Role of NLRP3 inflammasome in inflammatory bowel diseases

Inflammasomes are multiprotein intracellular complexes which are responsible for the activation of inflammatory responses. Among various subtypes of inflammasomes, NLRP3 has been a subject of intensive investigation. NLRP3 is considered to be a sensor of microbial and other danger signals and plays a crucial role in mucosal immune responses, promoting the maturation of proinflammatory cytokines interleukin 1β(IL-1β) and IL-18. NLRP3 inflammasome has been associated with a variety of inflammatory and autoimmune conditions, including inflammatory bowel diseases(IBD). The role of NLRP3 in IBD is not yet fully elucidated as it seems to demonstrate both pathogenic and protective effects. Studies have shown a relationship between genetic variants and mutations in NLRP3 gene with IBD pathogenesis. A complex interaction between the NLRP3 inflammasome and the mucosal immune response has been reported. Activation of the inflammasome is a key function mediated by the innate immune response and in parallel the signaling through IL-1β and IL-18 is implicated in adaptive immunity. Further research is needed to delineate the precise mechanisms of NLRP3 function in regulating immune responses. Targeting NLRP3 inflammasome and its downstream signaling will provide new insights into the development of future therapeutic strategies.

Linarin ameliorates innate inflammatory response in an experimental dry eye model via modulation of the NLRP3 inflammasome

Objective To investigate the effect and mechanism of linarin(LA) in an experimental dry eye model.Methods LA or vehicle was applied in two dry eye models: an in vitro hyperosmotic stress model and an in vivo desiccating stress(DS) murine model. The viability of human corneal epithelial cells(HCECs) was measured using a cell counting kit(CCK-8).Tear secretion was assessed using the phenol red cotton test. The tear break-up time(TBUT) was recorded using 0.1% liquid fluorescein sodium. Corneal epithelial permeability was evaluated through Oregon green dextran(OGD) staining.Conjunctival goblet cells were counted using periodic acid-Schiff(PAS) staining. Terminal deoxynucleotidyl transfer d UTP nickend labeling(TUNEL) staining was used to quantify apoptotic cells in both models. The expression of Ki-67 was measured in HCECs in the cell model while that of matrix metalloproteinase(MMP)-3 and-9 was measured in the murine model through immunofluorescence staining. Real-time quantitative PCR(RTqPCR) was performed to assess the expression of MMP-3 and MMP-9 in the corneal epithelium and NLRP3, ASC, Caspase-1,interleukin(IL)-1β, IL-18, and tumor necrosis factor(TNF)-α in the conjunctiva. The protein expression levels of NLRP3, ASC,Caspase-1, IL-1β, and IL-18 in the conjunctiva were assessed via Western blot.Results In the in vitro model, treatment of HCECs with LA showed no toxicity, increased proliferation, and reduced apoptosis. In the murine model, compared to the control, LA significantly increased tear production and TBUT, improved OGD staining, and increased the number of goblet cells. Topical treatment of LA to mice provided decreased expression of MMP-3, MMP-9, TNF-α, and apoptotic corneal epithelium. Topical administration of LA also suppressed the NLRP3 inflammasome in the dry eye disease(DED) murine model by decreasing the expression of NLRP3, ASC, Caspase-1, IL-1β, and IL-18 in the conjunctiva.Conclusion Our findings support the safety and efficacy of LA in the treatment of DED. LA alleviated corneal epithelial damage and suppressed NLRP3 inflammasome-mediated immunity in the conjunctiva in a murine model of DED.

