The Research On The Neuroprotective Mechanism Of Copper Oxide-loaded Electrospun Scaffold By Regulating Copper Homeostasis After Mild Traumatic Brain Injury

Objective:Traumatic Brain Injury(TBI)induces progressive neuroinflammation and degeneration,resulting in different types of programmed cell deaths,which in turn leads to motor,memory and cognitive emotional dysfunction,is already became the most common injury in clinical and forensic practice.One of the cell deaths that takes place during the acute stage of traumatic brain injury is pyroptosis.The main outcome is the release of inflammatory mediators after cell death to initiate an inflammatory response.According to certain theories,the copper is thought to be crucial in the prevention of inflammation and oxidative stress,and the imbalance of copper homeostasis is linked to the progression of many diseases.However,the effect of copper on TBI has not been reported yet.The purpose of this study is to explore the role of copper and its homeostasis in mild TBI,and how the copper oxide-loaded electrospun scaffold achieved its neural protection by regulating pyroptosis and copper homeostasis.and detected the change of copper ion concentration in the injured cortex after 1 day by inductively coupled plasma mass spectrometry(ICPMS),and then detected the copper ion concentration in the injured cortex by western blot experiment.Methods:In this research group,the mild TBI model was first established and copper concentration in ipsilateral cortex after 1 day was detected by inductively coupled plasma mass spectrometry(ICPMS),and then western blot experiments were conducted to detect the presence of Copper-transporting ATPase 1(ATP7A),copper transporter 1(CTR1),Tyrosine hydroxylase(TH),Copper chaperone for superoxide dismutase(CCS)and Superoxide dismutase 1(SOD1)at different timepoint after TBI(12 h,1 d,2 d,3 d,7 d and 14 d).1 d,3 d,and 7 d were used for immunofluorescence experiment to observe the expressions of ATP7A in neurons,astrocytes and microglia in cortex.On 1 d after TBI,we observed the co-localization of CCS and SOD 1 with neurons,astrocytes and microglia,as well as the expression of recombinant human copper transporter protein 1(Atox 1)and human cytochrome c oxidase 17(COX17)in neurons.In order to explore whether exogenous copper have neuroprotective effect on mild TBI,CuO-loaded electrospun scaffold was prepared by adding copper oxide particles into the mixed solution of polycaprolactone(PCL)and gelatin(Gel).Its characterization was analyzed by Fourier transform infrared spectrum analyzer,scanning electron microscope,and water contact angle test,and that antibacterial function is teste by antibacterial experiment.Copper oxide loaded electrospun scaffold was selected as the delivery of copper to research t he effect of exogenous copper on neuronal pyroptosis.The injured cortex on 1 d after in situ delivery was detected by western blot,and the expression levels of NOD-like receptor family pyr in domain containing 3(NLRP3),pyroptosis-related protein gasderminD(GSDMD),caspasel and apoptosis-related spot-like protein(ASC)to determine the optimal implantation time window and concentration.To further verify the role of copper in mild TBI,the copper chelator agent Tetrathiomolybdate(TTM)was treated to investigate the role of exogenous copper in pyroptosis and neuroprotective function by molecular biology(western blot and immunofluorescence were used to detect the cellular localization of the above-mentioned pyroptosis-related proteins),histomorphology(Nissl staining)and behavioral tests(Wire-Grip Test,Morris Water Maze,and Open Field Test).Finally,To verity whether exogenous copper affected total copper content in blood and tissues,detecting the total copper concentration in blood and cortex after exogenous coppertreating by ICPMS.Finally,the expressions of ATP7A,CTR1,CCS,SO1 and TH in the cortex and their localization to neurons were detected by western blot experiment and immunofluorescence,to explore whether the exogenous copper achieved the neuroprotective effects by regulating copper homeostasis after mild TBI.Results:(1)After mTBI,the concentration of copper in ipsilateral cortex was decreased,and the expression levels of CTR1 and ATP7A in ipsilateral cortex showed an increasing trend and were still higher than the baseline level on 14 d.CCS,SOD1 and TH all peaked around 12 h-1 d and then gradually declined,and basically returned to the baseline level on 14 d.In normal cortex ATP7A was mainly located in microglia and decreased 1d after mTBI and restored to baseline level in 3d;After TBI,the expression of ATP7A in neuronal cells increased until the baseline level was restored on 7 d.Among the astrocytes,the ATP7Apositive cells gradually increased from 3 d to 7 d.ATP7A is mainly expressed in neurons 1 d afterTBI.In normal cortex CCS was more expressed in microglia and astrocytes,and SOD1 was mainly located in microglia.After TBI,CCS was mainly expressed in neurons and microglia,and SOD1 was mainly located in neurons and astrocytes.The expression levels of COX17 and Atoxl in the injured cortical neurons were increased after TBI.(3)After loading CuO,the mechanical properties and morphological characteristics of PCL electrospinning did not change significantly,and its hydrophilicity was decreased.The release of copper exhibited the characteristics of sustained-release.The implantation of CuO-loaded electrospun scaffold could reduce the pyroptosis of neurons and alleviate the deformation of neurons by treat with exogenous copper.The optimal treat time window and concentration were 6 h after mTBI and 0.5%CuO,respectively.Besides,exogenous copper improved motor dysfunction,spatial memory ability and cognitive affective disorder,the protective effect of exogenous copper dispeared after TTM treatment.(4)After mild TBI,total copper in ipsilateral cortex was decreased,which is restoring after exogenous copper-treating with blood copper increasing,and copper chelator agent TTM could reduce this trend.Moreover,Exogenous copper could reduce the expressions of ATP7A,TH,CCS and SOD1 in the cortex after mTBI,which mainly in neurons,while TTM could offset this trend,indicating that exogenous copper restored the homeostasis of copper in cells.Conclusion:(1)After mTBI,the concentration of copper in ipsilateral cortex was decreased,and the expression of copper protein in cortex showed showed a time-dependent upward trend,with total copper level in ipsilateral cortex was decreased,showing a trend of increasing copper metabolism level,which indicated the existence of copper homeostasis imbalance and increased copper demand.(2)The CuO-loaded electrospun scaffold achieved slow,continuous and low-dose copper supply to the brain,and it was confirmed from the cellular,histopathological and behavioral tests that CuO-loaded electrospun scaffold could reduce neuronal pyroptosis after mild TBI,and play a neuroprotective role.(3)Exogenous copper could increase copper concentration in blood,restore the total copper level in cortex and the copper metabolism level in neurons after mild brain injury to recover copper homeostasis in cell.