纤维状α-突触核蛋白聚集体激活NLRP3炎症小体的机制研究

目的 探索纤维状α-突触核蛋白（α-synuclein）聚集体激活NLRP3炎症小体诱导神经炎症的机制。方法 构建纤维状α-synuclein聚集体,采用纤维状α-synuclein聚集体刺激BV-2小胶质细胞,检测白介素（IL）-1β、IL-18、IL-6和肿瘤坏死因子α(TNF-α)等相关炎症因子和NLRP3、caspase-1、ASC等蛋白的表达和mRNA水平评价NLRP3炎症小体激活;采用乳酸脱氢酶释放（LDH）实验检测细胞焦亡的发生。机制研究部分,检测Toll样受体（TLR）2和TLR4的激活,并分别加入TLR2和TLR4抑制剂C29和TAK-242检测对纤维状α-synuclein聚集体诱导的NLRP3炎症小体激活的影响及核转录因子-κB(NF-κB)的入核情况。结果 Westernblot和硫黄素T染色实验结果显示,纤维状α-synuclein聚集体成功制备。纤维状α-synuclein聚集体刺激BV-2小胶质细胞24 h后可激活NLRP3炎症小体,表现为IL-1β释放增加,相关蛋白NLRP3、caspase-1表达升高,N LR P3、ASC和I L-1β的m R NA...     

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。     

灯盏花乙素对J774A.1巨噬细胞中ATP诱导的炎症小体活化和细胞焦亡的影响

目的以LPS致敏的小鼠巨噬细胞J774A.1作为炎症细胞模型,研究灯盏花乙素对ATP诱导的炎症小体活化和细胞焦亡的影响及其机制。方法利用碘化丙锭(PI)染色法检测LPS+ATP诱导的小鼠J774A.1巨噬细胞发生细胞焦亡的情况;免疫印迹法检测细胞裂解液和上清中IL-1β、caspase-1、HMGB1等蛋白的表达水平;基于微珠的免疫测定法(CBA)检测细胞上清中IL-1β的分泌水平。结果 ATP能够明显诱导LPS致敏的J774A.1巨噬细胞中caspase-1活化、成熟IL-1β(17 ku)和HMGB1释放至培养上清中,并诱导细胞焦亡;而灯盏花乙素预处理能够剂量依赖性地抑制ATP诱导的caspase-1活化以及成熟IL-1β、HMGB1的释放,并抑制细胞焦亡;同时,经腺苷酸环化酶抑制剂MDL12330A和蛋白激酶A(PKA)抑制剂H89处理,可以逆转灯盏花乙素对ATP诱导的细胞焦亡的抑制作用。结论灯盏花乙素通过调节PKA活性,抑制NLRP3炎症小体的活化与细胞焦亡,从而发挥抗炎作用。     

雷公藤甲素对类风湿关节炎成纤维样滑膜细胞线粒体自噬、NLRP3炎症小体活化和细胞焦亡的影响

目的 探究雷公藤甲素对类风湿关节炎成纤维样滑膜细胞（FLSs）线粒体自噬、NOD样受体蛋白3(NLRP3)炎症小体活化和细胞焦亡的影响。方法 将FLSs细胞和类风湿关节炎FLSs细胞进行传代培养。将类风湿性关节炎FLSs细胞采用0、10、20、40 ng/mL雷公藤甲素进行处理。采用蛋白质印迹法（Western blotting）检测线粒体自噬相关蛋白LC3B、p62、PHB2、NLRP3的蛋白表达。采用酶联免疫吸附试验（ELISA）检测炎症因子白细胞介素（IL）-1β和IL-18的水平。采用流式细胞术检测细胞焦亡情况,并采用Western blotting法检测焦亡相关蛋白GSDMD和GSDMD-N表达。结果 采用10、20、40ng/mL雷公藤甲素处理类风湿关节炎FLSs后,雷公藤甲素各剂量组均能显著减弱类风湿关节炎FLSs细胞的线粒体自噬相关蛋白LC3B的表达,上调p62和PHB2的表达;NLRP3表达水平均显著降低,IL-1β和IL-18水平显著降低;细胞焦亡率显著降低;GSDMD蛋白表达显著上升,GSDMD-N蛋白表达下降（P<0.05、0.01、0.001）,且呈剂量...     

