HSP90? Regulates Microglial M1 Polarization Associated To CNS Neuroinflammation In Heat Stroke

BackgroundHeat stroke?HS?is one of the most serious and harmful diseases in heat illness.Its clinical symptoms are hyperthermia,central nervous system dysfunction and rhabdomyolysis,disseminated intravascular coagulation?DIC?,systemic multiple organ dysfunction syndrome?MODS?,systemic inflammatory response syndrome?SIRS?,and so on,with an associated mortality of approximately 15⁵0%.As central nervous system damage is the main clinical feature of heat stroke,the brain becomes one of the main target organs of heat stroke.Therefore,the research on the mechanism of central nervous system injury with prevention and treatment strategies of heat stroke has attracted much attention.At present,it is generally believed that the mechanism of nerve cells injury caused by heat stroke is the direct attack of hyperthermia on nerve cells,and the secondary injury of nerve cells caused by cerebral edema,cerebral ischemia and hypoxia during heat stroke.The prevention and treatment strategies of brain injury in HS mainly include physical effort such as hyperbaric oxygen and cooling,as well as biological researches such as targeted functional protein and stem cell transplantation.Recently,with the continuous exploration of the pathological mechanisms of heat stroke,it has been found that the occurrence and development of inflammation in the central nervous system?CNS?of heat stroke is closely related to the severity of brain injury.However,due to the lack of deep and systematic studies on the molecular mechanism of CNS inflammation in heat stroke,which limited the clinical treatment of brain injury in heat stroke.Microglia,an important effector cells of CNS inflammation,are activated in many CNS diseases,such as brain trauma,cerebral ischemia and neurodegenerative diseases,manifesting M1 phenotypic activation,which has the characteristics of pro-inflammatory responses.However,the mechanism of microglia involved in CNS inflammation and CNS injury is still unclear.Therefore,to explore the mechanism of microglial M1 activation in CNS inflammation and CNS injury,studies targeting microglia may be a new breakthrough in the treatment of CNS injury in heat stroke,and the exploration of effective neuroprotective drugs,which have pivotal roles in maintaining the health of the general population and the professional population.Heat shock protein?HSP?,a group of genetically conserved stress proteins,can induced by hyperthermia,hypoxia,cold,trauma,infection and other stressors.Heat stress can induce HSP expression in microglia.Recently,many studies have confirmed that HSP is involved in the regulation of immune response and control of inflammatory response.Therefore,during the process of hyperthermia induced the M1 microglial phenotype,which may attribute to the regulation of HSP.HSP90 is the most widely reported pro-inflammatory regulatory factor,in sepsis,arthritis,enteritis,multiple sclerosis,lupus erythematosus and other inflammatory diseases can recruit a variety of inflammatory signaling molecules.The biological function of HSP90 depends on its isoforms of HSP90?and HSP90?.It was found that Hsp90?may be related to tumorigenesis,enhanced cell cycle regulation,and rapid protection of cell stress,while HSP90?may be related to cell function regulation such as cancer and inflammation.Therefore,we speculate that Hsp90?is involved in the regulation of M1 microglia stimulated by hyperthermia in the course of heat stroke.Therefore,this thesis based on heat stroke mice model,observed the central nervous system pathological changes and inflammatory mediators production,detected the expression of microglial M1 markers.Using an in vitro model of heat shock microglia,the expression of M1 markers,inflammatory factors and HSPs were detected.The role of HSP90 and its isoforms in the regulation the M1 microglia in heat shock was demonstrated in inhibitor intervention experiments.The mechanism of HSP90?regulation of M1microglia was systematically explored via the MAPK,STAT3,NF-?B inflammatory signaling pathways.These results will provide experimental basis for clinical intervention of HSP90?in the treatment of CNS inflammatory injury in heat stroke.Methods1,Male healthy BALB/c mice of 12 weeks old,were continuously exposed to thier core temperature reached 42.7°C in an environmental simulation chamber with a temperature of 42.0°C and 60%humidity.The mice were divided into normal control group?Con?,Heat stroke recovery 0 h group?HS-0h?,3 h group?HS-3h?,6 h group?HS-6h?,24 h group?HS-24h?,72 h group?HS-72h?,Ganetespib?Gan?group and HS+Gan group?at the time point of 6 h recovery?.The core temperature and body weight loss of each group were observed and the death was recorded.The brain tissue was collected at the corresponding time point and the pathological changes were observed by hematoxylin-eosin?HE?staining.Analyze the correlation between central nervous system inflammation and brain injury