Paeonol inhibits NLRP3 mediated inflammatory reaction in rat endothelial cells by elevating hyperlipidemic rats plasma exosomal miRNA-223

OBJECTIVE Atherosclerosis(AS) is featured as a chronic inflammatory disease of vascular stenosis.Paeonol(Pae) is a natural phenolic compounds isolated from a traditional Chinese medicine, Cortex Moutan, which exhibits anti-AS effects in vitro and in vivo. In this study, we aimed to investigate whether the anti-AS efficacy of Pae was regulated through inhibiting NLRP3 inflammasome activityvia elevating hyperlipidemic rats plasma exosomalmicro RNA-223(miR-223). METHODS The Sprague-Dawley rats was induced by a high-fat diet, which was used as AS models.AS aortic pathological morphological in AS mice was examined by HE staining, and serum TC and TG levels were determined by automatic chemistry analyzer. Rat aortic endothelial cells(RAECs) were used during the whole study. After oral administration of Pae, we isolated exosomes from hyperlipidemic rats plasma(Pae-Exos) by ultracentrifugation and characterized by transmission electron, nanoparticle tracking analysis, dynamic light scattering and Western blotting.The activity of RAECs was detected by CCK-8 and trypan blue staining method. IL~(-1)β and IL-6 levels were detected by ELISA method. The expression of miR-223 was detected by q PCR, and the expression of NLRP3, ASC, caspase-1, and ICAM-1 was detected by Western blotting. RESULTS In vivo experiments confirmed that Pae could effectively reduce serum TC and TG levels and decrease serum IL~(-1)β and IL-6 levels, which demonstrated that Pae restricted AS development in hyperlipidemia rats. Both CCK-8 and trypan blue staining showed that the survival rate of RAECs in the PaeExos co-incubation group was higher than that in the model group. We also confirmed via real-time q PCR that Pae-Exos suppressed the expression of the inflammatory cytokines IL~(-1)β and IL-6. Accordingly, Pae-Exos dose-dependently increased the survival rate of RAECs and reduced inflammatory response. Furthermore, compared with the model group, Pae-Exos more successfully increased the expression of miR-223 and inhibited IL~(-1)β and IL-6 expression, which implied that Pae-exo may inhibited the inflammatory response of RAECs by increasing the content of miR-223. Subsequently, we found that Pae-Exos reduced the expressions of NLRP3, ASC, caspase-1 and ICAM-1, which indicated that Pae-Exos may reduced RAECs inflammation by suppressing NLRP3 signaling pathway via promoting miR-223 expression.CONCLUSION Pae can inhibit the downstream NLRP3 inflammatory corpuscle signaling pathway by increasing the level of miR-223 in plasma Exos of hyperlipidemic rats, providing new insights into the anti-atherosclerosis activity of Pae.     

基于NLRP3/GSDMD信号通路研究人参皂苷Rb1对高糖诱导的胰岛β细胞焦亡的影响

目的: 研究人参皂苷Rb1(GRb1)抑制高糖诱导的胰岛β细胞焦亡及对NLRP3/GSDMD信号通路的调节机制。方法: 高糖(50 mmol·L^-1)诱导大鼠胰岛细胞瘤细胞(rat insulinoma cells, INS-1)焦亡模型,并构建NLRP3过表达质粒转染INS-1细胞,采用倒置显微镜观察GRb1对细胞形态变化的影响;采用ELISA法检测GRb1对细胞上清液中乳酸脱氢酶(LDH)、白细胞介素-1β(IL-1β)、胰岛素水平的影响;采用Western blot法和qRT-PCR法检测GRb1对INS-1细胞中核苷酸结合域样受体蛋白3(nucleotide binding domain like receptor protein 3, NLRP3)和切割蛋白D(gasdermin D, GSDMD)表达的影响。结果: 在高糖环境下,GRb1可减轻INS-1细胞形态学改变,显著增加INS-1细胞胰岛素分泌,降低细胞上清液中IL-1β、LDH水平(P＜0.05),且呈剂量依赖性;GRb1呈剂量依赖性抑制高糖诱导的INS-1细胞中GSDMD及上游调节因子NLRP3的表达(P＜0.05);过表达NLRP3后可显著逆转高糖环境下GRb1对INS-1细胞的部分保护性作用(P＜0.05)。结论: GRb1能够改善高糖诱导的INS-1细胞焦亡,减轻炎症反应,其机制可能与抑制NLRP3/GSDMD信号通路有关,从而防治糖尿病及其并发症的发生。     