五味子丙素抑制脂多糖诱导心肌细胞的炎症反应与 细胞焦亡的作用及机制研究

目的:探究五味子丙素(SchC)对脂多糖(LPS)诱导心肌细胞HL-1炎症反应与细胞焦亡的影响。方法:培养小鼠心肌细胞HL-1,将其分为空白对照组、模型组(LPS)、LPS+SchC组。SchC预处理1 h后,分别以LPS刺激24 h或48 h,ELISA法提取细胞培养液上清液,检测细胞因子IL-1β、HMGB1和TNF-α的分泌,MTT法收集细胞检测细胞活力,Western Blot检测cleaved-Caspase1、GSDMD-N和NLRP3的表达;将HL-1细胞分为空白对照组、模型组(LPS)、LPS+siGSDMD(GSDMD siRNA)组、LPS+siGSDMD+SchC组,给药预处理1 h后,LPS刺激24 h,收集细胞培养液上清液,ELISA法检测IL-1β的分泌,MTT法检测细胞活力。结果:与LPS组比较,LPS+SchC组细胞活力得到改善(P<0.05),IL-1β、HMGB1和TNF-α的分泌均显著降低(P<0.05);Western Blot结果表明,与LPS组比较,LPS+SchC组的cleaved-Caspase1、GSDMD-N段和NLRP3蛋白水平显著降低(P<0.05);ELISA和MTT结果表明,与LPS组比较,LPS+siGSDMD组IL-1β的分泌显著降低(P<0.05)且细胞活力显著得到改善(P<0.01),然而LPS+siGSDMD组与LPS+siGSDMD+SchC组的IL-1β分泌水平和细胞活力并无显著性差异。结论:五味子丙素能有效缓解LPS诱导HL-1的炎症反应和细胞焦亡。     

IVF/ICSI助孕后早期流产患者蜕膜组织焦亡相关因子表达水平的研究

目的 探讨体外受精（IVF）/卵胞浆内单精子注射（ICSI）助孕妊娠后早期流产患者蜕膜组织中焦亡相关因子的表达及作用。方法 选取行IVF/ICSI助孕妊娠后早期流产患者102例（研究组），同期因意外妊娠行人工流产术的患者100例（对照组），收集蜕膜组织。实时荧光定量PCR(qRT-PCR)法及Western blot法检测蜕膜组织中焦亡相关因子Nod样受体蛋白3(NLRP3)、白细胞介素（IL）-1β、胱天蛋白酶1(Caspase1)、gasdermin D(GSDMD)mRNA及蛋白表达，TUNEL法检测蜕膜组织中细胞焦亡情况，Pearson法分析蜕膜组织中细胞焦亡率与NLRP3、IL-1β、Caspase1、GSDMD蛋白水平的相关性。结果 与对照组相比，研究组蜕膜组织中NLRP3、IL-1β、Caspase1、GSDMD mRNA及蛋白水平、蜕膜组织中细胞焦亡率升高（P<0.05）；研究组患者蜕膜组织中细胞焦亡率与NLRP3、IL-1β、Caspase1、GSDMD蛋白表达水平均呈正相关（r分别为0.429、0.474、0.498、0.540，均P<0.05）。结论 ...     

大黄素调控组蛋白乙酰化促进HpG2肝癌细胞焦亡及凋亡的发生

目的 研究天然产物大黄素是否能够影响HpG2肝癌细胞中组蛋白乙酰化水平,进而加速肝癌细胞焦亡和凋亡,为肝癌的治疗提供新的靶点。方法 CCK-8法检测不同浓度大黄素对Hp G2细胞活力的影响;生物信息学分析TCGA数据库中肝癌患者组蛋白乙酰化相关基因表达情况,验证候选基因赖氨酸乙酰基转移酶2A(KAT2A)与细胞凋亡通路的相关性;实时荧光定量PCR(q PCR)检测Hep G2细胞与L02细胞KAT2A m RNA水平;酶联免疫吸附试验(ELISA)检测大黄素对Hp G2细胞中组蛋白乙酰转移酶(HAT)、组蛋白去乙酰转移酶(HDAC)、白细胞介素(IL)-1β、IL-18的影响;流式细胞术检测大黄素对肝癌细胞凋亡的影响;Western blot检测细胞凋亡、细胞焦亡相关蛋白B淋巴细胞瘤-2(Bcl-2)、Bcl-2-相关X蛋白质(Bax)、NOD样受体热蛋白结构域相关蛋白3(NLRP3)、胱天蛋白酶1(Caspase-1)、Gasdermin家族成员D N端(GSDMD-N)及KAT2A的表达情况。结果 大黄素能降低Hp G2细胞活性,半抑制浓度(IC₅₀)95%置...     

