The Role Of NLRP3 Inflammasome In The Comorbidity Of Depression And Diabetes

Background and Objective:As economic and social developments accelerate,people are suffering from more pressures and challenges.Unfortunately,long-term stress exposure may pose a huge threat to human health both physically and mentally,and even lead to a variety of chronic diseases regarding the cardiovascular,metabolic,digestive,and neuropsychiatric systems.According to the report on the global burden of disease?GBD?,chronic non-communicable diseases have become a major contributor to the GBD.In the notorious list of GBD,depressive disorder and diabetes have been ranked third and fourth?only after low back pain and headache syndrome?;however,in China,they ranked the second and sixth,respectively.More importantly,these two diseases are highly comorbid,also seriously hamper clinical diagnosis and treatment,as well as the qualities of the patients'life.Recently,researches on the relationship between depression and diabetes have attracted great interests,but the underlying mechanism still remains unclear.Abundant studies indicate that inflammatory cytokines including IL-1?play an important role in multiple chronic diseases.For example,IL-1?can damage pancreatic B cells through endoplasmic reticulum and mitochondrial stresses,and then results in a sharp decrease in insulin secretion.In addition,IL-1?can also promote serine phosphorylation of IRS-1 and induce insulin resistance in metabolic organs such as liver and adipose tissue,thereby participates in the pathogenesis of both T1DM and T2DM.As to the research of depression,pro-inflammatory cytokines like IL-1?may participate in the pathophysiological process of depression via multiple pathways including activating HPA axis,disrupting kynurenine pathway,promoting glutamate excitotoxicity,and influencing neuroplasticity.It is worth noting that clinical researches also find that depressive patients have higher levels of IL-1?in blood and cerebrospinal fluid than healthy volunteers.Besides,applications of antidepressants can significantly rescue the upregulation of proinflammatory cytokines.In fact,Caspase-1,which acts as a vital component of the inflammasome complex,is indispensable for the maturation of IL-1?.Considering that NLRP3 inflammasome often plays an anti-infective role in the innate immune system,it is currently believed to be the most important one among the known types of inflammasomes.However,when it is over-activated,it may trigger a systemic inflammatory cascade that leads to various diseases.As was reported,higher expressions of NLRP3 inflammasomes could be detected in the monocytes from both diabetic and depressive patients.Furthermore,researchers across the world unanimously confirm that danger signals such as glucocorticoids,hyperglycemia,and hyperlipidemia can activate NLRP3inflammasomes.Accordingly,we wonder that whether the chronic mild inflammatory response,which centered on NLRP3 inflammasome activation,would be the critical linkage connecting stress,depression,and diabetes.In order to unveil the exact mechanisms underlying the comorbidity of depression and diabetes,we carried out several experiments.We do hope this study will provide valuable targets for preventions and treatments of the comorbidity,as well as offer a novel therapeutic strategy.Methods:1.The role of NLRP3 inflammasome in CUMS-induced depressive-like behaviorAt first,a CUMS paradigm was applied to establish a depression mouse model,then the concentrations of serum corticosterone and proinflammatory cytokines in each mouse were detected.Meanwhile,the stressed mice were administered with NLRP3 inflammasome inhibitor glyburide or antidepressant fluoxetine.In order to evaluate the effect of glyburide and fluoxetine,depressive-like behaviors,as well as hippocampal glial cells and NLRP3inflammasomes were analyzed.2.The role of NLRP3 inflammasome in CUMS-induced glucose metabolism disorder comorbid with depressive-like behaviorFirstly,we detected several glucose metabolism indicators including the insulin resistance index and islet secretion function of the model mice.Afterward,the hippocampal insulin signaling pathway was investigated.In order to confirm the exact causes of these abnormalities,the structure and function of islet tissue were analyzed.Besides,we observed the infiltration of inflammatory cells in pancreatic islets.Finally,the expression and activation of NLRP3 inflammasome in the pancreas were determined and then compared with those in the hippocampus.3.The influence of in vitro high-glucose environment on the NLRP3 inflammasomeFirstly,we established a high glucose environment in vitro,which helped investigate the effect of high glucose on the activation of microglial NLRP3 inflammasome in BV-2 cell lines.Secondly,we examined both the upstream signal??TLR4/MyD88/NF-?B?and signal??ROS/PKR/P2X7R/TXNIP?,as well as the related activation signaling pathways.In order to confirm the underlying mechanism of inflammasome activation in the high-glucose model,we used various specific inhibitors targeting different signaling molecules.Thirdly,the most efficient NLRP3 inflammasome inhibitor,namely MCC950,was applied in the high-glucose model,then its inhibitory effect was analyzed and also compared with those of the upstream signaling blockers.4.The role of NLRP3 inflammasome in depressive-like behavior induced by hyperglycemia in vivoAt first,to establish a mouse model of hyperglycemia,they were injected intraperitoneally with high-dose STZ.Afterward,we