The Role Of Endoplasmic Reticulum-mitochondria Crosstalk At The Early Phase Of Traumatic Brain Injury

Objective: Traumatic brain injury(TBI)is one of the most common diseases in neurosurgery,which has the characteristics of high mortality and high disability rate,and causes serious harm to human health.Although the domestic treatment of TBI is gradually mature,the research on secondary neuroinflammation and apoptosis after TBI is in a bottleneck stage.The physical structure that can form interaction between endoplasmic reticulum and mitochondria is called MAMs(Mitochondria-associated membranes).MAMs is closely related to cell inflammation and apoptosis.Our team’s previous experiments have shown that MAMs is essential for early intracellular homeostasis in TBI,providing a platform for material transport between endoplasmic reticulum and mitochondria.MAMs reaches its peak at 6 hours after TB and plays an important role in different signal pathways to maintain cell activity.However,little is known about the MAMs structure in the pathological effects of TBI.The purpose of this study was to explore the effects of early changes of MAMs on neurofunction,blood-brain barrier leakage,neuroinflammation,oxidative stress and apoptosis after nerve injury in mice with traumatic brain injury,and to explore the changes of related pathways in the structure of MAMs by inhibiting the expression of PACS-2 protein on MAMs by PACS-2 si RNA,so as to provide a new idea for the treatment of craniocerebral injury in the future.Methods: 1.The establishment of CCI model and HE staining of paraffin sections of brain tissue.2.We observed the transfection effect of mice 6 hours after TBI by transfection PACS2 siRNA,before TBI.3.We used m NSS score to explore the changes of neurological function in mice caused by the destruction of MAMs integrity after TBI.4.We examined the permeability of EB and the dry-wet weight ratio of brain tissue 3 days after TBI in mice to explore the change of blood-brain barrier permeability caused by the disturbance of MAMs formation after TBI.5.We detected the results of WB in brain tissue of mice after TBI to explore the change of calcium homeostasis caused by the destruction of MAMs integrity after TBI.6.We detected the results of WB in brain tissue of mice after TBI to explore the changes after UPR activation caused by the destruction of MAMs integrity after TBI.7.We detected the results of WB in brain tissue of mice after TBI and DCFH-DA probe technique to explore the changes of inflammatory reaction and ROS caused by the destruction of MAMs integrity after TBI.8.We detected the TUNEL staining of cortical tissue and WB results of brain tissue 3 days after TBI in mice to explore the change of nerve cell activity caused by the destruction of MAMs integrity after TBI.Results: 1.HE staining of mouse brain tissue showed that after being hit by CCI,the cortex was seriously damaged,and the brain tissue around the trauma showed edema and necrosis.2.Western blotting and immunofluorescence techniques showed that the expression of PACS-2 and MFN2 protein in TBI+PACS-2 si RNA group was lower than that in TBI group,and the double staining rate of PACS-2 and Neu N in TBI+PACS-2 si RNA group was lower than that in TBI group,indicating that the formation of MAMs structure decreased and the interaction between endoplasmic reticulum and mitochondria decreased 6 hours after TBI.3.The m NSS score showed that the m NSS score of the mice hit by CCI increased rapidly on the first day after operation,and then gradually improved.The recovery rate of TBI+PACS-2 si RNA group was more significant than that of TBI group and TBI+NC group on the 3rd,5th and 7th day after traumatic brain injury,indicating that the neurological function of mice could be improved by blocking the function of related proteins on MAMs after TBI.4.The permeability of blood-brain barrier in TBI+PACS-2 si RNA group was significantly lower than that in TBI group by detecting EB permeability and brain dry-wet weight ratio on the 3rd day after TBI,which indicated that the permeability of blood-brain barrier in MAMs group was lower than that in TBI mice due to the disturbance of MAMs formation after TBI.5.The changes of IP3R1,GRP75,VDAC1 and Sigma1 r proteins detected by immunoblotting showed that the calcium transport between endoplasmic reticulum and mitochondria in TBI+PACS-2 si RNA group was significantly lower than that in TBI group,which further indicated that the formation of MAMs structure and calcium transport between brain endoplasmic reticulum and mitochondria decreased 6 hours after TBI.6.The protein expressions of PERK,eif2 α,ATF4 and GRP78 detected by immunoblotting showed that the activation of UPR in TBI+PACS-2 si RNA group was significantly lower than that in TBI group after endoplasmic reticulum stress,which indicated that the formation of MAMs structure decreased 6 hours after TBI,and the activation of UPR decreased after endoplasmic reticulum stress.7.The protein expression of IL-1 β and TNF α detected by Western blotting showed that the inflammatory reaction in the brain tissue of the TBI+PACS-2 si RNA group was significantly lower than that of the TBI group,which indicated that the production of MAMs structure and the inflammatory reaction in the brain tissue decreased 6 hours after TBI.The changes of ROS in brain tissue detected by DCFH-DA probe technique showed that the content of ROS in brain tissue of TBI+PACS-2 si RNA group was significantly lower than that of TBI group,which indicated that the formation of MAMs structure and oxidative stress in brain tissue decreased 6 hours after TBI.8.Western blotting and immunofluorescence techniques showed that the apoptosis of brain tissue in TBI+PACS-2 si RNA group was significantly lower than that in TBI group,which indicated that the production of MAMs structure decreased 6 hours after TBI and improved the apoptosis of brain tissue in TBI mice after 3 days.Conclusion: By inhibiting mouse PCAS-2 protein,it can block the function of related proteins on MAMs,cause the destruction of MAMs integrity,reduce the early interaction of endoplasmic reticulum mitochondria,and improve neurological function,blood-brain barrier leakage,mitochondrial calcium overload,early UPR activation,early inflammation,ROS production and brain apoptosis after severe TBI.