瓜子金皂苷己抑制IL-1β的释放及其机制研究

目的研究瓜子金皂苷己(polygalasaponin F,PS-F)对LPS诱导的BV2小胶质细胞炎症反应中炎性因子IL-1β释放的影响及其作用机制。方法实验分为空白组、LPS模型组、LPS+PS-F (0.10、1.00、10.00μmol·L~(-1))给药组。运用ELISA法检测培养液上清中IL-1β的分泌量;real-time PCR检测IL-1βmRNA的表达;Western blot检测IL-1β、NLRP3、caspase-1、ASC、caspase-11的蛋白表达量。结果 ELISA、real-time PCR和Western blot结果均表明,PS-F能有效抑制LPS诱导BV2小胶质细胞释放炎性因子IL-1β(P<0.01,P<0.05);Western blot结果表明,PS-F可以下调NLRP3、caspase-1、ASC、caspase-11的蛋白表达,其中ASC、caspase-11所得结果差异有统计学意义(P<0.05)。结论 PS-F可以有效抑制LPS诱导BV2细胞炎症反应中炎性因子IL-1β的释放,且与下调NLRP3炎性小体、抑制caspase-11的激活有关。     

中介素通过激活AMPK信号通路对脂多糖诱导巨噬细胞极化的影响

目的探讨中介素(intermedin,IMD)对脂多糖(lipopolysaccharide,LPS)诱导小鼠单核巨噬细胞系RAW 264.7极化的影响及其作用机制。方法RAW 264.7细胞随机分为对照组、LPS组、LPS+IMD组、LPS+IMD+CC(AMPK抑制剂Compound C)组。Real time-PCR法检测TNF-α、CD86、iNOS、Arg^-1、CD206 mRNA表达,Western blot法检测p-AMPK、AMPK、TNF-α、IL-6和IL^-10蛋白表达,流式细胞术检测巨噬细胞亚型,ELISA法检测培养基上清IL-6和TNF-α浓度。结果与对照组及LPS组比较,IMD处理可增加AMPK磷酸化水平,增加p-AMPK/AMPK比值;与对照组相比,LPS诱导可导致巨噬细胞发生M1极化,M1型标志分子CD86、TNF-α及iNOS mRNA表达升高,M2型标志分子CD206、Arg^-1 mRNA表达降低,上调促炎因子TNF-α、IL-6表达,降低抑炎因子IL^-10表达,使M1型细胞数量增加,细胞上清中TNF-α、IL-6分泌增加;而IMD处理可抑制LPS诱导的M1极化,AMPK抑制剂Compound C组处理可在一定程度上拮抗这一作用。结论IMD通过激活AMPK信号通路抑制LPS诱导的巨噬细胞M1型极化。     

DAPT对慢性社会挫败应激诱导小鼠抑郁样行为的作用及机制北大核心CSCD

目的探究DAPT对慢性应激诱导的小鼠抑郁样行为的作用及机制。方法C57BL/6J小鼠给予慢性社会挫败应激(chronic social defeat stress,CSDS)处理10 d以建立抑郁模型,药物处理组小鼠每天腹腔注射DAPT 5 mg·kg^(-1)。通过社会接触、糖水偏爱、旷场、强迫游泳和悬尾实验来评价小鼠的抑郁样行为;采用Western blot检测小鼠海马NLRP3、ASC、caspase-1、TNF-α、p62、Atg7、Atg5和Beclin1蛋白的表达,运用ELISA检测小鼠海马IL-1β和IL-18的表达水平。结果CSDS诱导小鼠出现明显的抑郁样行为,同时增加了小鼠海马NLRP3、ASC、caspase-1的表达和炎症因子TNF-α、IL-1β和IL-18的水平,另外,CSDS处理小鼠海马自噬相关蛋白Atg7、Atg5和Beclin1表达明显减少,而p62的表达则明显增加。DAPT处理能明显改善CSDS诱导的小鼠抑郁样行为,并能抑制NLRP3、ASC、caspase-1、TNF-α、IL-1β、IL-18以及p62蛋白表达的增加和Atg7、Atg5和Beclin1蛋白表达的减少。结论DAPT能够改善慢性应激诱导的小鼠抑郁样行为,其机制可能与其抑制NLRP3炎症小体激活和上调自噬功能有关。     

