Effect And Mechanism Of ALDH2 On Regulation Of Mitochondrial Division And Fusion In Sepsis Associated Encephalopathy

Objective: To observe the effect of acetaldehyde dehydrogenase 2(ALDH2)on the permeability of mouse blood-brain barrier(BBB)and brain microvascular endothelial cells(BMECs)induced by LPS.And to explore the potential mechanism of mitochondrial fission and fusion in the brain endothelial barrier.Methods: 1)C57BL/6J mice were divided into four groups(n=15 in each group): Sham,sepsis(LPS),sepsis+ALDH2 agonist Alda-1 group(LPS+Alda-1),and sepsis +ALDH2inhibitor Daidzin group(LPS+Daidzin).The sepsis model was established by intraperitoneal injection of 10 mg/kg LPS.Brain tissue damage was observed by neuroreflex score,HE staining,brain water content and Evans blue content.The pathological changes of brain tissue and mitochondrial morphology were observed by transmission electron microscope.Mitochondrial function was measured by ATP kit.The content of 4-HNE was detected by ELISA,and the oxidative stress index was detected by MDA,SOD and CAT kits.The expressions of ALDH2,tight junction proteins ZO-1 and Occludin,mitochondrial fusion proteins OPA1 and Mfn2,and fission proteins Drp1 and Fis1 were detected by Western blot.2)Cell experiments: The b End.3 brain microvascular endothelial cells was treated with LPS for 12 h to simulate the sepsis brain injury,the groups are as follows: 1)Normal control group: control group(Con),LPS injury group(LPS),LPS+Alda-1 group,LPS+Daidzin group,LPS+Drp1 inhibitor Mdivi-1 group(LPS+Mdivi-1)and LPS+Mdivi-1+Daidzin group.Cell viability was detected by CCK8,transendothelial cell resistance and FITC-Dextran leakage were used to detect BBB permeability.The content of 4-HNE was detected by ELISA,and the oxidative stress index was detected by MDA,SOD and CAT kits.ATP kit was used to detect mitochondrial function,JC-1 assay was used to detect mitochondrial membrane potential,Mitochondrial morphological changes were detected by Mito Tracker Red.DHE probe was used to detect the content of ROS,Mito SOX was used to detect the expression of mitochondrial ROS.Western blot was used to detect the expressions of ALDH2,ZO-1,Occludin,OPA1,Mfn2,Drp1 and Fis1 proteins.Results: 1)Animal experiments: Compared with Con group,the pathological changes of abnormal brain tissue,brain water content,Evans blue content,serum 4-HNE and MDA contents were increased in LPS group,but the activities of SOD,CAT and the concentration of ATP were decreased(P<0.01,P<0.05).The protein expression of ALDH2,OPA1,Mfn2,ZO-1 and Occludin were decreased(P<0.01,P<0.05),and the expression of Drp1 and Fis1 proteins were increased(P<0.05).Compared with the LPS group,the abnormal brain tissue lesions,brain water content,Evans blue content,serum4-HNE and MDA content were decreased,while the activities of SOD and CAT(P<0.01)and ATP concentration were increased(P<0.05)in the LPS+Alda-1 group.The protein expressions of ALDH2,ZO-1,Occludin,OPA1 and Mfn2 were increased,while the protein expressions of Drp1 and Fis1 were decreased(P<0.01,P<0.05).Compared with the LPS+Alda-1 group,the LPS+Daidzin group showed opposite results.2)Cell experiments: Compared with the Con group,the cell viability(P<0.01),transendoendothelial resistance TEER(P<0.05),mitochondrial membrane potential JC-1,SOD,CAT activities and ATP concentration reduction(P<0.01)in the LPS group were significantly decreased(P<0.01).The leakage of FITC-Dextran,the fluorescence intensity of intracellular and mitochondrial ROS,the contents of 4-HNE and MDA in the supernatant of cells were increased(P<0.01).The protein expressions of ALDH2,ZO-1,Occludin,OPA1 and Mfn2 were decreased,while the protein expressions of Drp1 and Fis1 were increased(P<0.01,P<0.05).Compared with the LPS group,the cell viability,transendoendothelial electrical resistance TEER,mitochondrial membrane potential JC-1,SOD and CAT activities and ATP concentration were significantly increased in the LPS+Alda-1 and LPS+Mdivi-1 groups(P<0.01).The leakage of FITC-Dextran,the fluorescence intensity of intracellular and mitochondrial ROS,the content of 4-HNE and MDA in the supernatant of cells were decreased(P<0.01).The protein expressions of ALDH2,ZO-1,Occludin,OPA1 and Mfn2 were increased,while the protein expressions of Drp1 and Fis1 were decreased(P<0.01,P<0.05).Compared with LPS+Alda-1 group,LPS+Daidzin group showed opposite results.In addition,compared with LPS+Mdivi-1group,the cell viability,transendoendothelial electrical resistance TEER,mitochondrial membrane potential JC-1,SOD,CAT and ATP were significantly decreased in LPS+Mdivi-1+Daidzin group(P<0.01).However,the leakage of FITC-Dextran,intracellular and mitochondrial ROS fluorescence intensity,4-HNE and MDA contents in the supernatant of the cells were increased(P<0.01).The protein expressions of ALDH2,ZO-1,Occludin,OPA1 and Mfn2 were decreased,and the protein expressions of Drp1 and Fis1 were increased(P<0.01,P<0.05).Conclusion: 1)Activation of ALDH2 can reduce oxidative stress in sepsis-associated encephalopathy,maintain mitochondrial homeostasis,and play a protective role in endothelial barrier.2)Inhibition of mitochondrial fission by Mdivi-1 can reduce the production of oxidative stress and improve the permeability of brain microvascular endothelial cells induced by LPS.3)Activation of ALDH2 plays a role in LPS-induced endothelial barrier injury,which may be related to the inhibition of mitochondrial fission.