Research On The Mechanism Of Protein Nanoparticle Induced Osmotic Pressure Modify Pulmonary Edema In Acute Respiratory Distress Syndrome

Objective:Acute respiratory distress syndrome(ARDS),caused by noncardiogenic pulmonary edema(PE),contributes significantly to Coronavirus 2019(COVID-19)associated morbidity and mortality.It is characterized by an inflammatory reaction,increased vascular permeability,and pulmonary edema.Intracellular protein nanoparticle-related osmotic pressure has a pivotal role in edema,but the exact mechanism in pulmonary edema remain unclear.Herein,we explored the effect of transmembrane osmotic pressure gradients in pulmonary edema using a FRET-based IF optical tension probe.Methods:The intracellular tension for intermediate filament protein Vimentin was evaluated using the fluorescence resonance energy transfer(FRET)-based tension probes and cpstFRET analysis.The freezing point Osmometer and Nanosight NS300 were used to measure the cytoplasmic osmotic pressure(OP)and the count rate of cytoplasmic nanoparticles,respectively,in order to explore the mechanism of PN-OP in pulmonary edema.The change of cytoskeleton and the production of NLRP3 inflammasome were detected by Immunofluorescence,to analyze the source of protein nanoparticles.Intracellular calcium or chloride ions were measured using Fura-2AM,MQAE and ENG dyes,exploring the relationship between ions and PN-OP.The activation of TRPC6 and TRPM4 was examined by whole-cell patch-clamp experiments.Based on PN,Ca2+signaling and cation,screened effective therapeutic drugs.The tight junction formation was assessed by the measurement of transendothelial electrical resistance(TEER)and the permeability in vivo was determined via the changes of Evans Blue(EB)injected into C57BL/6 mice.The above experiments were used to detect the PN-OP treated with Alb,to explore the transmembrane OP gradients of alveolar epithelial cells and further assessed the potential effects of four selected drugs on it.Mouse model of intratracheally instillation-induced ARDS was established to confirm the mechanisms studied in vitro.Results:1.The effectiveness of the created vimentin fluorescence probe was established and tested.2.Cytoplasmic OP,intracellular PN number,calcium,chlorine,and sodium ion levels were all increased by AngⅡ or BK treatment.3.Intracellular OP,protein nanoparticles,and IF tension were downregulated by stabilization of the microfilament and microtubule cytoskeleton.4.Protein nanoparticles and OP can be greatly downregulated by inhibiting the NLRP3 inflammasome.Caspase-1 activity and IL-1 release were both markedly reduced.5.AngⅡ and BK caused cell membrane hyperpolarization and decreased the mitochondrial membrane potential.Protein nanoparticles and OP can both be drastically downregulated by inhibiting the intracellular calcium signal.6.The TRPC6 current increased by AngⅡ or BK and affected the time-course development of the current and Ca2+inflow.Inhibition of PNs and NLRP3 inflammasome reduced TRPC6 current,but had no effect on the DAG content after AngⅡ or BK stimulation.7.Compared with the AngⅡ or BK groups,the Ca2+inhibitors partly reduced the TRPM4 currents and intracellular Na+levels.8.Based on intracellular PN,Ca2+,and Na+,the most effective combination of drugs was SennosideA+Tranilast+SAR7334+Glibenclamide.The intracellular structure of MFs and MTs,PN-OP,the levels of NLRP3 inflammasomes,intracelular Ca2+and Na+levels,lung epithelial cell permeability and alveolar clearance were significantly improved by the drug combination.9.Drug combination notably downregulated the Alb-induced the transmembrane osmotic gradients.10.Drug-combination treatment could improve pulmonary edema elicited by AngⅡ or BK stimuli in vivo.Conclusion:Angiotensin-Ⅱ-and bradykin-induced increases in intracellular protein nanoparticle(PN)-OP were associated with inflammasome production and cytoskeletal depolymerization.Intracellular protein nanoparticle production also resulted in hyperpolarization and L-VGCCinduced calcium signals,which differed from diacylglycerol-induced calcium increment via TRPC6 activation.Both pathways involve voltage-dependent cation influx and OP upregulation via SUR1-TRPM4 channels.Meanwhile,intra/extracellular PN-induced OP gradients across membranes upregulated pulmonary endothelial and alveolar barrier permeability.Attenuation of intracellular PN,calcium signals,and cation influx by drug combinations effectively relieved intracellular OP and pulmonary endothelial nonselective permeability,and improved epithelial fluid absorption and pulmonary edema.This study reveals that the mechanism of inflammasome induced PN-OP in pulmonary edema,and provides a new view of mechanism in in ARDS.