The Alzheimer's disease-associated gene TREML2 modulates inflammation by regulating microglia polarization and NLRP3 inflammasome activation

Triggering receptor expressed on myeloid cells-like 2(TREML2)is a newly identified susceptibility gene for Alzheimer's disease(AD).It encodes a microglial inflammation-associated receptor.To date,the potential role of mic roglial TREML2 in neuroinflammation in the context of AD remains unclear.In this study,APP/PS1 mice were used to investigate the dynamic changes of TREML2 levels in brain during AD progression.In addition,lipopolysaccharide(LPS)stimulation of primary microglia as well as a lentivirus-mediated TREML2 overexpression and knockdown were employed to explore the role of TREML2 in neuroinflammation in the context of AD.Our res ults show that TREML2 levels gradually increased in the brains of AP P/PS1 mice during disease progression.LPS stimulation of primary microglia led to the release of inflammato ry cytokines including interleukin-1β,inte rleukin-6,and tumor necrosis factor-a in the culture medium.The LPS-induced mic roglial release of inflammatory cytokines was enhanced by TREML2 overexpression and was attenuated by TREML2 knoc kdown.LPS increased the levels of mic roglial M1-type polarization marker inducible nitric oxide synthase.This effect was enhanced by TREML2 overexpression and ameliorated by TREML2 knockdown.Furthermore,the levels of microglial M2-type polarization markers CD206 and ARG1 in the primary microglia were reduced by TREML2 overexpression and elevated by TREML2 knockdown.LPS stimulation increased the levels of NLRP3 in primary microglia.The LPS-induced increase in NLRP3 was further elevated by TREML2 overexpression and alleviated by TREML2 knockdown.In summary,this study provides the first evidence that TREML2 modulates inflammation by regulating microglial polarization and NLRP3 inflammasome activation.These findings reveal the mechanisms by which TREML2 regulates microglial inflammation and suggest that TREML2 inhibition may represent a novel therapeutic strategy for AD.

Vav1 promotes inflammation and neuronal apoptosis in cerebral ischemia/reperfusion injury by upregulating microglial and NLRP3 inflammasome activation

Microglia,which are the resident macrophages of the central nervous system,are an important part of the inflammatory response that occurs after cerebral ischemia.Vav guanine nucleotide exchange factor 1(Vav1) is a guanine nucleotide exchange factor that is related to microglial activation.However,how Vav1 participates in the inflammato ry response after cerebral ischemia/reperfusion inj ury remains unclea r.In this study,we subjected rats to occlusion and repe rfusion of the middle cerebral artery and subjected the BV-2 mic roglia cell line to oxygen-glucose deprivatio n/reoxygenation to mimic cerebral ischemia/repe rfusion in vivo and in vitro,respectively.We found that Vav1 levels were increased in the brain tissue of rats subjected to occlusion and reperfusion of the middle cerebral arte ry and in BV-2 cells subjected to oxygen-glucose deprivation/reoxygenation.Silencing Vav1 reduced the cerebral infarct volume and brain water content,inhibited neuronal loss and apoptosis in the ischemic penumbra,and im p roved neurological function in rats subjected to occlusion and repe rfusion of the middle cerebral artery.Further analysis showed that Vav1 was almost exclusively localized to microglia and that Vav1 downregulation inhibited microglial activation and the NOD-like receptor pyrin 3(NLRP3) inflammasome in the ischemic penumbra,as well as the expression of inflammato ry facto rs.In addition,Vov1 knoc kdown decreased the inflammatory response exhibited by BV-2 cells after oxygen-glucose deprivation/reoxyge nation.Taken together,these findings show that silencing Vav1 attenuates inflammation and neuronal apoptosis in rats subjected to cerebral ischemia/repe rfusion through inhibiting the activation of mic roglia and NLRP3 inflammasome.

