Tubastatin A’s Regulation Of Platelet Cytoskeletal Motility Affecting On NETs Formation

Objective: NETs are a type of extracellular traps released by neutrophils.However,the pathophysiological mechanism of NETs formation disorder is still not clear enough in the acute and critical illness,and there are no effective interventions yet.Recent studies have found that dysregulation of platelet activation is an important factor inducing excessive release of NETs from neutrophils in vivo.Therefore,this project aims to further explore the intrinsic link between platelet activation and neutrophil extracellular trap formation by investigating the effect of HDAC inhibitors on activated platelets morphology and secretion function as well as their ability to induce NETs formation.Method: In this experiment,neutrophils as well as fresh platelets were separated from the blood of well adults.Platelets were cultured in vitro and pretreated with platelet activator,and the pretreated platelets and neutrophils were co-cultured for subsequent experiments.Sytox blue,a extracellular fluorescent nucleic acid dye was added to the medium.The fluorescence intensity of the cell suspension Sytox blue in each well of the 96-well plate was repeatedly measured with a fluorescence microplate during constant temperature culture.The measured data were used to plot the time-fluorescence intensity kinetic curve as a dynamic monitoring and quantitative analysis of NETs formation.The ability of different inducers to induce NETs was evaluated by comparing the trend of fluorescence intensity over time in different treatment groups and the increase in fluorescence intensity.The specimens were immunofluorescence stained for qualitative analysis of NETs formation.The pretreated platelet suspension supernatant was drawn to detect the concentration of PF4 secreted by platelets to reflect the secretion function of platelets.Immunofluorescence staining of the cytoskeleton was performed after platelet culture,and the structure of platelet microtubules was observed under the microscope.By comparing the concentration of platelet PF4 and the nodes of the platelet cytoskeleton in different treatment groups,the effects of different treatments on platelet function were analyzed.Results: After stimulation by PMA,the regularity of neutrophils releasing extracellular DNA changed,the amount of DNA released increased,and the network structure of DNA attached with NE was observed by immunofluorescence,indicating that the neutrophils isolated in this experiment had the physiological activity of producing NETs.The regularity of the release of extracellular DNA from neutrophils co-cultured with activated platelets has changed,and the amount of DNA released has increased.Immunofluorescence can be seen on the network structure of DNA attached with NE,indicating that activated platelets can induce the release of NETs from neutrophils.These changes were also observed in co-culture with activated platelets treated with HDAC6 inhibitor,indicating that activated platelets treated with HDAC6 inhibitor can still induce neutrophils to release NETs.Compared with activated platelets without inhibitor treatment,activated platelets treated with HDAC6 inhibitor have different rules and reduced release of extracellular DNA from neutrophils,indicating that HDAC6 inhibitor can downregulate the ability of activated platelets to induce neutrophils to release NETs.The concentration of PF4 in the supernatant of platelet suspension pretreated with platelet activator increased and was higher than that in the supernatant of activated platelet suspension treated with HDAC6 inhibitor.The concentration of PF4 in the supernatant of the latter was higher than that of PF4 in the supernatant of platelet suspension without stimulant,indicating that HDAC6 inhibitor failed to completely block the secretion function of activated platelet PF4 and only had a weakening effect.The activated platelet cytoskeleton treated by HDAC6 inhibitor can restore the morphology of some platelets when they are not activated,indicating that HDAC6 inhibitor can change the morphology of platelet cytoskeleton.Conclusion: Tubastatin A can inhibit the ability of activated platelets to induce neutrophils to release NETs.The mechanism may be that HDAC6 specifically inhibits the movement of activated platelet α-tubulin,weakens the secretion function of platelets,and thus reduces the generation of NETs.