PFOS facilitates liver inflammation and steatosis: An involvement of NLRP3 inflammasome-mediated hepatocyte pyroptosis

Perfluorooctane sulfonate (PFOS) is a fluorinated organic pollutant with substantial accumulation in mammalian liver tissues. However, the impact of chronic PFOS exposure on liver disease progression and the underlying molecular mechanisms remain elusive. Herein, we for the first time revealed that micromolar range of PFOS exposure initiates the activation of NLR pyrin domain containing 3 (NLRP3) inflammasome to drive hepatocyte pyroptosis. We showed that 5 mg/kg/day PFOS exposure may exacerbated liver inflammation and steatosis in high-fat diet (HFD)-fed mice with concurrently elevated expression of NLRP3 and caspase-1. PFOS exposure resulted in viability impairment and LDH release in BRL-3A rat liver cells. 25–100 μM concentrations of PFOS exposure activated the NLRP3 inflammasome, leading to consequent GSDMD cleavage, IL-1β release and the initiation of pyroptosis in a dose-dependent manner, whereas treatment with 10 μM NLRP3 inhibitor MCC950 abrogated this effect. Moreover, pretreatment of 5 mM ROS scavenger N-acetyl-L-cysteine (NAC) ameliorated PFOS-induced NLRP3 inflammasome activation and pyroptosis. Collectively, our data highlight a pivotal role of pyroptotic death in PFOS-mediated liver inflammation and metabolic disorder.     

Celastrol inhibits microglial pyroptosis and attenuates inflammatory reaction in acute spinal cord injury rats

Pyroptosis pathway is closely related to inflammation. However, Celastrol effect on pyroptosis pathway after spinal cord injury (SCI) are poorly understood. We studied the anti-inflammatory and neuroprotective effects of Celastrol on acute spinal cord injury in rats, and its anti-inflammatory effects on lipopolysaccharide (LPS)/ATP-induced microgliosis. Our results show that Celastrol can improve the recovery of hindlimb motor function after SCI in Sprague-Dawley (SD) rats, and reduce the cavity area of spinal cord injury along with the neuronal loss. Celastrol simultaneously reduced the activation of microglia (especially M1 microglia) in the spinal cord, inhibited the pyroptosis-related proteins (NLRP3 ASC Caspase-1 GSDMD), reduced the release of TNF-α IL-1β and IL-18 inflammatory factors, and increased the release of IL10 cytokines. In vitro studies showed that Celastrol reduced the toxicity resulting from the administration of LPS with ATP to BV-2 cells, inhibited the pyroptosis-related proteins (NLRP3 Caspase-1 GSDMD), and inhibited the release of corresponding inflammatory factors. Finally, Celastrol can inhibit the expression of NFκB/p-p65 in vitro and in vivo. Our results show that Celastrol can attenuate the inflammatory response of the spinal cord after SCI, which is associated with inhibition of microglial activation and pyroptosis pathway. Further study to explore the use of Celastrol to treat SCI is warranted.     

益母草碱抑制NLRP3炎症小体过度激活调控巨噬细胞M1/M2表型分化

目的 研究益母草碱对脂多糖(LPS)诱导小鼠腹腔巨噬细胞免疫应答影响及相关机制.方法 分离小鼠腹腔巨噬细胞,用脂多糖和益母草碱预处理24 h,MMT法检测巨噬细胞活性;Griess法检测NO释放量;ELISA法检测IL-1β、IL-18、IL-6、TNF-α的释放量;RT-PCR法检测NLRP3、ASC、caspase...     