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。     

基于TLR4/NF-κB/NLRP3信号通路研究三叶香茶菜含药血清对枯否细胞活化的影响

目的 研究三叶香茶菜含药血清(isodon ternifolius-containing serum，ITS)通过Toll样受体4(TLR4)/核因子κB(NF-κB)/NOD样受体蛋白3(NLRP3)信号通路对脂多糖(lipopolysaccharide，LPS)诱导的大鼠原代肝枯否细胞(Kupffer cell，KC)活化的影响。方法 分离培养大鼠原代KC，将LPS诱导的大鼠原代KC分为空白对照组、模型对照组、空白血清组、阳性对照组(秋水仙碱含药血清组)、ITS组、TLR4阻断剂组、TLR4阻断剂+ITS组。MTT法检测不同浓度ITS对KC增殖活性的影响；ELISA法检测KC细胞上清液白介素-1β(IL-1β)、白细胞介素-18(IL-18)、肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)含量；荧光定量聚合应(PCR)、Western blotting和免疫荧光检测KC中TLR4/NF-κB/NLRP3信号通路中TLR4、核因子κB抑制蛋白α(IκBα)、半胱肽氨酸蛋白酶-1(Caspase-1)、NLRP3 mRNA和TLR4、IκBα、磷酸化IκBα(p-IκBα)、Caspase-1、NLRP3、NF-κBp65蛋白表达情况。结果 与模型对照组相比，各药物组KC上清液中IL-1β、IL-18、TNF-α和IL-6含量，以及细胞中TLR4、IκBα、Caspase-1、NLRP3 mRNA和TLR4、IκBα、p-IκBα、Caspase-1、NLRP3、NF-κBp65蛋白的表达均下调或降低(P<0.05或P<0.01)；与TLR4阻断剂组比较，TLR4阻断剂+ITS组上述多数指标的改善更加明显。结论 三叶香茶菜可能通过下调TLR4/NF-κB/NLRP3信号通路抑制KC活化，减少炎性因子的表达和释放，从而减轻肝脏炎症损伤。     

C/EBPβ通过pim-1介导足细胞损伤的作用机制

目的　探讨CCAAT增强子结合蛋白β（C/EBPβ）/丝氨酸/苏氨酸激酶1（pim-1）/NOD样受体热蛋白结构域相关蛋白3（NLRP3）轴介导小鼠肾脏足细胞损伤的作用机制。方法　体外培养及转染足细胞后分为Control组、siRNA-NC组（用siRNA-NC转染足细胞）、siC/EBPβ组（用siC/EBPβ慢病毒转染足细胞）、Vector-NC组（空载体转染足细胞）和pim-1-OE组（pim-1过表达慢病毒转染足细胞）。脂多糖（LPS）与腺苷三磷酸（ATP）刺激足细胞后分为LPS+ATP组、LPS+ATP+siRNA-NC组、LPS+ATP+siC/EBPβ组、LPS+ATP+siC/EBPβ+Vector-NC组和LPS+ATP+siC/EBPβ+pim-1-OE组。实时荧光定量PCR（qPCR）检测C/EBPβ和pim-1 mRNA水平。Western blot检测C/EBPβ、pim-1、NLRP3、p20半胱氨酸蛋白酶（Caspase）-1、Gasdermin D（GSDMD）、p17白细胞介素（IL）-1β蛋白水平。酶联免疫吸附试验（ELISA）检测炎性因子IL-1β和IL-6水平。染色质免疫共沉淀（chip）实验及双荧光素酶报告基因实验检测C/EBPβ与pim-1基因启动子结合。结果　与Control组和siRNA-NC组相比,siC/EBPβ组的C/EBPβ及pim-1 mRNA水平明显下降（P＜0.01）。与Control组相比,LPS＋ATP组NLRP3、p20Caspase-1、p17IL-1β蛋白水平及IL-1β、IL-6水平明显升高（P＜0.01）。与LPS+ATP+siRNA-NC组相比,LPS+ATP+siC/EBPβ组NLRP3、p20Caspase-1、p17IL-1β蛋白水平及IL-1β、IL-6水平降低（P＜0.01）。chip实验及双荧光素酶报告基因实验显示C/EBPβ可与pim-1基因启动子结合。与Control组和Vector-NC组比较,pim-1-OE组pim-1 mRNA水平明显升高（P＜0.05）。与LPS+ATP+siC/EBPβ+Vector-NC组相比,LPS+ATP+siC/EBPβ+pim-1-OE组NLRP3、p20Caspase-1、p17IL-1β、GSDMD蛋白水平及IL-1β、IL-6水平升高（P＜0.05）。结论　C/EBPβ/pim-1/NLRP3轴可能参与足细胞损伤,并作为LN治疗的潜在靶点。     

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     

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.     