隐丹参酮对骨髓来源巨噬细胞功能的影响

目的研究隐丹参酮对骨髓巨噬细胞功能的影响。方法激光共聚焦检测巨噬细胞吞噬功能;庆大霉素保护法检测巨噬细胞杀菌能力;q-PCR法检测TNF-α、IL-6、IL-10的表达水平;Western blot检测NF-κB通路;LPS(lipopolysaccharides)及IL-4刺激检测巨噬细胞分型。结果隐丹参酮能够增强巨噬细胞吞噬功能及杀菌能力;促进抗炎因子IL-10并抑制促炎因子TNF-α及IL-6的表达;阻断NF-κB信号通路;诱导巨噬细胞向M2型分化。结论隐丹参酮能促进巨噬细胞向M2型分化,并上调骨髓巨噬细胞的免疫功能。     

桃叶珊瑚苷通过调节小胶质细胞M1/M2极化抑制神经炎症

目的:探究桃叶珊瑚苷(aucubin)对脂多糖(LPS)诱导建立的体外神经炎症模型的影响。方法:用LPS诱导N9细胞激活建立体外神经炎症模型，加入桃叶珊瑚苷处理细胞24 h,对细胞上清一氧化氮(NO)含量和细胞活力进行检测，显微镜拍摄细胞形态，免疫荧光法检测小胶质细胞特异标志物Iba-1水平，Flowsight检测CD11b水平和CD86/CD206比值，并用试剂盒检测IL-4,IL-10,TGF-β,IL-1β,IL-6,TNF-α水平。结果:与模型对照组比较，桃叶珊瑚苷组可有效改善细胞形态，降低细胞上清NO含量，降低细胞标志物CD11b水平和CD86/CD206比值，并调节炎症因子的释放。结论:桃叶珊瑚苷可能是通过调控炎症因子的释放，促进小胶质细胞由M1型向M2型转化，从而抑制N9小胶质细胞的激活，最终抑制LPS诱导的神经炎症。     

香青兰总黄酮对RAW264.7巨噬细胞泡沫化及炎症反应的调控作用北大核心CSCD

目的研究香青兰总黄酮(total flavonoids of Dracocephalum moldavica L.,TFDM)对氧化低密度脂蛋白(oxidized low density lipoprotein,ox-LDL)诱导的小鼠单核巨噬细胞白血病细胞(RAW264.7)泡沫化及炎症的影响,进一步阐明TFDM抗动脉粥样硬化(atherosclerosis,AS)的作用机制。方法体外培养RAW264.7巨噬细胞,采用ox-LDL刺激诱导使其成为泡沫细胞,TFDM(25、50、100 mg·L^(-1))及辛伐他汀(10μmol·L^(-1))进行干预,油红O染色法观察胞内脂滴的聚集情况,CCK-8法检测细胞活力,活性氧试剂盒测定ROS的生成,实时荧光定量PCR测定细胞中NF-κB、NLRP3、caspase-1、IL-18和IL-1βmRNA的表达,免疫蛋白印迹法检测巨噬细胞中IκBα、NF-κB p65、NLRP3、pro-caspase-1、caspase-1、IL-1β以及IL-18蛋白的表达,ELISA法检测TNF-α和IL-10的表达。结果TFDM可以减少泡沫巨噬细胞的形成,降低炎症因子IL-1β、IL-18和TNF-α的表达,增加抑炎因子IL-10的表达;并且下调NF-κB p65、NLRP3、pro-caspase-1、caspase-1蛋白的表达,上调IκBα的蛋白表达。结论TFDM能够减轻巨噬细胞的泡沫化,抑制炎症因子的表达,从而可能延缓动脉粥样硬化的发展进程。其作用机制可能是通过抑制NF-κB途径,减少ox-LDL诱导的RAW264.7细胞中炎症介质的产生。     

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...     