Ameliorative effect of selective NLRP3 inflammasome inhibitor MCC950 in an ovalbumin-induced allergic rhinitis murine model

Allergic rhinitis (AR) is a complex IgE-mediated nasal allergic and inflammatory disease. Nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3) is essential in the process of allergic and inflammatory responses. MCC950 is a selective NLRP3 inhibitor. However, its role and mechanism in AR remains undetermined. The present study aimed to explore the effect and mechanism of MCC950 on an ovalbumin (OVA) induced mouse model of AR. The AR BALB/c mice were constructed using OVA and administrated intranasally with MCC950. Concentrations of OVA-specific IgE, histamines and leukotrienes C4 (LTC4) in serum, and OVA-specific IgE, ECP, IFN-γ, IL-4, IL-5, IL-13, IL-1β and IL-18 in nasal lavage fluid (NLF) were assayed by enzyme-linked immunosorbent assay (ELISA). Inflammatory cells were counted in NLF. HE and PAS staing were used for evaluating eosinophils and goblet cells. Immunohistochemistry (IHC) staining were employed to evaluate immunolabeling of NLRP3, Caspase-1, ASC, IL-1β and IL-18 in nasal mucosas of mice. Real-time PCR was conducted to assay NLRP3, Caspase-1, ASC, IL-1β and IL-18 mRNA levels. In vitro studies, western blotting, real-time PCR and ELISA were performed to evaluate the effects and mechanisms of OVA and NLRP3 inhibitor MCC950 on spleen mononuclear cells. We found significant downregulation of sneezing, nasal rubbing, inflammatory cytokines, inflammatory cells and NLRP3, Caspase-1, ASC, IL-1β and IL-18 expression in MCC950 treated mice compared with untreated AR mice. In spleen mononuclear cells culture and stimulation experiment, NLRP3, Caspase-1, ASC, IL-1β and IL-18 levels were upregulated by OVA but inhibited by MCC950. In conclusion, MCC950 could effectively exert its ameliorative effect in murine AR by inhibiting NLRP3 and leads to reduction of Caspase-1, ASC, IL-1β and IL-18, resulting in the attenuation of the allergic and inflammatory responses.     

Kanglexin,a novel anthraquinone compound,protects against myocardial ischemic injury in mice by suppressing NLRP3 and pyroptosis

OBJECTIVE To investigate the myocardial protection effect of a novel anthraquinone compound(4,5-dihydroxy-7-methyl-9,10-anthraquinone-2-ethyl succinate) named Kanglexin(KLX) in vivo and in vitro. METHODS Male C57 BL/6 mice were pre-treated either with KLX(20, 40 mg·kg~(-1)·d~(-1), ig) or vehicle for 7 consecutive days prior to ligation of coronary artery to induce permanent MI. Neonatal mouse cardiomyocytes(NMCMs) were exposed to hypoxia or LPS to detect the myocardial protection effect of KLX. Echocardiographic and myocardial infarct size were used to evaluate the effect of KLX on MI treated mice. Electron microscopic examination and H&E staining were preformed to determine morphological changes of each group. Western blotting and q PCR were used to detect the expressions of pyroptosis relative proteins. The numbers of TUNEL and PI-positive cells were measured to assess pyroptotic cell death.RESULTS KLX reduced myocardial infarct size and LDH release and improved cardiac function as compared to vehicle-treated mice 24 h after MI. We found that MI triggered NLRP3 inflammasome activation leading to conversion of IL-1β and IL-18 into their active mature forms in the heart. We also showed that MI induced pyroptosis, as evidenced by increased DNA fragmentation, mitochondrial swelling and cell membrane rupture as well as increased levels of several pyroptosis-related proteins, including GSDMD, N-GSDMD and cleaved caspase-1. All these detrimental alterations were prevented by KLX administration. In hypoxia-or LPS-treated NMCMs,we showed that 10 μmol·L~(-1) KLX decreased the elevated levels of TUNEL-and PI-positive cel s as wel as pyroptosisrelated proteins. CONCLUSION KLX prevents MI-induced cardiac damages and cardiac dysfunction at least partly through attenuating NLRP3 and subsequent cardiomyocyte pyroptosis, and it is worthy of more rigorous investigations for its potential for alleviating ischemic heart disease.     