Anti-inflammatory effect of Yam Glycoprotein on lipopolysaccharide-induced acute lung injury via the NLRP3 and NF-κB/TLR4 signaling pathway

Acute lung injury (ALI) is a common lung disease accompanied by acute and persistent pulmonary inflammatory response syndrome, which leads to alveolar epithelial cells and capillary endothelial cell damage. Yam glycoprotein, separated from traditional Chinese yam, has been shown to have anti-inflammatory and immunomodulatory effects. In this experiment, we mainly studied the therapeutic effect and mechanism of a glycoprotein on the lipopolysaccharide (LPS)-induced ALI mice. An oral glycoprotein method was used to treat the mouse ALI model induced by LPS injection in the peritoneal cavity. Afterward, we measured the wet/dry (W/D) ratio, the activity of myeloperoxidase (MPO), the oxidative index superoxide dismutase (SOD), malondialdehyde (MDA), glutathione peroxidase (GSH-PX) and the production of inflammatory cytokines interleukin-1β (IL-1β), tumour necrosis factor-α (TNF-α), and interleukin-6 (IL-6) to evaluate the effect of yam glycoprotein on lung tissue changes. We examined the protein expression of TLR4, ASC, NF-κBp65, p-NF-κBp65, Caspase-1, IκB, NLRP3, p-IκB, and β-actin by western blot analysis. Immunohistochemical analyses of NLRP3 and p-p65 in lung tissue were carried out to assess the mechanism of glycoprotein action. This result suggests that glycoprotein markedly depressed LPS-induced lung W/D ratio, MPO activity, MDA content SOD and GSH-Px depletion, and the contents of inflammatory cytokines IL-1β, IL-6, and TNF-α. Moreover, glycoprotein blocked TLR4/NF-κBp65 signaling activation and NLRP3inflammasome expression in LPS-induced ALI mice. As this particular study shows, glycoprotein has a safeguarding effects on LPS-induced ALI mice, possibly via activating NLRP3inflammasome and TLR4/NF-κB signaling pathways.     

基于NLRP3-IL-1β-NF-κB信号轴研究野菊花总黄酮对 脂多糖诱导HK-2细胞炎性反应及自噬的影响

摘要：目的 探究野菊花总黄酮（TFC）对脂多糖（LPS）诱导HK-2细胞炎性反应及自噬的影响,并初步探究其可 能的作用机制。方法 体外培养人肾小管上皮HK-2细胞,将细胞分为阴性对照（Control）组、LPS处理（LPS）组、TFC 预处理（TFC）组、NLRP3激活预处理（4-MET）组、TFC联合4-MET预处理（TFC+4-MET）组。CCK-8法检测细胞增殖 情况;Hoechst 33342染色观察各组细胞形态变化;免疫印迹法检测各组细胞核苷酸结合域样受体蛋白3（NLRP3）、凋 亡相关斑点样蛋白（Asc）、半胱氨酸天冬氨酸蛋白酶1（caspase-1）、p-IκB、IκB、自噬相关蛋白（Beclin1）、微管相关蛋 白轻链3Ⅱ（LC3Ⅱ）、LC3Ⅰ、蛋白表达情况;ELISA法检测细胞中白细胞介素（IL）-1β、IL-18水平;透射电镜观察细胞 自噬情况。结果 与Control组相比,LPS组细胞增殖率降低,细胞核破碎情况加重,细胞凋亡率升高,NLRP3、Asc、 caspase-1蛋白表达升高、IκB蛋白磷酸化水平升高,IL-1β、IL-18水平升高,LC3Ⅱ/Ⅰ水平、Beclin-1蛋白表达降低 （P＜0.05）。与LPS组相比,TFC组细胞增殖率升高,细胞核破碎减轻,细胞凋亡率降低,NLRP3、Asc、caspase-1蛋白 表达降低、IκB蛋白磷酸化水平降低,IL-1β、IL-18水平降低,细胞自噬程度增加,LC3Ⅱ/Ⅰ水平、Beclin-1蛋白表达升 高;4-MET组细胞增殖率降低,细胞凋亡率升高,细胞核破碎情况加重,NLRP3、Asc、caspase-1蛋白表达升高、IκB蛋 白磷酸化水平升高,IL-1β、IL-18水平升高,细胞自噬程度降低,LC3Ⅱ/Ⅰ水平、Beclin-1蛋白表达降低（P＜0.05）。 TFC+4-MET组细胞增殖率、细胞自噬程度、LC3Ⅱ/Ⅰ水平、Beclin-1蛋白表达高于4-MET组,细胞核破碎情况减轻, 细胞凋亡率、NLRP3、Asc、caspase-1蛋白表达、IκB蛋白磷酸化、IL-1β、IL-18水平低于4-MET组（P＜0.05）。结论 野菊花总黄酮可能通过抑制NLRP3-IL-1β-NF-κB信号通路活化,进而抑制脂多糖诱导HK-2细胞炎性反应,诱导 细胞自噬     

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.     

