Study On The Mechanism Of HMGB1 Activating Platelet NLRP3 Inflammasome In Severe Heatstroke

Background and objective:Heat stroke is a disease that seriously threatens the life of the military and civilians.The performance of the central body temperature exceeds 40℃.Severe heatstroke often presents with multiple organ dysfunction syndrome(MODS),which is characterized by central nervous system dysfunction.Mortality is high.Recent studies have found that microthrombi and inflammatory network responses based on the damage of vascular endothelial cells are the underlying pathogenesis of multiple organ dysfunction in severe heat stroke.Platelet activation plays an important regulatory role in the network of inflammation and thrombosis.Whether it participates in the regulation of severe heat stroke microthrombosis and inflammatory response network and its possible mechanisms is still unclear.The inflammasome is a multi-protein complex with a molecular weight of approximately 700 kDa,consisting of Nlrp3(NACHT-LRR-PYD-containing protein3),adaptor speck-like protein containing a CARD and effector protein Caspase-1(The composition of cysteine aspartic acidspecific protease)is involved in the initiation of inflammatory responses in a variety of infectious and non-infective disease states.The study found that there is a molecular mechanism of platelet NLRP3 Inflammasome activation in dengue fever.The clinical changes and pathological features of severe heat stroke platelets are similar to those of dengue fever.The existence of similar molecular mechanisms of inflammatory bodies in platelets has not been reported.HMGB1 is one of the important dangerous-associated molecular pattern(DAMPs)molecules,and studies have demonstrated that HMGB1 can activate NLRP3 Inflammasome based on reactive oxygen species(ROS)mechanism.Our previous study found that plasma HMGB1 was significantly elevated in the early stage of heat stroke and was negatively correlated with the prognosis.In addition,our team found that HMGB1-induced activation of Nlrp3 inflammatory mediators in hepatocytes involved in severe heat stroke liver injury.Based on this,this study intends to investigate the activation of platelet NLRP3 Inflammasome by HMGB1 and its related molecular mechanisms,in order to elucidate the possible mechanism of platelet involvement in severe heat stroke thrombotic network reactions.In this study,a rat model of severe heat stroke was used to observe the changes in platelet count,structural and function,to detect the expression and activation of platelet NLRP3 Inflammasome,and to analyze the molecular mechanism of activation of HMGB1-receptors-ROS in the NLRP3 Inflammasome of severe heat stroke platelets.The effects of HMGB 1 activating platelet NLRP3 Inflammasome on platelet count and activation in rats with severe heatstroke were also investigated.The purpose of this study was to elucidate the possible molecular mechanism of HMGB1 activation of severe NLRP3 Inflammasome in severe heat stroke and to provide a possible molecular mechanism for revealing the pathological process of severe heat stroke platelet activation.Method:1.Study the dynamic changes of number,structure and function of platelets caused by Heat StrokeSprague Dawley rats were randomly divided into 5 groups:normal temperature group(Sham),severe heat stroke(HS)rewarming 0 h group(HS-0 h),severe heat stroke rewarming 3 h group(HS-3 h),severe Heatstroke rewarming 6 h group(HS-6 h)and severe heatstroke rewarming 9 h group(HS-9 h).The normal temperature group was placed at room temperature[temperature(22.0±0.5)℃ and humidity(50.0%±5.0%)]and each group of severe heat stroke was placed in a artificial climate incubator[temperature(39.5±0.2)℃ and humidity(60.0%±5.0)%)],take rectal temperature(Tr)as the core temperature(Tc),Tc up to 43 ℃ as the standard of severe heat stroke.The following indicators were measured in each group:changes in body weight and Tc before and after heat stroke;Using a blood cell counter to detect platelet counts,mean platelet volume(MPV)and platelet distribution width(PDW);Platelet was isolated by PRP method and its purity was identified;transmission electron microscopy was used to observe the platelet ultrastructure;flow cytometry was used to detect the change of platelet activation marker CD62P;The maximum platelet aggregation rate was measured with a platelet aggregation tester and the platelet function(PF)was measured with the Sonoclot platelet function