Dexmedetomidine inhibits pyroptosis by down-regulating miR-29b in myocardial ischemia reperfusion injury in rats

ObjectiveDexmedetomidine (DEX) was reported to protect heart against ischemic-reperfusion (IR) but the mechanism herein remains elusive. This study aims to explore the mechanism of DEX on pyroptosis induced by myocardial ischemic reperfusion (MIR).MethodsMIR rat models were established and injected DEX or miR-29b agomir/antagomir separately. The possible effect of DEX or miR-29b on myocardial cells was assessed according to measurement on creatine kinase-MB (CK-MB), cardiac troponin I (cTnI), interleukin-1β (IL-1β) and interleukin-18 (IL-18), myocardial infarction size, myocardial injury and apoptosis. Western blot determined the expression levels of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing CARD (ASC) and cleaved-caspase-1. Hypoxia/reoxygenation (H/R) cell model was established. The lactate dehydrogenase (LDH) content released by myocardial cells was examined. The relation between miR-29b and FoxO3a was confirmed by dual luciferase reporter gene assay. FoxO3a or ARC level was elevated in H/R myocardial cells to detect its effect on pyroptosis.ResultsMIR rat models were successfully established, in which cell pyroptosis was triggered as evidenced by increased expression levels of NLRP3, ASC and cleaved-caspase-1. Rats with DEX precondition had attenuated cell pyroptosis and ameliorated inflammatory response. FoxO3a was a target of miR-29b. MiR-29b agomir or miR-29b antagomir could inhibit or promote the protective effect of DEX on MIR. Overexpression of FoxO3a/ARC axis could suppress myocardial pyroptosis induced by H/R.ConclusionDEX could ameliorate MIR injury (MIRI) and H/R injury in rats and inhibit H/R induced pyroptosis in myocardial cells via down-regulating miR-29b to activate FoxO3a/ARC axis.     

灯盏花乙素通过调节线粒体融合-分裂平衡抑制氧糖剥夺/复糖复氧诱导的N2a细胞焦亡

目的: 探讨灯盏花乙素对氧糖剥夺/复氧复糖(OGD/R)诱导的N2a细胞焦亡的影响及作用机制。方法: 建立N2a细胞OGD/R损伤模型,设立对照组、模型组、灯盏花乙素给药组和线粒体分裂抑制剂Mdivi-1组。通过CCK-8检测N2a细胞增殖能力,检测各组细胞培养基中LDH和IL-1β水平,通过JC-1探针检测线粒体膜电位,Western blot检测线粒体融合分裂关键蛋白(Drp-1、Mfn-1、Mfn-2、OPA-1)和细胞焦亡标志蛋白(Caspase-1、NLRP-3和GSDMD)表达。结果: 与对照组相比,OGD/R可显著降低N2a细胞活力,灯盏花乙素可明显提高N2a细胞活力。与模型组相比,灯盏花乙素可减少LDH的释放,降低Caspase-1和IL-1β水平,提高线粒体膜势能,降低Drp-1的表达水平,上调Mfn-1、Mfn-2和OPA-1的表达,并减少细胞焦亡关键蛋白NLRP-3和GSDMD的表达。进一步给予Drp-1抑制剂Mdivi-1,与模型组相比,Mdivi-1可明显提高线粒体膜势能,减少Caspase-1、NLRP-3和GSDMD的表达。结论: 灯盏花乙素可通过抑制线粒体过度分裂,改善线粒体融合-分裂失衡与功能异常,进而缓解OGD/R所致N2a细胞焦亡。     

去肾交感神经对2型糖尿病大鼠血管内皮细胞自噬及NLRP3活化的影响

目的 探讨去肾交感神经（RDN）对2型糖尿病（T2DM）大鼠血管内皮细胞自噬及核苷酸结合寡聚化结构域受体蛋白3（NLRP3）炎症小体活化的影响。方法 采用高脂饮食联合小剂量链脲佐菌素（STZ）腹腔注射制备T2DM大鼠模型。采用随机数字表法分为对照组（CON组）、糖尿病对照组（T2DM组）、双侧假手术组（Sham组）、双...     

