The Protective Effect And Mechanism Of Humnan Neural Stem Cells Secretome On Neuronal Endoplasmic Reticulum Stress In Traumatic Brain Injury

BackgroundTraumatic brain injury（TBI）is a group of diseases mainly caused by falls from heights,traffic accidents,etc.It is one of the leading causes of death in adolescents and children.According to statistics,69 million patients suffer from TBI for various reasons every year,and it is considered a serious health problem and medical burden worldwide.Despite this,there is no recognized treatment for TBI.Stem cells,especially neural stem cells（NSCs）,have gained much attention as emerging therapeutic interventions for various Central nervous system diseases and injuries such as spinal cord injury,multiple sclerosis,ischemic stroke,and TBI.The results of several of these studies have demonstrated the positive protective effects of neural stem cell product-neural stem cell secretome（NSC-S）on neurological injury,and their multiple repair capacity and improvement of injured neuronal structure and function have been observed in a large number of animal models.In addition,they are easy to isolate,have low immunogenicity,are widely available,and lack ethical controversy,and thus are expected to be desirable materials for TBI treatment.ObjectivesTo investigate NSC-S’effects on behavioral improvement,histological repair,and inhibition of endoplasmic reticulum stress（ER stress）in TBI rats.In vitro,the improvement effect of NSC-S on the thapsigargin（TG）-induced ER stress model of PC12 cells and its specific mechanism were further explored.Methods（1）The Feeney free-fall method was used to establish the TBI rat model,and the success of the model was assessed by the modified neurological severity score（m NSS）.（2）The behavioral improvement of NSC-S on TBI rats was assessed by m NSS neurological severity score,beam walking test,hang wire test,grid stepping test,open field test,and elevated cross maze test.（3）The effect of NSC-S on tissue repair in TBI rats was observed by hematoxylin-eosin staining（HE staining）and Nissl staining.（4）The effect of NSC-S on the morphological recovery of neuronal endoplasmic reticulum in TBI rat brain tissue was observed by transmission electron microscope（TEM）;the changes of NSC-S on ER stress-related protein levels in TBI rat brain tissue were detected by western blot;the effect of NSC-S on neuronal apoptosis in TBI rat brain tissue was detected by immunofluorescence,the effect of NSC-S on neuronal apoptosis in TBI rat brain tissue was detected by Western blot.（5）The ER stress cell model was established in vitro by using TG-injured PC12 cells,and the protective effects of NSC-S on cell damage,ER stress,and apoptosis in the ER stress cell model were observed by MTT assay,immunofluorescence assay,flow cytometry,full-wavelength spectrophotometer,TEM and Western blot assay,respectively.（6）PERK activator CCT020312 was added and its effects on ER stress-related protein levels and apoptosis were detected by Western blot assay and flow cytometry assay,respectively.To observe the effects of NSC-S on cell injury,ER stress,and apoptosis in ER stress cell model by flow cytometry,immunofluorescence staining,Western blot,and TEM and to investigate the mechanisms.（7）Detection of NSC-S components using liquid chromatography-tandem mass spectrometry（LC-MS/MS）,biochemical analysis to explore the specific substances that exert cytoprotective effects,and validation in an in vitro endoplasmic reticulum stress cell model using an exogenous recombinant protein pure product of this substance.Results（1）A rat model of TBI was successfully established,and the m NSS neurological function score was moderate TBI model.（2）Compared with the TBI group,NSC-S treatment significantly improved the performance of m NSS neurofunction score,beam walking experiment,hang wire test,grid stepping test,open field test,and elevated cross maze test in rats.（3）Compared with the TBI group,NSC-S treatment significantly reduced the size of the brain tissue damage area in the rat model（P<0.05）.（4）Compared with the TBI group,NSC-S treatment significantly restored the morphology of neuronal endoplasmic reticulum in rat brain tissue,the levels of stress-related GRP78,CHOP,p-PERK,p-e IF-2α,and ATF4 proteins（P<0.05）were reduced,and neuronal apoptosis.（5）Compared with the TG group treatment,co-culture treatment with NSCs significantly restored PC12 cell viability（P<0.05）,cell axon length,and reduced cell ROS toxicity（P<0.05）.The Ca²⁺-ATPase activity（P<0.05）,endoplasmic reticulum morphological structure,cellular Ca2+level（P<0.05）,and ER stress-related GRP78,CHOP,p-PERK,p-e IF-2αand ATF4 protein levels（P<0.05）were significantly restored.（6）Compared with the NSCs co-culture group,the treatment of PERK activator CCT020312 significantly increased the level of PERK signaling pathway protein（P<0.05）and apoptosis level.（7）LC-MS/MS detected NSC-S components and found that the recombinant protein calcium-binding protein（calumenin,CALU）in NSC-S significantly restored TG-induced PC12 cell morphology,cell Ca²⁺level（P<0.05）,endoplasmic reticulum probe fluorescence intensity,ER stress-related GRP78,CHOP,p-PERK,p-e IF-2α,ATF4 protein levels（P<0.05）and apoptosis levels.（8）Compared with the TG group treatment,CALU recombinant protein significantly restored the activity of Ca²⁺-ATPase in PC12 cells（P<0.05）,reduced the overall ubiquitination level of cells,and reduced the expression of EDEM1,HERP1,HRD1,Derlin-1,Derlin-2 and Derlin-3 m RNA in PC12 cells（P<0.05）.Conclusions（1）NSC-S restores ER stress,reduces tissue damage,and promotes neurological recovery in a rat model of TBI.NSC-S reduced histological damage in a TBI rat model.（2）NSC-S alleviates ER stress induced by TG in PC12 cells through CALU protein and activates the PERK signaling pathway to inhibit neuron apoptosis.