analyzer.2.Experimental study on assembly and activation of platelet NLRP3 Inflammasome in rats with severe heatstrokeThe expression of platelet Nlrp3 and activated Caspase-1:platelet Nlrp3 and activated Caspase-1 in rats with severe heatstroke increased as rewarming time prolonged.Platelet NLRP3 Inflammasome assembly:Laser confocal microscopy showed that colocalization of Nlrp3 and ASC proteins were present in platelets 9 hours after heat stroke onset;NLRP3 Inflammasome activation:the engagement of Nlrp3 with ASC and the activation of Caspase-1 represented activation of inflammasome.With the onset of severe heat stroke,the Nlrp3 and ASC junctions and Caspase-1 activation were detected in platelets of rats,and gradually increased with rewarming time and peaked at 9 hours.3.The role of HMGB1 and its key receptors in initiating platelet NLRP3 Inflammasome in severe heat strokeELISA was used to detect the changes of serum HMGB1 concentration in Sham,HS-0 h,HS-3 h,HS-6 h and HS-9 h;to observe the effect of HMGB1 on activation of platelet inflammasome in severe heat stroke:ethyl pyruvate(EP)and Anti-HMGB1-specific antibody(a-HMGB1)pretreated rats to detect HS-9 h platelet count,CD62P,Nlrp3 and activated Caspase-1 expression;Select HMGB1 receptors for activating severe heat stroke in platelet inflammatory bodies:Detection the dynamic changes of HMGB1 specific receptors TLR2(toll like receptor 2),TLR4(toll like receptor 4),and RAGE on platelet surface at different time points(Sham,HS-0 h,HS-3 h,HS-6 h,HS-9 h);Rats were pretreated with anti-TLR4 specific antibody a-TLR4,anti-RAGE specific antibody a-RAGE,and a-TLR4+a-RAGE to detect HS-9 h platelet count,CD62P,Nlrp3,and activated Caspase-1 expression.Inhibition of Caspase-1 activation on platelet count and CD62P expression changes:The rats were pretreated with ac-YVAD-cmk and the HS-9 h platelet count and CD62P expression were measured.4.Role of ROS in platelet HMGB1/NLRP3 Inflammasome pathway in rats with severe heat strokeMitox-SOX(mitochondrial superoxide),DHE(dihydroethidium),and DCF-DA(2’,7’-dichlorofluorescein diacetate)fluorescent probes were used to detect dynamic changes of platelet mitochondrial superoxide anion(mitochondrial O2·-),cytoplasmic O2·-and hydrogen peroxide(H2O2)at Sham,HS-0 h,HS-3 h,HS-6 h,and HS-9 h in rats with severe heat stroke;Platelet ROS expression induced by HMGB1 in rats with severe heatstroke:rats were pretreated with EP and a-HMGB1 to detect changes in platelet mitochondrial O2·-,O2·-,and H2O2 expression;Effect of ROS on the expression of platelet Nlrp3 and activated Caspase-1:rats were pretreated with N-acetyl-L-cysteine(NAC)to detect HS-9 h platelet count,CD62P,Nlrp3 and activate Caspase-1 expression.Results:1.Study the dynamic changes of number,structure and function of platelets caused by Heat StrokeIn the severe heatstroke group,the body weight loss was 28.3±3.7g after heat stroke,and the body weight of the Sham group was reduced by 5.5±1.2g at the same time;the Tc of the severe heatstroke group was increased by 6.3±0.2℃.There was no change in body temperature in the Sham group.Compared with the Sham group,there was a statistically significant difference in weight loss and Tc between the rats with severe heatstroke(P<0.001).The PRP method was used to isolate platelets.The purity of the isolated platelets was determined by platelet-specific antibody CD61-FITC(94±2)%.Rat platelet count decreased in a time-dependent manner(P<0.01):Sham group(814.3 ± 17.8*109/L),HS-0 h group(625.7 ± 17.8*109/L),HS-3 h group(521.3 ± 11.9*109/L),HS-6 h(417.3±6.1*109/L)and HS-9 h(259.3±124.1*109/L).Changes in MPV and PDW in rats:MPV and PDW in the Sham,HS-0 h,HS-3 h,HS-6 h groups were all within the normal range and there was no statistical difference.There was a significant increase in the HS-9 h group of MPV(7.1±0.4fL,P<0.001)and PDW(17.5±0.3,P<0.001).Transmission electron microscopy revealed that as the rewarming time prolonged,the platelet ducts of rats gradually expanded,vacuoles increased,mitochondrial membranes swollen and ambiguous,and autophagosomes increased.Platelet vacuolar/platelet ratio progressively increased(P<0.05):Sham group(1.7±0.3%),HS-0 h group(4.2±0.3%),HS-3 h group(6.7±0.2%),HS-6 h group(9.6±1.7%)and HS-9 h group(19.1 ± 3.2%).The rat platelet activation marker CD62P increased in a time-dependent manner(P<0.05):Sham group(1.7±0.3%),HS-0 h(4.2±0.3%),HS-3 h group(6.7±0.2%),HS-6 h group(9.6±1.7%)and HS-9 h group(19.1 ± 3.2%).The maximum