCI was evaluated by the detection of brain water content.6.The behavioral changes of mice after CCI were detected by neurological severity score（NSS）,rotating rod test and Morris Water Maze（MWM）.7.Lentivirus vector transfection was used to upregulate or downregulate the expression of Parkin in neurons8.Cell damage was determined by lactate dehydrogenase（LDH）and cell viability,as well as propidium iodide（PI）staining.9.The expression of related genes was observed at the transcription level and translation level by RNA sequence,real-time quantitative PCR,Western blot（WB）and immunofluorescence experiment.10.Apoptosis was observed by terminal d UTP nick end labeling（TUNEL）staining and caspase-3 activation rate.11.The production of intracellular reactive oxygen species（ROS）was evaluated by 2,7-dichlorodihydrofluorescein diacetate（H₂DCFDA）.12.Malondialdehyde（MDA）and 4-hydroxynonenal（4-HNE）were measured to evaluate the lipid peroxidation level.13.The mitochondrial membrane potential was evaluated by Tetramethylrhodamine Methyl Ester（TMRM）staining;The level of mitochondrial energy metabolism was evaluated by measuring adenosine triphosphate（ATP）level.14.The morphology of mitochondria in neurons was observed by transmission electron microscope.15.The primary cortical neurons of CAG-RFP-EGFP-LC3 transgenic mice were used to visualize autophagy flux,and the effect of Parkin on autophagy flow after TNI was observed.16.The changes of mitophagy were detected by LV-mito-Keima.17.The activation of ferroptosis signal was evaluated by Fe²⁺level and GSH content detection.【Result】The first part:changes and role of Parkin after TBIAfter both CCI and TNI,the expression level of Parkin was upregulated and peaked at 24h after injury.In CCI model,overexpression of Parkin reduced brain tissue injury and brain function defect;knockout of Parkin aggravated brain tissue injury and brain function defect.In TNI model,overexpression of Parkin reduced mechanical neuronal injury.Interference with Parkin aggravated mechanical neuronal injury.Further studies found that Parkin can reduce apoptosis,oxidative stress and protect mitochondrial function.The second part:mechanism of Parkin after TBIIn CCI model,RNA-sequence suggested that Parkin may play a role through signal pathways such as phagosome and inflammation.Further study on TNI model showed that Parkin could regulate mitochondrial quality control,inhibiting mitochondrial division and mitophagy weaken the neuroprotective effect of Parkin;Parkin can inhibit NLRP3inflammasome signal pathway,and inhibiting NLRP3 can reduce the aggravating effect of injury caused by Parkin interference;Parkin can inhibit the activation of ferroptosis signal,and inhibiting ferroptosis can reduce the aggravating effect of injury caused by Parkin interference.The third part:construction of Parkin recombinant protein and its role after TBIA recombinant Parkin protein was successfully constructed,which can cross the cellmembrane and increase the level of Parkin in neurons.In TNI model,Parkin recombinant protein can reduce neuronal toxicity caused by mechanical injury and inhibit neuronal apoptosis,suggesting that Parkin recombinant protein can reduce TBI.【Conclusion】The above studies found that the expression level of Parkin increased after TBI,which can play a neuroprotective role.This protective effect is exerted by affecting mitochondrial quality control,inhibiting NLRP3 inflammatory signal pathway and ferroptosis signal.On this basis,we successfully constructed a Parkin recombinant protein,which can reduce mechanical neuronal injury.These results provide potential intervention targets and new ideas for the diagnosis,treatment and clinical transformation of TBI.