Norisoboldine, a natural aryl hydrocarbon receptor agonist, alleviates TNBS-induced colitis in mice, by inhibiting the activation of NLRP3 inflammasome

Although the etiology of inflammatory bowel disease is still tmcertain, increasing evidence indicates that the excessive activation of NLRP3 inflammasome plays a major role. Norisoboldine （NOR）, an alkaloid isolated from Radix Linderae, has previously been demonstrated to inhibit inflammation and IL-1,8 production. The present study was to examine the effect of NOR on colitis and the underlying mechanism related to NLRP3 inflammasome activation. Our results showed that NOR alleviated colitis symptom in mice induced by 2, 4, 6-trinitrobenzene sulfonic acid （TNBS）. Moreover, it significantly reduced expressions of cleaved IL-lfl, NLRP3 and cleaved Caspase-1 but not ASC in colons of mice. In THP-1 cells, NOR suppressed the expressions of NLRP3, cleaved Caspase-1 and cleaved IL-1β but not ASC induced by lipopolysaccharide （LPS） and adenosine triphosphate （ATP）. Furthermore, NOR could activate aryl hydrocarbon receptor （AhR） in THP-1 cells, inducing CYP1A1 mRNA expression, and promoting dissociation of AhR/HSP90 complexes, association of AhR and ARNT, AhR nuclear translocation, XRE reporter activity and binding activity of AhR/ARNT/XRE. Both siAhR and a-naphthoflavone （a-NF） markedly diminished the inhibition of NOR on NLRP3 inflammasome activation. In addition, NOR elevated Nrf2 level and reduced ROS level in LPS- and ATP-stimulated THP-1 cells, which was reversed by either siAhR or α-NF treatment. Finally, correlations between activation of AhR and attenuation of colitis, inhibition of NLRP3 inflammasome activation and up-regulation of Nrf2 level in colons were validated in mice with TNBS-induced colitis. Taken together, NOR ameliorated TNBS-induced colitis in mice through inhibiting NLRP3 inflammasome activation via regulating AhR/Nrf2/ROS signaling pathway.

NLRP3炎症小体在代谢性疾病中的研究进展

代谢性疾病是指机体内物质代谢或能量代谢异常引发的一类疾病,代谢性因素介导的长时间免疫炎症反应会对相应组织的生理功能造成严重损伤。NLRP3炎症小体是一种多聚体胞质蛋白复合物,可以识别细胞内外多种病原体和危险损伤信号导致caspase-1的激活,从而促进炎症细胞因子IL-1β和IL-18的成熟和释放。本文就NLRP3炎症小体激活机制及其在代谢性疾病如糖尿病、肥胖、痛风、动脉粥样硬化、高血压疾病中发挥的重要作用进行综述,旨在为其进一步研究提供依据。

NLRP3炎性体与代谢性疾病的研究进展

代谢性疾病是由体内氨基酸、葡萄糖和脂质代谢紊乱引起的一类疾病,慢性炎症反应是其重要特征之一.Nod样受体蛋白3(Nod-like receptor protein 3,NLRP3)炎性体是位于细胞内的一种蛋白质复合体,主要功能为活化半胱氨酸天冬氨酸蛋白酶1(caspase-1)以间接调控白介素1β(IL-1β)、IL-18和IL-33等的成熟和分泌.NLRP3炎性体是炎性体相关研究的热点,多种内源性或外源性危险信号通过激活这一蛋白质复合体上调炎性因子的表达水平,从而促进多种代谢性疾病的发生发展.本文对NLRP3炎性体的结构、功能、调节以及在代谢性疾病中的作用做一综述,以期为代谢性疾病的防治提供新靶点.

RRx-001 ameliorates inflammatory diseases by acting as a potent covalent NLRP3 inhibitor

The NLRP3 inflammasome plays a crucial role in innate immune-mediated inflammation and contributes to the pathogenesis of multiple autoinflammatory,metabolic and neurodegenerative diseases,but medications targeting the NLRP3 inflammasome are not available for clinical use.RRx-001 is a well-tolerated anticancer agent currently being investigated in phase III clinical trials,but its effects on inflammatory diseases are not known.Here,we show that RRx-001 is a highly selective and potent NLRP3 inhibitor that has strong beneficial effects on NLRP3-driven inflammatory diseases.RRx-001 inhibits the activation of the canonical,noncanonical,and alternative NLRP3 inflammasomes but not the AIM2,NLRC4 or Pyrin inflammasomes.Mechanistically,RRx-001 covalently binds to cysteine 409 of NLRP3 via its bromoacetyl group and therefore blocks the NLRP3-NEK7 interaction,which is critical for the assembly and activation of the NLRP3 inflammasome.More importantly,RRx-001 treatment attenuates the symptoms of lipopolysaccharide(LPS)-induced systemic inflammation,dextran sulfate sodium(DSS)-induced colitis and experimental autoimmune encephalomyelitis(EAE)in mice.Thus,our study identifies RRx-001 as a new potential therapeutic agent for NLRP3-driven diseases.