Pinellia pedatisecta lectin exerts a proinflammatory activity correlated with ROS-MAPKs/NF-κB pathways and the NLRP3 inflammasome in RAW264.7 cells accompanied by cell pyroptosis

Pinellia pedatisecta, a widely used herb in Chinese medicine, has proinflammatory toxicity related to its Pinellia pedatisecta lectin (PPL), but the mechanism is still unknown. However, for safer use, it is necessary to clarify its proinflammatory mechanism. Herein, we studied the mechanism in RAW264.7 cells. PPL decreased the mitochondrial membrane potential (MMP) and increased the outflow of calcium, accompanied by the overproduction of reactive oxygen species (ROS), which resulted in the activation of the MAPK and NF-κB pathways and the release of IL-1β. The maturation of IL-1β relied on caspase-1 p20, the active caspase-1, as demonstrated by adding caspase-1 inhibitor. While caspase-1 was associated with the activation of the NLRP3 inflammasome, we further found that the stimulation of PPL also contributed to the activation. In addition, TXNIP was downregulated, whereas NLRP3/caspase-1 p20/ASC was upregulated, and there was binding of TXNIP with NLRP3. There was also binding of NLRP3 with ASC and caspase-1. Further, we found that N-acetylcysteine (NAC), an ROS scavenger, could inhibit the PPL-stimulated activation of these pathways and the release of IL-1β. Moreover, PPL led to cell pyroptosis with pyknotic nuclei and plasma membrane rupture, which could be inhibited by NAC. All of these findings demonstrated an important role of ROS in the inflammation caused by PPL. Taken together, our data provide new mechanistic insights into the possible endogenous signaling pathways involved in the inflammation of RAW264.7 cells, stimulated by PPL.     

Scutellarin inhibits caspase-11 activation and pyroptosis in macrophages via regulating PKA signaling

Inflammatory caspase-11 senses and is activated by intracellular lipopolysaccharide (LPS) leading to pyroptosis that has critical role in defensing against bacterial infection, whereas its excess activation under pathogenic circumstances may cause various inflammatory diseases. However, there are few known drugs that can control caspase-11 activation. We report here that scutellarin, a flavonoid from Erigeron breviscapus, acted as an inhibitor for caspase-11 activation in macrophages. Scutellarin dose-dependently inhibited intracellular LPS-induced release of caspase-11p26 (indicative of caspase-11 activation) and generation of N-terminal fragment of gasdermin D (GSDMD-NT), leading to reduced pyroptosis. It also suppressed the activation of non-canonical nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome as evidenced by reduced apoptosis-associated speck-like protein containing a CARD (ASC) speck formation and decreased interleukin-1 beta (IL-1β) and caspase-1p10 secretion, whereas the NLRP3-specific inhibitor MCC950 only inhibited IL-1β and caspase-1p10 release and ASC speck formation but not pyroptosis. Scutellarin also suppressed LPS-induced caspase-11 activation and pyroptosis in RAW 264.7 cells lacking ASC expression. Moreover, scutellarin treatment increased Ser/Thr phosphorylation of caspase-11 at protein kinase A (PKA)-specific sites, and its inhibitory action on caspase-11 activation was largely abrogated by PKA inhibitor H89 or by adenylyl cyclase inhibitor MDL12330A. Collectively, our data indicate that scutellarin inhibited caspase-11 activation and pyroptosis in macrophages at least partly via regulating the PKA signaling pathway.KEY WORDS: Caspase-11, Macrophages, Gasdermin D, Pyroptosis, Scutellarin, PKA signaling     

The mechanisms of NLRP3 inflammasome/pyroptosis activation and their role in Parkinson's disease