溪黄草水提物对高糖诱导的大鼠HBZY-1细胞炎症反应和氧化应激的抑制作用以及对TLR 4/NF-κB/NLRP 3通路的作用

目的：溪黄草水提物（RWE）对高糖诱导的大鼠HBZY-1细胞炎症反应和氧化应激的抑制作用以及对TLR 4/NF-κB/NLRP 3通路的作用。方法：用高糖（HG,30 mmol·L^-1）孵育大鼠肾小球细胞株HBZY-1细胞,MTT法测定不同浓度RWE（5,10,20 mg·mL^-1）对HG环境下HBZY-1细胞增殖的影响,测定RWE对HG诱导的HBZY-1细胞氧化应激水平和炎性细胞因子表达的影响,同时测定其对细胞TLR4/NF-κB/NLRP3信号通路的影响。结果：HG能诱导HBZY-1细胞增殖,提高MDA、IL-6、IL-1β、TNF-α水平和TLR 4/NF-κB/NLRP 3信号通路的表达,降低SOD和GSH水平;RWE减弱细胞的增殖能力,降低MDA、IL-6、IL-1β和TNF-α的水平,提高SOD和GSH水平,抑制TLR 4/NF-κB/NLRP 3信号通路的表达。结论：RWE对HG诱导的HBZY-1细胞具有保护作用,其作用机制主要是抑制炎症反应和氧化应激,TLR 4/NF-κB/NLRP 3信号通路可能参与RWE对细胞的保护作用。     

Melatonin prevents endothelial cell pyroptosis via regulation of long noncoding RNA MEG3/miR-223/NLRP3 axis

OBJECTIVE Atherosclerosis(AS) is an inflammatory disease linked to endothelial dysfunction.Melatonin is reported to possess substantial anti-inflammatory properties, which has proven to be effective in AS. Emerging literature suggests that pyroptosis plays a critical role during AS progression. However, whether pyroptosis contributes to endothelial dysfunction and the underlying molecular mechanisms remained unexploited.This study was designed to investigate the antipyroptotic effects of melatonin in atherosclerotic endothelium and to elucidate the potential mechanisms. METHODS ApoE-/-mice were fed a high-fat diet to establish an atherosclerotic model, then divided into normal diet(ND) group, normal diet+melatonin(ND+melatonin) group, high fat diet(HFD)group and high fat diet+melatonin(HFD+melatonin) group.After 12 weeks, HE and oil Red O staining were used to detect the formation of atherosclerosis; qRT-RCR and Western blotting were used to detect the expression of NLRP3, ASC, IL-1β, IL-18, GSDMD, NF-κB, miR-223 and MEG3 in aortic endothelium; The luciferase assay was used to detect the binding of mi R-223 to MEG3. Human aortic endothelial cells(HAECs) were pretreated with ox-LDL. After melatonin treatment, qRT-RCR was used to detect the expression of mi R-223 and MEG3. Western blotting was used to detect NLRP3, ASC, c-caspase1,p-caspase1, GSDMD expression. In addition, after overexpressing MEG3 and knocking out mi R-223, the pyroptosis of HAECs was also detected. RESULTS We found intragastric administration of melatonin for 12 weeks markedly reduced the atherosclerotic plaque in aorta.Meanwhile, melatonin also attenuated the expression of pyroptosis-related genes, including NLRP3, ASC,cleaved caspase1, NF-κB/GSDMD, GSDMD N-termini,IL-1β, and IL-18 in aortic endothelium of melatonin-treated animals. Consistent antipyroptotic effects were also observed in ox-LDL-treated human aortic endothelial cells(HAECs). We found that lnc RNA MEG3 enhanced pyroptosis in HAECs. Moreover, MEG3 acted as an endogenous sponge by sequence complementarity to suppress the function of mi R-223 and to increase NLRP3 expression and enhance endothelial cel pyroptosis. Furthermore, knockdown of mi R-223 blocked the antipyroptotic actions of melatonin in ox-LDL-treated HAECs. CONCLUSION Our results suggest that melatonin prevents endothelial cell pyroptosis via MEG3/mi R-223/NLRP3 axis in atherosclerosis, and therefore, melatonin replacement might be considered a new strategy for protecting endothelium against pyroptosis, thereby for the treatment of atherosclerosis associated with pyroptosis.