screened the model mice and then measured their depression-like behavioral indicators.Secondly,based on the aforementioned methods,MCC950 was used to intervene the hyperglycemic mice,then its possible improving effect was assessed.Additionally,the levels of serum corticosterone and proinflammatory cytokines in the model mice were determined.Finally,the activation of glial cells and NLRP3 inflammasomes in the hippocampus were detected and analyzed.Results:1.Chronic stress induced depressive-like behavior in mice1.1 After 12 weeks of CUMS,model mice displayed a depressive-like phenotype,whereas daily i.p.injection of either glyburide or fluoxetine could effectively alleviate it.1.2 CUMS could induce the overactivation of the HPA axis and then initiated moderate inflammatory responses,while glyburide had a protective effect.1.3 CUMS induced the activation of hippocampal microglia rather than astrocytes.However,neither glyburide nor fluoxetine would ameliorate it.1.4 CUMS prompted the activation of NLRP3 inflammasome and the production of IL-1?possibly via upregulation the expression of TXNIP in the hippocampus.Meanwhile,both glyburide and fluoxetine could abort the process.2.CUMS induced glucose metabolism disorder comorbid with depressive-like behavior in mice2.1 After the 12-week CUMS procedure,the stressed mice demonstrated multiple glucose metabolism malfunctions such as abnormalities of insulin secretion and tolerance,as well as the elevated fasting insulin levels.Inflammasome inhibitor glyburide partially ameliorated the phenotype of impaired insulin tolerance.2.2 The hippocampal insulin signaling pathway of the model mice was disturbed,and we observed that the phosphorylation of AKT1 at Ser473 was inhibited.Interestingly,glyburide could improve the disturbance to a certain extent.2.3 CUMS reduced the proportion of B cells in islet tissues,both glyburide and fluoxetine had protective effects on it.2.4 CUMS promoted the accumulation of F4/80-positive macrophages in islet tissues,glyburide could effectively improve this circumstance.2.5 CUMS might induced the activation of NLRP3 inflammasome and the production of IL-1?in the pancreas possibly by upregulating TXNIP levels,which was similar to the central nervous system.Nevertheless,both glyburide and fluoxetine could inhibit the process.3.High-glucose environment activated the NLRP3 inflammasome in BV-2 microglial cell line3.1 In vitro high-glucose environment?50 mM?activated microglial NLRP3inflammasome,independent of the elevated osmotic pressure,and also promoted the synthesis and secretion of IL-1?.3.2 In vitro high-glucose environment promoted the phosphorylation of NF-?B p65subunit,which was the key molecule of inflammasome activation Signal?.Nevertheless,the classical TLR4/MyD88 signaling pathway was not involved in this process.3.3 The specific inhibitor of NF-?B could effectively block the activation of NLRP3inflammasome and the production of IL-1?induced by the high-glucose environment.3.4 In vitro high-glucose environment increased the production of ROS and the phosphorylation of PKR,as well as upregulated the expressions of P₂X₇R and TXNIP,which were all vital switches of inflammasome activation Signal?.3.5 Both P₂X₇R or PKR-specific inhibitors abrogated the activation of NLRP3inflammasome and the production of IL-1?induced by high glucose in vitro.3.6 ROS scavenger NAC efficiently decreased the phosphorylation of PKR and the expression of TXNIP,thus inhibited the high glucose-induced NLRP3 inflammasome activation and IL-1?production.However,NAC could not change the expression of P₂X₇R.3.7 The novel inhibitor named MCC950 effectively inhibited the high glucose-induced activation of NLRP3 inflammasome and the production of IL-1?in vitro.4.In vivo hyperglycemia induced depressive-like behavior in mice4.1 A single i.p.injection of high-dose STZ would induce a diabetic?hyperglycemic?mouse model by impairing islet B cells.The inhibitor of NLRP3 inflammasome MCC950failed to affect the blood glucose significantly.4.2 STZ-induced hyperglycemic model mice exhibited depressive-like behavior,and MCC950 was effective in alleviating this phenotype.4.3 The IL-1?concentrations in serums of hyperglycemic mice were increased,and MCC950 could significantly ameliorate it.4.4 The microglia in the hippocampus of hyperglycemic model mice were activated,and MCC950 had an inhibitory effect on it.In addition,astrocytes showed no obvious alterations.4.5 The NLRP3 inflammasome in the hippocampus of hyperglycemic model mice was activated,and the expression of IL-1?was also enhanced,but MCC950 could impede them somehow.Conclusions:Collectively,chronic unpredictable mild stress could activate the NLRP3inflammasome systemically,which in turn induced depressive-like behavior comorbid with glucose metabolism disorders in mice.Besides,high-glucose environment would initiate the activation of inflammasome especially in microglia,thereby induced depressive-like behavior in mice.Moreover,specific inhibitors targeting NLRP3 inflammasome?glyburide and MCC950?significantly alleviated the comorbidity of depression-and diabetes-like phenotypes caused by multiple stressors.Herein,the above evidence suggests that the activation of NLRP3 inflammasome and the following systematic chronic low-grade inflammation may be a vital mechanism underlying the comorbidity of depression and diabetes.This study contributes new evidence to the modern concept of integral medicine,and also provides a promising target for precise clinical diagnosis and treatment.