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。     

丙泊酚对BV-2小胶质细胞活化的影响

目的探讨丙泊酚对脂多糖(LPS)诱导的BV-2小胶质细胞活化的影响及其可能机制。方法体外培养小鼠BV-2小胶质细胞,随机分为LPS 1μg/ml组(A组)、丙泊酚30μM组(B组)、LPS 1μg/ml+丙泊酚30μM组(C组)和空白对照组(D组)。采用RT-PCR检测各组细胞中IL-1β和TNF-αmRNA表达量,Western blot检测总糖原合成酶激酶-3β(GSK-3β)和磷酸化GSK-3β(p-GSK-3β)的蛋白表达水平。结果 A、C组IL-1β、TNF-α及p-GSK-3表达量均较D组明显增加(P<0.05或P<0.01)。与A组相比,C组IL-1β和TNF-αmRNA表达量降低,而p-GSK-3β蛋白表达量增加(P<0.05)。结论丙泊酚30μM能在体外减轻LPS诱导的BV-2小胶质细胞释放IL-1β和TNF-α的水平,此作用可能与抑制GSK-3β活性有关。     

木犀草素通过HIF-1α抑制糖酵解调控M1型巨噬细胞极化

目的 基于低氧诱导因子1α(hypoxia inducible factor-1α,HIF-1α)介导糖酵解途径研究木犀草素(luteolin)调控M1型巨噬细胞极化的分子机制。方法 RAW264.7细胞随机分为对照组(M0)和脂多糖(LPS)联合干扰素γ(IFN-γ)诱导组(M1);之后将M1组分为Luteolin治疗组、2-DG(糖酵解抑制剂)组、Luteolin+2-DG组、Luteolin+DMOG(HIF-1α激活剂)组。Western blot检测iNOS、Arg-1和HIF-1α蛋白表达;流式细胞仪检测巨噬细胞亚型;ELISA检测细胞IL-6和IL-10浓度;Real-time PCR检测GLUT1、HK2、PFK1、PK和HIF-1α mRNA表达。结果 与M1组比较,Luteolin治疗组以及Luteolin和2-DG共培养组糖酵解相关因子HIF-1α、GLUT1、HK2、PFK1和PK表达水平降低;细胞葡萄糖消耗增加、乳酸分泌量减少。M1型巨噬细胞标记物iNOS、CD86和IL-6表达降低,M2型巨噬细胞标记物Arg-1、CD206和IL-10表达增高。而DMOG能...     

Biochanin A protects lipopolysaccharide/D-galactosamine-induced acute liver injury in mice by activating the Nrf2 pathway and inhibiting NLRP3 inflammasome activation

Biochanin A, an isoflavone existed in red clover and peanuts, has been reported to possess a wide spectrum of pharmacological activities, such as anti-inflammatory and antioxidant effects. However, the protective effects and mechanism of biochanin A on liver injury have not been reported. In this study, acute liver injury was induced by intraperitoneal injection of lipopolysaccharide (LPS) and d-galactosamine (D-GalN). Biochanin A was administrated 1 h prior to LPS/D-GalN challenge. Serum ALT, AST, IL-1β, and TNF-α levels, hepatic malondialdehyde (MDA), GPx, SOD, and Catalase contents, tissue histology, IL-1β, TNF-α, NLRP3, and Nrf2 expression were detected. The results showed that serum ALT, AST, IL-1β, and TNF-α levels and hepatic MDA content increased after LPS/GalN treatment. These changes were attenuated by biochanin A. Meanwhile, biochanin A dose-dependently up-regulated the expression of Nrf2 and HO-1. Biochanin A also inhibited hepatic IL-1β and TNF-α expression in a dose-dependent manner. Biochanin A did not inhibit LPS/D-GalN-induced hepatic NLRP3, ASC, and caspase-1 expression. However, the interaction of NLRP3 with ASC and caspase-1 were inhibited by biochanin A. In addition, LPS/D-GalN-induced up-regulation of thioredoxin-interacting protein (TXNIP) and interaction between TXNIP and NLRP3 were also inhibited by biochanin A. In conclusion, biochanin A protected against LPS/GalN-induced liver injury by activating the Nrf2 pathway and inhibiting NLRP3 inflammasome activation.     

