The Function Of Endoplasmic Reticulum And Mitochondria Crosstalk After Traumatic Brain Injury

Objective:The high disability and mortality rate of TBI(traumatic brain injury),which is a serious threat to the health of Chinese people.Although the level of TBI treatment has been significantly improved in recent years,the mortality rate of TBI is still at a high level,so it is urgent to find more effective treatment methods.The abnormality of nerve function after TBI is mainly related to the death of neurons and the loss of neural function network.It is an important way to break through the bottleneck of treatment by rebuilding the steady state of nerve cells,reducing the death of nerve cells and the destruction of neural network connections,and restoring the functional connections between nerve cells.In recent years,the interaction between endoplasmic reticulum and mitochondria,as the key subcellular response of cell homeostasis regulation,has attracted the attention of scholars at home and abroad because of its important role in cell homeostasis reconstruction,damage resistance and survival.The membrane contact site between endoplasmic reticulum and mitochondria is called MAMs(Mitochondria-associated membranes),which is the main functional unit of the interaction between endoplasmic reticulum and mitochondria.In the study of Alzheimer’s disease,lateral sclerosis of spinal cord and other neurodegenerative diseases,it has been found that a variety of proteins located on MAMs are involved in lipid metabolism,calcium signal transduction,inflammatory response and other pathophysiological processes in nerve cells.In this experiment,we aim to explore the changes and pathophysiological mechanism of the interaction between endoplasmic reticulum and mitochondria after traumatic brain injury,in order to provide effective theoretical support for the treatment of traumatic brain injury.Methods:1.HE staining in mouse brain tissue after TBI and the mouse TBI model was established by CCI(Controlled cortical impact);2.The change of interaction between endoplasmic reticulum and mitochondria in brain tissue of mice after TBI was observed by electron microscope;3.The expression of mfn2 and pacs2 was detected by immunofluorescence staining and Western blot,these proteins are the key factors regulating the interaction between endoplasmic reticulum and mitochondria;4 The expression of calcium channel protein such as IP3R1 σ-1R,GRP75 and VDAC1 on MAMs was detected by Western Blot;5.the ratio of Bcl-2/Bax and the expression of Cytc were detected by Western Blot after TBI.To investigate the relationship between MAMs and mitochondrial dysfunction;6.The changes of P-perk-P-eif2 α-ATF4 and GRP78 in endoplasmic reticulum stress pathway were detected by Western Blot.;7.D etection of apoptotic proteins of Chop and Caspase-12 downstream of endoplasmic reticulum stress pathway by Western Blot and determining the effect of MAMs on endoplasmic reticulum.8.The expression of inflammatory factor IL-1 β and TNF-α and the changes of ROS were detected by Western Blot and DCFH-DAprobe.Results:The results showed that,after traumatic brain injury,the formation of MAMs decreased at first and then increased rapidly,reached the peak at 6 hours after injury,and then decreased gradually;The electrophoretic results of calcium channel protein IP3R1/σ-1R/GRP75/VDAC1 on MAMs showed that when the interaction between the two organelles increased,a stable Ca2+channel was formed between endoplasmic reticulum and mitochondria,and Ca2+transport was enhanced.The determination of Bcl-2/Bax and Cytc,which caused the change of mitochondrial permeability,found that the enhancement of Ca2+transport led to the destruction of Ca2+balance in mitochondria,which led to mitochondrial dysfunction and mitochondrial permeability changes.The changes of the expression of endoplasmic reticulum stress pathway protein P-perk/P-eif2 α/ATF4 and GRP78 showed that the increase of MAMs formation did not lead to mitochondrial dysfunction,but also led to the activation of endoplasmic reticurm stress pathway.Further detection of the expression of apoptosis proteins Chop and Caspase-12,which is downstream of endoplasmic reticuhrm stress pathway showed that the enhancement of their interaction could lead to the activation of endoplasmic reticulum stress pathway,and then induce apoptosis.The results of IL-1β and TNF-α expression and ROS detection showed that MAMs not only had an important effect on neuronal apoptosis after traumatic brain injury,but also played an important role in the activation of inflammatory factors and the occurrence of oxidative stress in the early stage after injury.Conclusion:Based on the above data the interaction between endoplasmic reticulum and mitochondria was strengthened and the formation of MAMs was increased in the early stage of brain injury.The transport of Ca2+ between endoplasmic reticulum and mitochondria was enhanced.The destruction.of Ca2+ balance led to mitochondrial dysfunction and mitochondrial permeability change.At the same time,it could lead to the activation of endoplasmic reticulum stress pathway and the release of apoptosis factors.In addition,MAMs not only has an important effect on neuronal apoptosis after traumatic brain injury,but also plays an important role in the activation of inflammatory factors and the occurrence of oxidative stress in the early stage after injury.