TMEM16F may be a new therapeutic target for Alzheimer's disease

TMEM16F is involved in many physiological processes such as blood coagulation,cell membrane fusion and bone mineralization.Activation of TMEM16F has been studied in various central nervous system diseases.High TMEM16F level has been also found to participate in microglial phagocytosis and transformation.Microglia-mediated neuroinflammation is a key factor in promoting the progression of Alzheimer’s disease.However,few studies have examined the effects of TMEM16F on neuroinflammation in Alzheimer’s disease.In this study,we established TMEM16F-knockdown AD model in vitro and in vivo to investigate the underlying regulatory mechanism about TMEM16F-mediated neuroinflammation in AD.We performed a Morris water maze test to evaluate the spatial memory ability of animals and detected markers for the microglia M1/M2 phenotype and NLRP3 inflammasome.Our results showed that TMEM16F was elevated in 9-month-old APP/PS1 mice.After TMEM16F knockdown in mice,spatial memory ability was improved,microglia polarization to the M2 phenotype was promoted,NLRP3 inflammasome activation was inhibited,cell apoptosis and Aβplaque deposition in brain tissue were reduced,and brain injury was alleviated.We used amyloid-beta(Aβ_(25-35))to stimulate human microglia to construct microglia models of Alzheimer’s disease.The levels of TMEM16F,inducible nitric oxide synthase(iNOS),proinflammatory cytokines and NLRP3 inflammasome-associated biomarkers were higher in Aβ_(25-35) treated group compared with that in the control group.TMEM16F knockdown enhanced the expression of the M2 phenotype biomarkers Arg1 and Socs3,reduced the release of proinflammatory factors interleukin-1,interleukin-6 and tumor necrosis factor-α,and inhibited NLRP3 inflammasome activation through reducing downstream proinflammatory factors interleukin-1βand interleukin-18.This inhibitory effect of TMEM16F knockdown on M1 microglia was partially reversed by the NLRP3 agonist Nigericin.Our findings suggest that TMEM16F participates in neuroinflammation in Alzheimer’s disease through participating in polarization of microglia and activation of the NLRP3 inflammasome.These results indicate that TMEM16F inhibition may be a potential therapeutic approach for Alzheimer’s disease treatment.