platelet aggregation rate in rats decreased in a time-dependent manner(P<0.05):Sham group(73.5+2.0%),HS-0 h group(56.5± 1.6%),HS-3 h group(46.3±0.9%),HS-6 h group(40.5±0.9%)and HS-9 h group(33.5±1.7%).With the prolonged rewarming time,the PF value of platelet function was progressively decreased(P<0.05):Sham group(3.72±0.06),HS-0 h group(3.03 ±0.03),HS-3 h group(2.84±0.07),HS-6 h group(2.28±0.07)and HS-9 h group(1.28±0.28).2.Experimental study on assembly and activation of platelet NLRP3 Inflammasome in rats with severe heatstrokeThe expression of platelet Nlrp3 and activated Caspase-1:Platelet Nlrp3 and activated Caspase-1 in rats with severe heatstroke increased as rewarming time prolonged.Platelet NLRP3 Inflammasome assembly:Laser confocal microscopy showed that colocalization of Nlrp3 and ASC proteins was present in platelets 9 hours after severe heat stroke onset;NLRP3 Inflammasome activation:the engagement of Nlrp3 with ASC and the activation of Caspase-1 represented activation of inflammasome.With the occurrence of severe heat stroke,the Nlrp3 and ASC junctions and Caspase-1 activation were detected in platelets of rats,and gradually increased with rewarming time and peaked at 9 hours.3.The role of HMGB1 and its key receptors in initiating platelet NLRP3 Inflammasome in severe heat strokeThe serum HMGB1 concentration in rats with severe heat stroke increased progressively with prolonged rewarming time(P<0.01):Sham group(0 pg/ml),HS-0 h group(65.8± 15.5 pg/ml),HS-3 h group(1265.3± 163.6 pg/ml),HS-6 h(1602 ±71.3 pg/ml)and HS-9 h(1827.8 ± 106.7 pg/ml).Inhibition of HMGB1 inhibited the decrease of platelet count(P<0.05),inhibited the increase of CD62P(P<0.01),and decreased the expression of Nlrp3 and activated Caspase-1 in HS-9 h rats(P<0.05).Effects of TLR4 and RAGE receptors on platelet count,activation,expression of Nlrp3 and activated Caspase-1:platelet surface receptor TLR2 rises early in rats with severe heatstroke and increases with time(P<0.01):Sham group(12.8)±1.8%),HS-0 h group(20.7± 1.3%),HS-3 h group(25.7±0.7%),HS-6 h group(32.8±0.6%)and HS-9 h group(42.4±3.6%);The platelet surface receptor TLR4 in the rats with heatstroke increased gradually with prolonged rewarming time(P<0.01):Sham group(2.5± 1.5%),HS-0 h(5.7±0.8%)group,HS-3 h group(9.9± 1.0%)HS-HS group(15.5± 1.8%)and HS-9 h(22.9±4.1%);platelet surface receptor RAGE increased in a time-dependent manner in rats with severe heat stroke(P<0.05):Sham group(10.2± 1.8%)),HS-0 h group(20.5±2.7%),HS-3 h group(24.6± 1.2%),HS-6 h group(29.1±0.3%)and HS-9 h group(35.7±4.7%).Inhibition of TLR4 and RAGE inhibited the decrease of platelet count in HS-9 h rats,inhibited the increase of CD62P,and decreased the expression of Nlrp3 and activated Caspase-1.Inhibition of Caspase-1 inhibited the decrease of platelet count and the increase of CD62P expression in HS-9 h.4.Role of ROS in platelet HMGB1/NLRP3 Inflammasome pathway in rats with severe heat strokeDynamic expression changes of platelet mitochondrial O2·-,cytoplasmic O2·-and H2O2 in rats with severe heatstroke:The mitochondrial O2·-in platelets of rats with severe heat stroke increased as the rewarming time prolonged(P<0.05),and the average fluorescence value of Mito-SOX:Sham group(183.7±52.4),HS-0 h group(276.3±35.8),HS-3 h group(276.3±35.8),HS-6 h group(437.7±16.3)and HS-9 h group(722.7+22.7);The expression of cytoplasmic O2·-in the platelets of rats with heatstroke increased with prolonged rewarming time,and the average fluorescence value of DHE increased with rewarming time(P<0.05):Sham group(2273.0±500.2),HS-0 h group(3535.7±)359.5),HS-3 h group(4516.7±189.5),HS-6 h group(5667.7±256.0)and HS-9 h group(7420.3±409.1);H2O2 in platelets of rats with severe heatstroke increased with prolonged rewarming time,The average fluorescence value of DCF-DA increased with rewarming time(P<0.05):Sham group(206.3±31.8),HS-0 h group(277.7±14.2),HS-3 h group(344.0±26.9),HS-6 h group(381.0±3.0)and HS-9 h(420.3 ± 19.1);Platelet ROS expression induced by HMGB1 in rats with severe heatstroke:inhibition of HMGB1 HS-9 h platelets Mitochondrial O2·-,cytoplasmic O2·-,H2O2 expression decreased;ROS on platelet count,activation,Nlrp3 and activation of Caspase-1 expression:inhibition of ROS can inhibit the HS-9 h platelet count decreased,inhibit the increase of CD62P,so that Nlrp3 and reduced expression of activated Caspase-1.Conclusion:HMGB1-TLR4/RAGE may activate the severe heat stroke platelet NLRP3 Inflammasome through ROS pathway.