Parkinson's disease (PD) is a typical neurodegenerative disease and the pathological feature of which is the death of dopamine neurons in the substantia nigra region. At present, neuronal death caused by inflammatory cytokine-mediated neuroinflammation is being extensively studied. The nucleotide-binding oligomerization domain-, leucine-rich repeat and pyrin domain-containing 3 (NLRP3) inflammasome is an inflammatory complex existing in microglia. Its activation promotes the secretion of the inflammatory cytokine interleukin-1β/18 (IL-1β/18) and induces pyroptosis, a type of cell death that possesses the potential for inflammation, to rupture microglia to further release IL-1β. In this review we focus on the mechanisms of activation of the NLRP3 inflammasome and pyroptosis and their inflammatory effects on the development of PD. In addition, we focus on some inhibitors of NLRP3 inflammatory pathways to alleviate the progression of PD by inhibiting central inflammation and provide new therapeutic strategies for the treatment of PD.     

Gasdermin D-mediated pyroptosis in myocardial ischemia and reperfusion injury: Cumulative evidence for future cardioprotective strategies

Cardiomyocyte death is one of the major mechanisms contributing to the development of myocardial infarction (MI) and myocardial ischemia/reperfusion (MI/R) injury. Due to the limited regenerative ability of cardiomyocytes, understanding the mechanisms of cardiomyocyte death is necessary. Pyroptosis, one of the regulated programmed cell death pathways, has recently been shown to play important roles in MI and MI/R injury. Pyroptosis is activated by damage-associated molecular patterns (DAMPs) that are released from damaged myocardial cells and activate the formation of an apoptosis-associated speck-like protein containing a CARD (ASC) interacting with NACHT, LRR, and PYD domains-containing protein 3 (NLRP3), resulting in caspase-1 cleavage which promotes the activation of Gasdermin D (GSDMD). This pathway is known as the canonical pathway. GSDMD has also been shown to be activated in a non-canonical pathway during MI and MI/R injury via caspase-4/5/11. Suppression of GSDMD has been shown to provide cardioprotection against MI and MI/R injury. Although the effects of MI or MI/R injury on pyroptosis have previously been discussed, knowledge concerning the roles of GSDMD in these settings remains limited. In this review, the evidence from in vitro, in vivo, and clinical studies focusing on cardiac GSDMD activation during MI and MI/R injury is comprehensively summarized and discussed. Implications from this review will help pave the way for a new therapeutic target in ischemic heart disease.     

NLRP3炎性体在心肌缺血再灌注损伤中作用的研究进展 #br#

炎性体是一种大分子多蛋白复合体,是固有免疫的重要组成成分。核苷酸结合寡聚化结构域受体蛋白3（NLRP3）炎性体是目前被研究得最多的炎性体。NLRP3炎性体由NLRP3、凋亡相关斑点样蛋白（ASC）及pro-半胱氨酸天冬氨酸特异性蛋白酶（caspase）-1组成。当细胞受到刺激时,NLRP3被激活并能够通过结合蛋白ASC招募procaspase-1,形成NLRP3炎性体。在NLRP3炎性体的作用下,pro-caspase-1被激活成为caspase-1,最终导致白细胞介素（IL）-1β和IL-18的成熟与分泌,引起一系列炎症反应。NLRP3炎性体在动脉粥样硬化、2型糖尿病、癌症等多种疾病中有重要作用,并且越来越多的证据表明NLRP3炎性体亦在心肌缺血再灌注损伤中发挥重要作用。本文介绍了NLRP3炎性体的基本结构和功能,并综述了NLRP3炎性体在心肌缺血再灌注损伤中作用的研究进展。     

