Study On The Mechanism Of Intracellular Tension Regulating Blood-brain Barrier Function

Objective:The blood brain barrier(BBB)is a natural physical barrier that strictly controls the entry and exit of substances in the brain.Its damage and dysfunction may lead to a variety of central nervous system diseases,but the tough structure of the BBB is hard for drugs to reach their targets for treatment.Brain microvascular endothelial cells are the main component of the blood-brain barrier.The tight junction between endothelial cells(Occludin,ZO1,etc.)and the dense membrane formed by adhesion junction proteins are the key factors that lead to the low permeability of the blood-brain barrier.In addition,the normal osmotic pressure gradient between brain tissues is also an important factor in maintaining the integrity of the blood-brain barrier.In this study,we established the human brain microvascular endothelial cell(HBMEC)injury model and SD rat blood-brain barrier injury model to explore roles of Occludin/ZOl tension in BBB lesion,and to identify the physical mechanism of intracellular osmotic pressure regulating membrane fluidity and non-selective permeability of blood-brain barrier,providing a new research idea for drug transport in brain diseases.Methods:We used the principle of fluorescence resonance energy transfer(FRET)and molecular cloning technology to construct Occludin/ZO1 angular fluorescence tension detection probe,and transfected it into human microvascular endothelial cells(HBMEC),and converted the intracellular mechanical changes into visible optical signals to detect the intracellular tension changes stimulated by a variety of BBB injury factors.(1)In this experiment,human vascular endothelial growth factor(VEGF),glutamate(Glu),hydrogen peroxide(HP),and histamine(His)were used to stimulate HBMEC to simulate the human brain microvascular endothelial cell injury model;(2)Laser confocal technology was used to analyze the effectiveness and motility of the tension probe,and to detect the Occludin/ZO1 tension changes in the HBMEC damage model and their mutual mechanical effects;(3)The changes of cell permeability in HBMEC damage model were analyzed by measuring TEER value.(4)The change of intracellular osmotic potential energy was analyzed by detecting the cytoplasmic osmotic pressure and the size and distribution of cytoplasmic solution protein nanoparticles.(5)Fluorescence Recovery After Photobleaching(FRAP)was used to detect the membrane fluidity changes of Occludin in HBMEC under different drug treatments and different osmotic pressures.(6)established the blood brain barrier injury model of SD rats,and studied the effect of cytoskeleton stabilizer on non-selective permeability of BBB in vivo by measuring the content of Evans blue in brain tissue.(7)Screening of relevant Chinese medicine monomers for calcium ion and chloride ion imaging experiments to further clarify the new mechanism affecting the non-selective permeability of BBB.Results:1.The newly constructed fluorescent tension probe is effective and can be used to detect intracellular Occludin/ZO1 tension changes in the HBMEC injury model.Stimulation of VEGF and Glu can enhance intracellular Occludin/ZO1 tension,while stimulation of HP and His can reduce intracellular Occludin/ZO1 tension.2.1n the HBMEC damage model,BBB damage factors treatment can increase the content of ions in the cytoplasm,activate the microfilaments and microtubule depolymerization factors to induce the formation of intracellular protein nanoparticles and increase the related osmotic pressure.3.Depolymerizing agents of microfilaments,microtubules,and intermediate filaments increased the tension of Occludin/ZO1 in HBMEC cells,the membrane fluidity of Occludin protein,and the permeability of HBMEC cells.4.Microfilaments and microtubule stabilizers could induce the recovery of intracellular tension,antagonize the increase of ions and osmotic pressure induced by BBB damage factor,and restore the permeability of endothelial cells.5.1n the SD rat BBB injury model,microfilament microtubule stabilizers may block the non-selective permeability of the BBB model in vivo.6.Treating HBMEC cells with traditional Chinese medicine monomers such as borneol,muscone,and asar ether can not only induce the up-regulation of intracellular protein nanoparticles-related osmotic pressure,but also increase the content of intracellular calcium and chloride ionsConclusion:The stimulation of extracellular BBB lesion factors leads to the change of intracellular Occludin/ZO1 tension,which may also lead to the depolymerization of MF or MT and the massive production of protein nanoparticles,and induce an increment of intracellular osmotic potential based on the Donnan effect,resulting in the upregulation of membrane fluidity and the increase of non-selective drug permeability.This study reveals the important physical mechanism of intracellular tension regulating blood-brain barrier dysfunction and its permeability,and provides new research ideas for improving targeted drugs to cross the blood-brain barrier to reach the central nervous system.