| Objective:Brain edema usually refers to the increase of water content in the brain,mainly due to the breakdown of blood-brain barrier(vasogenic brain edema)and the influx of exogenous solutions,as well as the swelling of nerve cells(cytotoxic brain edema).Among them,cytotoxic brain edema originates from the volume expansion of a large number of astrocytes.When the concentration of osmotic substances in cells increases,the lipid bilayer of cells can bear the increased osmotic potential energy.By stretching the cytoskeleton structure connected with the cell membrane,the volume of cells can be induced to expand and the morphology and structure of nerve cells can be changed.At the same time,the stretching of the cell membrane can activate the mechanical sensitive ion channels,leading to the influx of ions such as Ca2+and the change of the tension of microfilaments and microtubules by regulating the kinetic protein molecules.Glial fibrillary acidic protein(GFAP)is the main intermediate filament protein in astrocytes.Osmotic pressure,microfilament and microtubule tension cooperate with each other to induce changes in cell structure and morphology.In this study,we detected the changes of intracellular microfilament,microtubule structure tension,intermediate fibrillary acidic protein tension and osmotic pressure of cytoplasmic solution in the process of cellular edema,and identified the mechanism of mechanical regulation of neuronal edema,so as to reveal the molecular mechanism of cytotoxic brain edema related to the mechanical activity of neuronal cells.The combination of microfilament,microtubule depolymerization factor upstream protein Slingshot(SSH)and rotein phosphatasa 2A(PP2A)monomer inhibitors sennoside A,cantharidin and calcium ion inhibitors was used to reduce intracellular tension and treat brain edema.Methods:Based on the principle of fluorescence resonance energy transfer(FRET)and molecular cloning technology,the donor Cerulean and the recipient Venus fluorescent protein were linked to construct an angular tension probe of cpstFRET.The mechanical changes were converted into optical signals.At the same time,the fluorescent tension probes of AcpA,TcpT and GcpG were constructed and transferred to U87 and primary generation.Astrocyte induces its expression and is used to detect intracellular structural tension.This experiment(1)simulated neurocytotoxic edema model by glutamate stimulation;(2)analyzed the effectiveness of tension probe by laser confocal technique,and detected the changes of cell structural tension in neurocyte edema model and their mutual mechanical effects;(3)evaluated the effects of microfilament and microtubule tension on glial fibrillary acidic eggs by microfilament depolymerizer and its dynamic molecular inhibitor.Vector relationship of white traction force;(4)The effect of osmotic pressure induced by intracellular ions and protein nanoparticles on skeleton structural tension was studied by measuring the osmotic pressure of ions and colloids and the distribution of nanoparticles in cytoplasmic solution;(5)The structural tension was studied by using skeleton protein stabilizers,Chinese herbal monomers,ion channel inhibitors and clinical oxygen free radical scavengers.The relationship with cytotoxic brain edema and the effect of structural tension change on brain edema;(6)Establish the model of cytotoxic brain edema in SD rats,and clarify the mechanism of mechanical regulation of combined drugs under glutamate induction by hematoxylin-eosin staining.Results:The constructed AcpA,TcpT and GcpG fluorescent tension probes can effectively detect the changes of intracellular microfilament,microtubule tension and glial fibrillary acidic protein traction,and GcpG fluorescent tension probes can be used to detect the mechanical changes of cell swelling under extracellular stress.Through tension probe,we found that intracellular osmotic pressure,microfilament and microtubule force were involved in the regulation of GFAP tension.At the same time,in glutamate-induced astrocyte swelling model,GFAP tension was found to be involved in cell swelling,and stable microfilaments and microtubules could antagonize glutamate-induced astrocyte swelling.Glutamate stimulation can induce the depolymerization of microfilaments and microtubules by activating Cofilin and Stathmin-1.The activation of upstream calcium signals,SSH and PP2A also participate in the depolymerization of microfilaments or microtubules in glutamate-induced cytotoxic edema.Glutamate-induced cytoskeleton depolymerization of astrocytes can lead to the formation of protein nanoparticles in cells,which can then generate protein nanoparticles and induce the increase of intracellular osmotic potential energy.Therefore,the combination of nimodipine,edaravone and cinepazide maleate,as well as cantharidin and sennoside A,which are the traditional Chinese medicine monomers of microfilament and microtubule depolymerization factor inhibitors,can effectively reduce the tension of GFAP,thereby reducing cell volume swelling.Conclusion:This study focused on the vectorial relationship between the structural tension of intracellular microfilaments,microtubules and intermediate fibers during astrocyte swelling,especially the osmotic pressure induced by protein nanoparticles.Ion osmotic pressure and osmotic pressure induced by protein nanoparticles synergistically regulate the outward pull tension of intermediate fibers.Recovery of cytoplasmic potential energy is an ideal target for developing new drugs and treating brain edema.This study provides a deeper understanding of the progress of astrocyte swelling,and provides a new way of thinking for the treatment of astrocyte swelling. |