白藜芦醇对脂多糖诱导的人肺上皮细胞焦亡的保护机制研究

目的:探究白藜芦醇对脂多糖(LPS)诱导的人肺上皮细胞(又称BEAS-2B)增殖、炎症因子释放和焦亡的影响。方法:用CCK-8法检测LPS和LPS与白藜芦醇联用对BEAS-2B细胞增殖的影响;采用ELISA法检测细胞上清炎症因子肿瘤坏死因子(TNF-α)、白介素-6(IL-6)、白介素-1β(IL-1β)和白介素-18(IL-18)表达以及qPCR和Western blot检测焦亡基因NLRP3、Gasdermin D、Caspase-1、ELAVL1的表达;分析其对细胞活力、BEAS-2B细胞中细胞因子的含量及其焦亡相关基因、相关蛋白表达的影响。结果:LPS可以抑制BEAS-2B细胞的增殖,同时可以促进炎症因子的表达,经qPCR和Western blot检测结果表明LPS可以促进NLRP3、Gasdermin D、Caspase-1、ELAVL1基因的表达;其经用白藜芦醇处理48 h后,可以有效逆转LPS所引起的细胞增殖受抑制和炎症因子高表达的现象;此外,NLRP3、Gasdermin D、Caspase-1、ELAVL1的表达也部分受抑制。结论:白藜芦醇通过减少炎症因子的释放和NLRP3、Gasdermin D、Caspase-1、ELAVL1表达对LPS诱导的BEAS-2B焦亡起到一定的保护作用。     

小肠结肠炎综合征小鼠肠黏膜中NLRP3表达与炎症分子关系的研究

目的 探讨食物蛋白诱导的小肠结肠炎综合征（FPIES）小鼠肠黏膜细胞中炎症小体NOD样受体热蛋白结 构域相关蛋白3（NLRP3）表达水平,明确NLRP3异常与炎症分子及细胞焦亡的关联性。方法 利用卵清蛋白灌胃建 立食物蛋白诱导FPIES小鼠模型;实时荧光定量聚合酶链反应（qPCR）和Western blot方法检测肠黏膜细胞NLRP3、 炎症分子及细胞焦亡通路分子的表达水平;采用药物干预黏膜细胞,分别抑制和激活NLRP3,Western blot方法检测 肠黏膜细胞炎症分子及细胞焦亡通路分子表达的改变情况。结果 FPIES小鼠肠黏膜细胞中NLRP3、炎症分子及细 胞焦亡通路分子表达水平显著上调（P＜0.05）;激活NLRP3表达,可以诱导炎症分子及细胞焦亡通路分子表达显著 上调,抑制NLRP3表达,可以显著上调炎症分子转化生长因子（TGF）-β和肿瘤坏死因子（TNF）-α表达（P＜0.05）。结论 FPIES小鼠肠黏膜细胞NLRP3表达与炎症反应及细胞焦亡密切相关,是调控FPIES肠道病理表型的上游靶标分子。     

Lysophosphatidylcholine induces apoptosis and inflammatory damage in brain microvascular endothelial cells via GPR4-mediated NLRP3 inflammasome activation

Lysophosphatidylcholine (LPC), as the main active component of oxidized low-density lipoproteins (ox-LDLs), has significant effects in cerebrovascular disease. However, the complex mechanism by which LPC functions in brain microvascular endothelial cells (BMECs) is not clearly understood. In this study, BMECs were transfected with G protein-coupled receptor 4 (GPR4) siRNA or an NLRP3-overexpression plasmid, and GPR4 expression was identified by RT-qPCR and western blotting; IL-1β, IL-18, and IL-33 levels were evaluated by ELISA. Apoptosis was monitored by flow cytometry and Hoechst staining, while Caspase 3, ASC, NLRP3, and GPR4 protein expression were examined by western blotting. Our results showed that LPC significantly increased the levels of inflammatory cytokines (IL-1β, IL-18, and IL-33) and markedly induced apoptosis and NLRP3 inflammasome activation in BMECs. Moreover, LPC notably upregulated GPR4 in BMECs, and knockdown of GPR4 significantly attenuated the effects of LPC in BMECs. Above all, we also proved that LPC induced apoptosis and inflammatory injury in BMECs by causing GPR4 to activate NLRP3 inflammasomes. Therefore, GPR4-mediated activation of NLRP3 inflammasomes might be the underlying mechanism by which LPC promotes the progression of cerebrovascular disease. In summary we found that LPC is an important pathogenic factor in cerebrovascular disease, and can induce GPR4 to active NLRP3 inflammasomes.