The Mechanism Of Diabetic Encephalopathy Mediated By NLRP3 Inflammasome And Golgi Stress

Diabetes is one of the most common immuno-metabolic diseases,with the development of the society,change of lifestyle such as reduced excercise,increased longevity,the incidence of diabetes increased globally in developing countries.As reported,diabetes has become the third non-infectious disease threatening human health and life after cardiovascular disease and cancer.Diabetes encephalopathy is the most common diabetes complication with the symptom including mild reaction retardation,memory loss,serious cerebral thrombosis,cerebral apoplexy and senile dementia,has great impact on the life of diabetes people.It has been reported that diabetes encephalopathy is associated with neuronal apoptosis and microglia inflammation caused by high glucose,whereas the mechanism remains unclear.Studies also found that high glucose induces the activation of microglia which causes the release of inflammatory factors,ended by diabetes dementia.The activation of NLRP3 inflammasome is closely associated with neuroinflammation mediated by high glucose,so we first explored whether high glucose could activate the NLRP3 inflammasome via the ROS pathway,then we investigated the effect of TREM2 in the activation of NLRP3.After that,we explored whether high glucose can cause Golgi stress in high glucose induced encephalopathy and its signalling pathway.Finally,we screened the specific inhibitors targeting NLRP3 and GOLPH3,explored the anti-neuroinflammation effect of these inhibitors to develop new drugs for the treatment of diabetes encephalopathy.First,we found that microglia NLRP3 inflammasome activation mediates diabetic neuroinflammation via ROS/MAPKs/NF-κB pathway.BV2 cells were activated by high glucose as indicated by incresed release of proinflammatory cytokines IL-1β,IL-18,IL-6 and TNF-α(p≤0.001).Cytoplasm ROS was upregulated by high glucose in a dose-dependent manner while mitochondrial ROS was unaffected.Additionally,we found that high glucose induced the upregulation of the NLRP3 inflammasome,MAPKs,NF-κB pathways at 35 mmol/L for 12 h.Inhibition assay using specific inhibitors indicated that high glucose could stimulate microglia NLRP3 activation via ROS/JNK MAPK/NF-κB pathway.In mouse models of STZ induced diabetes mice,microglia NLRP3,ASC and caspase-1 proteins were highly expressed,and serum cytokines IL-1β,IL-6,IL-18 and TNF-α were significantly upregulated compared with that of control mice indicated by immunohistochemistry and ELISA(p≤0.001).So we concluded that high glucose could stimulate microglia NLRP3 inflammasome activation via ROS/JNK MAPK/NF-κB pathways in vitro and in vivo.Next,we found that TREM2 regulated high glucose induced microglia inflammation via NLRP3 signalling pathway.By using CRISPR-Cas9,Western blot,q PCR,ELISA,Pulldown and co-IP methods,we found that TREM2 was significantly increased(p≤0.01)as well as the proinflammatory cytokine IL-1β(p≤0.05)in high glucose induced BV2 cells,TREM2 knockout(KO)attenuated the pro-inflammatory cytokine IL-1β(p≤0.01),conversely,TREM2overexpression(OE)exacerbated IL-1β expression(p≤0.01).Further,we found that high glucose promoted the interaction of TREM2 with NLRP3,TREM2 KO abolished the interaction of TREM2 with NLRP3,while,TREM2 OE enhanced the interaction.Moreover,TREM2 KO dampened the high glucose induced NLRP3 inflammasomes activation,TREM2 OE augmented high glucose induced NLRP3 inflammasomes activation,indicating that high glucose enhanced the expression of TREM2 which led the activation of NLRP3 inflammasome.To further clarify if TREM2 regulated inflammation response was mediated by NLRP3 signaling pathway,we blocked NLRP3 inflammasome by NLRP3 KO and caspase1 inhibitor,the pro-inflammatory cytokine IL-1β were decreased(p≤0.01),high glucose induced the expression of TREM2 were not influenced.So we concluded that TREM2 modulated high glucose induced microglia inflammation via NLRP3 signaling pathway.Additionally,we found that high glucose could induce Golgi stress,indicated by fragments of Golgi apparutus,which can be reversed by knockout of NLRP3.High glucose promotes the interaction of NLRP3 and VPS35 and the interaction of VPS35 and GOLPH3,knockout of NLRP3 suppressed the expression of VPS35 and GOLPH3,knockout of VPS35 reduced the expression of GOLPH3 but not NLRP3,indicating that high glucose could induce the activation of NLRP3/VPS35/GOLPH3 pathway.Further,we eluciated the mechanism of the GOLPH3 mediated Golgi stress in high glucose condition.GST-pulldown and Co-IP experiments showed that high glucose promoted the interaction of GOLPH3 and Vimentin,knockout of GOLPH3 alleviated the expression of Vimentin.Knockout out of GOLPH3 and Vimentin both ameliorated high glucose induced Golgi fragments.In summary,our study demonstrated that high glucose regulates Golgi stress through NLRP3/VPS35/GOLPH3/Vimentin pathway.At last,we screened small molecular inhibitors targeting the NLRP3 and GOLPH3 to provide new therapeutic strategy for diabetes encephalopathy.The NLRP3 inhibitor zafirlukast(ZAF)and GOLPH3 inhibitor bromocriptine(BRO)were screened by Autodock Vina and Autodock 4.0 software.The maximum safe dose of ZAF was 5 μM and that of BRO was 2.5 μM verified by CCK8 experiment.ZAF combined with BRO could reduce the transcription and expression of microglia inflammatory factors IL-1β and IL-6 caused by high glucose(p≤0.001).ZAF combined with BRO could suppress the expression of NLRP3-related proteins and Golgi stress-related proteins induced by high glucose(p≤0.001).In STZ-induced diabetic encephalopathy mouse model,ZAF combined with BRO could alleviate the increase of blood glucose and weight loss of diabetic mice(p≤0.001),and alleviate the cognitive impairment of diabetic mice(p≤0.001).So we concluded that combination of small molecular inhibitors targeting NLRP3 and GOLPH3 selected by molecular docking alleviated high glucose-induced neuroinflammation in vitro and in vivo.Therefore,the molecular inhibitors targeting NLRP3 and GOLPH3 have great potential for use in the development of antidiabetes neuroinflammatory therapies.In summary,our findings suggested that high glucose could activate microglia NLRP3 inflammasome through the ROS/JNK MAPK/NF-κB pathway,which mediates the development of diabetic encephalopathy.Meanwhile,high glucose stimulates the interaction of TREM2 and NLRP3 to regulate NLRP3 activation.Next,we explored the Golgi stress response mediated by high glucose through the NLRP3/VPS35/GOLPH3/Vimentin pathway.Finally,we screened specific small molecule inhibitors targeting NLRP3 and GOLPH3,and found that the combination of inhibitors could reduce microglial inflammatory response in vitro and ameliorate diabetic cognitive impairment in STZ induced diabetes mice.Our study provides scientific basis for studying the pathogenesis of diabetic encephalopathy and drug development.