in heat stroke mice.2,Total RNA and protein were extracted from cerebral cortical tissue of each group.qRT-PCR were used to detect the mRNA levels of inflammatory factor TNF-?,IL-1?,IL-6,COX-2 and mPGES-1.ELISA/EIA was used to detect the secretion of TNF-?,IL-1?,IL-6and PGE₂ production.Investigate the changes of inflammatory response in heat stroke mice and heat shock,and the effects of inhibitors and esiRNA on anti-inflammatory regulation.3,The expression of M1 markers CD45,CD68,CD64 and CD16 was detected by Western blot,and the expression and localization of CD45 and CD68 were also detected by flow cytometry and immunofluorescence.Investigate the changes of M1 makers in heat stroke mice and heat shock microglial cells,and the regulatory effects of inhibitor and esiRNA intervention on M1 phenotype.4,Using immunoblotting to detect the expression of HSP90?,HSP90?and HSP70protein,and the expression and phosphorylation of ERK,JNK,p38,JAK2,STAT3,I?B-?,and p65.The expression of Iba-1,HSP90?and p-STAT3 were detected by immunofluorescence.Explore the regulatory mechanism of HSP90?in the M1 microglial phenotype via MAPK,STAT3 and NF-?B inflammatory signaling pathways.Results1,The HS mice were successfully acquired with core temperature of 42.7°C using42.0°C heat exposure.The mice entered a significant hypothermia depth period at 3 h recovery of heat stroke mice.And at 6 h of recovery,the pathological damage of the cerebral cortex,the body weight loss,the increase of mRNA and protein levels of inflammatory factor,the increased expression of M1 phenotypic markers were significantly.These changes accompany by an incomplete recovery at 24 h.These results demonstrated that the pathological CNS injury,inflammatory response,and M1 microglial polarization are predominant in heat stroke mice.2,Heat shock treatment with moderate heat exposure dose of 42°C for 1.5 h significantly induced the mRNA and protein expression of inflammatory factors,M1phenotypic markers,3 h and 6 h after heat shock,then both were gradualy decreased,and incomplete recovery at 12 h recovery.HSP27,HSP70,and HSP90 expression were increased differently.Results from inhibitor intervention experiments of HSP27,HSP70,and HSP90 indicated that microglial proinflammatory M1 phenotype was only regulated by HSP90 inhibition via Gan.3,HSP90 inhibitor Gan was found to inhibit the increase of phosphorylation expression of signal transduction effector molecules ERK,JAK2,and STAT3.ERK inhibition showed no change in heat shock N9 microglia,while inhibitor of JAK2 and STAT3 significantly suppressed the release of inflammatory cytokines.HSP90?and HSP90?esi transient silencing experiment suggest that Gan reverses increased secretion of inflammatory cytokines and the increase of JAK2 and STAT3 phosphorylation,via HSP90?but not HSP90?in N9 microglia following heat shock.4,After the intracerebral stereotactic injection of Gan targeted HSP90?,the cerebral edema and inflammatory pathological features in the cerebral cortex of heat stroke mice were alleviated.The mRNA and protein levels of inflammatory factor,fluorescence intensity and/nuclear translocation of M1 phenotypic marker and STAT3 phosphorylation were depressed.These results suggest that in vivo Gan intervention can inhibit the activation of microglial M1 phenotype through HSP90?–STAT3 pathway and alleviate inflammatory injury in central nervous system of heat stroke mice.Conclusion1,The pathological CNS injury,inflammatory response,and M1 microglial polarization are predominant in heat stroke mice,indicating that both of microglial M1phenotype and CNS inflammation were related to CNS injury during heat stroke.2,Heat shock significantly induced the mRNA and protein expression of inflammatory factors,and M1 phenotypic markers,showing an obvious activation of pro-inflammatory M1 phenotype of microglia.HSP90 inhibitor significantly reversed the increase of these changes,indicating that HSP90 play a pivatol role in pro-inflammatory M1 phenotype of microglia.3,Gan inhibits the increase ERK,JAK2,and STAT3 phosphorylation induced by heat shock,however,only inhibitors of JAK2 and STAT3 but not ERK showing a similar inhibition of pro-inflammatory response of M1 phenotype of microglia.Moreover,esi transient silencing of HSP90?but not HSP90?abolished the increased secretion of inflammatory cytokines and activation of downstream pro-inflammatory signaling pathway in heat shock microglia.These results demonstrated that HSP90?regulates M1 microglial polarization through JAK2–STAT3 pathway.4,After the injection of Gan targeted HSP90?into the brain,the cerebral edema and inflammation in the cerebral cortex of heat stroke mice were alleviated.The levels of mRNA and/or protein of inflammatory factor,M1 phenotypic marker,and STAT3phosphorylation were suppressed.The results suggest that in vivo Gan intervention can inhibit the activation of microglia M1 phenotype through HSP90?–STAT3 pathway and alleviate inflammatory injury in central nervous system of heat stroke.