NLRP3炎症小体与类风湿关节炎患者疾病活动度和骨代谢指标的关系及其诊断价值分析

目的:研究核苷酸结合寡聚化结构域样受体热蛋白结构域相关蛋白3(NLRP3)炎症小体与类风湿关节炎(RA)患者疾病活动度和骨代谢指标的关系及其诊断价值。方法:选取2021年5月至2022年9月山西医科大学第一医院收治的RA患者300例,根据28个关节疾病活动度(DAS28)评分将患者分为低度组(n=62)、中度组(n=104)、重度组(n=134)。检测并对比各组NLRP3、凋亡相关斑点样蛋白(ASC),半胱天冬酶-1(caspase-1)m RNA表达水平以及骨代谢指标。Pearson相关性分析NLRP3炎症小体指标与DAS28评分和骨代谢指标的相关性,受试者工作特征(ROC)曲线分析NLRP3,ASC,caspase-1对RA疾病活动严重程度的诊断价值。结果:重度组和中度组的血清NLRP3、ASC及Caspase-1mRNA高于低度组,且重度组高于中度组(P<0.05)。中度组和低度组的血清骨钙素(BGP)、甲状旁腺激素(PTH)及β-胶原特殊序列(β-CTx)水平明显低于重度组,且低度组又低于中度组(P<0.05)。中度组和低度组的血清25-(OH)维生素D3[25-(OH)D3]及Ⅰ型前胶原N-末端前肽(PINP)水平明显高于重度组,且低度组又高于中度组(P<0.05)。Pearson相关性分析发现,RA患者血清NLRP3炎症小体指标与DAS28评分、BGP、PTH及β-CTx均呈正相关,而与25-(OH)D3及PINP均呈负相关(均P<0.05)。ROC分析显示:NLRP3、NLRP3、ASC单独及联合应用对RA疾病活动严重程度的曲线下面积分别为0.770、0.733、0.739、0.828。结论:NLRP3炎症小体与RA患者与疾病活动度关系紧密,血清NLRP3炎症小体指标与DAS28评分、BGP、PTH及β-CTx均呈正相关,而与25-(OH)D3及PINP均呈负相关。NLRP3、ASC及Caspase-1水平联合检测对RA疾病活动严重程度具有较高的诊断价值。

高脂饮食致非酒精性脂肪性肝病大鼠小肠、肝脏中NOD样受体蛋白3及相关炎症因子的表达

目的 观察高脂饮食致非酒精性脂肪性肝病(NAFLD)大鼠小肠、肝脏中NOD样受体蛋白3(NLRP3)及相关炎症因子表达情况。方法 SD雄性大鼠20只适应性喂养1周后,随机分为正常组、模型组,每组10只,正常组普通饲料喂养,模型组高脂饲料喂养。8周末取材,观察肝HE及油红O染色病理切片,检测各组血清和肝脏中谷丙转氨酶(ALT)、谷草转氨酶(AST)、胆固醇(TC)、甘油三酯(TG)、白介素-18(IL-18)、白介素-1β(IL-1β)。RT-PCR、免疫组化检测肝、肠NLRP3表达。结果 与正常组相比,模型组大鼠肝脏表现出明显的脂肪变性和炎性病变,肝脏、血清中ALT、AST、TG显著升高(P<0.05),TC升高,差异不明显,IL-18、IL-1β显著升高(P<0.05);肝脏、小肠NLRP3表达量亦显著升高(P<0.05)。结论 NAFLD可引起大鼠小肠、肝脏中NLRP3及相关炎症因子上调。

核苷酸结合寡聚化结构域样受体蛋白3与非酒精性脂肪肝的相关性分析

目的探讨核苷酸结合寡聚化结构域样受体蛋白3(NLR family pyrin domain containing 3,NLRP3)与非酒精性脂肪肝(nonalcoholic fatty liver disease,NAFLD)的相关性。方法采用横断面调查研究,随机选取上海健康医学院附属周浦医院2017年1月体检人员84名,通过肝脏超声明确有无NAFLD,分为NAFLD组(27例)及正常对照组(57例),分析两组研究对象一般资料及肝功能、血脂、血糖、肌酐、尿酸等生化指标;运用ELISA双抗体夹心法检测外周血NLRP3水平。通过Logistic回归分析NLRP3与NAFLD的关系。结果NAFLD患病率为32.1%(27/84)。NAFLD组患者血清胆固醇、甘油三酯、低密度脂蛋白胆固醇、空腹血糖、丙氨酸氨基转移酶、肌酐及尿酸水平均高于正常对照组(P均<0.01),血清高密度脂蛋白胆固醇水平则低于正常对照组(P=0.023);NAFLD组血清NLRP3浓度为(5.1±1.8)μg/L,正常对照组为(3.9±1.4)μg/L,两组比较差异有统计学意义(t=6.221,P=0.016)。Logistic回归分析显示,NLRP3是NAFLD发生的风险因素(OR=1.537,P=0.021)。结论NLRP3水平在NAFLD患者血清中上调,与NAFLD的发生相关。