The Experimental Study Of HDAC Inhibitor’s Effect On NETs Formation Induced By Activated Platelets

Objective: It has already been demonstrated that NETs play an important role in pathophysiology of critical diseases including sepsis,ischemia reperfusion injury and stroke.However,there is no effective therapy targeting dysregulated NETs formation due to complicated mechanism of NETs formation in vivo.Accumulating studies in recent years have revealed that uncontrolled platelet activation acted as an important inductive factor in robust NETs formation in vivo.In previous study,our group found that HDAC inhibitor attenuated the elevation of a serum NETs marker in mice with septic shock and others found that HDAC participated in platelet activation.So this project tries to establish a technical system for NETs formation induced by activated platelets.It will provide technical support for further study on internal relations between platelet activation and NETs formation.What’s more,this project evaluated HDAC inhibitor’s effect on platelets’ secretion and capacity of NETs inducing,to provide new insight into the mechanism on which HDAC inhibitors protect multiple organ function.Method: Fresh platelets and neutrophils were isolated from venous blood of healthy individuals.Isolated platelets were treated with platelet activator in vitro and then transferred to neutrophils to induce NETs formation.An extracellular nucleic acid dye,Sytox green,was added in the medium and the fluorescent intensity of Sytox green was measured repeatedly during incubation.A fluorescent intensity-time kinetic curve based on the repeatedly measured data was established to monitor and quantitively analyze NETs formation.NETs formation was also qualitatively analyzed with live-cell imaging and immunofluorescence.To evaluate the capacity of NETs inducing in different inducers,the trend of fluorescent intensity changing with time and increase in fluorescent intensity after incubation was compared.What’s more,to evaluate the influence on platelet secretion by different treatment,the concentration of PF4 in supernatant of platelet after different treatment was measured by ELISA.Results: Neutrophils treated with PMA had different trend and more quantity of DNA release,formation of web-like structure in live-cell imaging and NE attached to web-like structure in immunofluorescent image,indicating that isolated neutrophils could be activated and produce NETs.Neutrophils incubated with preactivated platelets haddifferent trend and more quantity of DNA release,formation of web-like structure in live-cell imaging and NE attached to web-like structure in immunofluorescent image,indicating that preactivated platelets could induce NETs formation.Neutrophils incubated with preactivated platelets in presence of HDAC inhibitor also had such changes,indicating that platelets preactivated in presence of HDAC inhibitor still had the capacity of NETs inducing.Compared with platelets preactivated without HDAC inhibitor,platelets preactivated in presence of HDAC inhibitor induced different trend and less quantity of DNA release,indicating that HDAC inhibitor attenuated preactivated platelets’ capacity of NETs inducing.What’s more,platelets stimulated with platelet activator had higher PF4 in supernatant,indicating that isolated platelets could be further activated.Platelets preactivated in presence of HDAC inhibitor had less PF4 in supernatant than platelets preactivated without HDAC inhibitor,indicating that the secretion of PF4 by activated platelets was not blocked but attenuated by HDAC inhibitor.Conclusion: HDAC inhibitor could attenuate preactivated platelets’ capacity of NETs inducing,partially through down-regulating platelet secretion.