Inhibiting NLRP3 inflammasome with MCC950 ameliorates perioperative neurocognitive disorders,suppressing neuroinflammation in the hippocampus in aged mice

Perioperative neurocognitive disorders (PND) are characterized by deficits in cognitive functions in the elderly following anesthesia and surgery. Effective clinical interventions for preventing this disease are limited. Growing evidence demonstrates that activation of NOD-like receptor protein3 (NLRP3) inflammasome is involved in neurodegenerative diseases. We therefore hypothesized that activation of NLRP3 inflammasome is linked to neuroinflammation and the subsequent cognitive impairments that occurred in an animal model of PND. In this study, 18-month-old C57BL/6 mice were subjected to an exploratory laparotomy under isoflurane anesthesia to mimic clinical human abdominal surgery. For interventional studies, mice received NLRP3 specific inhibitor MCC950 (10 mg/kg) or the vehicle only intraperitoneally. Behavioral studies were performed at 6 and 7 d after surgery using open field and fear conditioning tests, respectively. Interleukin-1β (IL-1β), interleukin-18 (IL-18), tumor necrosis factor-α (TNF-α), ionized calcium-binding adaptor molecule-1 (IBA1) positive cells, glial fibrillary acidic protein (GFAP) positive cells, NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), and cleaved caspase-1 were measured at 3 days post-surgery. Brain-derived neurotrophic factor (BDNF) and postsynaptic density protein 95 (PSD95) were measured at 7 days post-surgery. Our data indicates that surgery-induced cognitive impairments were associated with significant increases in IL-1β, IL-18, TNF-α, NLRP3, ASC, cleaved caspase-1, IBA1-positive cells and GFAP-positive cells, and decreases in BDNF and PSD95 expression in the hippocampus. Notably, administration with MCC950 attenuated inflammatory changes and rescued surgery-induced cognitive impairments. Our study suggests that surgery induces neuroinflammation and cognitive deficits that are partly attributed to the activation of NLRP3 inflammasome in the hippocampus of aged mice.     

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.     

紫草素通过TLR4/NF-κB信号通路抑制由脂多糖诱导的巨噬细胞炎症研究

目的 研究紫草素抑制由脂多糖(LPS)诱导的巨噬细胞炎症的机制.方法 以淀粉培养基注射BALB/c小鼠腹腔,诱导并分离巨噬细胞,以APC标记F4/80染色,并用流式细胞仪检测分离巨噬细胞情况;用1 mg·L-1的LPS刺激上述分离的巨噬细胞,分组如下:DMSO溶剂对照组,LPS对照组,LPS+低剂量紫草素组(0.1 μmol·L-1);LPS+中剂量紫草素组(1 μmol·L-1);LPS+高剂量紫草素组(10 μmol·L-1);通过ELISA和实时定量PCR(qRT-PCR)方法分别测定各处理组培养上清液以及细胞中肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)和白细胞介素-10(IL-10)的表达情况;Western blot分别测定Toll样受体4(TLR4)和核因子-κB(NF-κB)的p65亚基磷酸化表达情况.结果 流式细胞检测可知,APC标记的F4/80染色巨噬细胞的纯度可达97.8%;ELISA和实时定量PCR结果显示,紫草素处理后可以抑制由LPS刺激细胞因子TNF-α、IL-1β和IL-6的表达(P<0.05),并增加IL-10的表达(P<0.05),且呈现出一定的浓度依赖性;Western blot结果显示,紫草素能下调由LPS刺激的TLR4的表达水平和NF-κB的p65亚基磷酸化表达.结论 紫草素的抗炎机制可能是通过TLR4介导的信号通路活化NF-κB,抑制IL-1β、IL-6和TNF-α的分泌,并促进IL-10的分泌.     

Protective effects of ethyl acetate extracts of Rimulus Cinnamon on systemic inflammation and lung injury in endotoxin-poisoned mice

Rimulus cinnamon is the dried twig of Cinnamomum cassia Presl. It is widely used in China for the treatment of inflammatory processes, amenorrhea, and other diseases. We aimed to study the protective effects of ethyl acetate extracts of R. cinnamon (EAE) on systemic inflammation and lung injury in endotoxin-poisoned mice. EAE was administered 5 d prior to lipopolysaccharide (LPS) challenge with 15?mg/kg LPS. The administration of EAE increased the levels of interferon-γ (IFN-γ) and decreased the levels of interleukin-18 (IL-18) and tumor necrosis factor-α (TNF-α) in the serum. Additionally, EAE relieved the pathological changes in the tissues of the lungs and spleen, and significantly reduced the number of neutrophils in the lung tissues. In addition, treatment with EAE decreased the mRNA expression of the NLR family, pyrin domain-containing protein 3 (NLRP3), caspase-1, and interleukin-1β (IL-1β) in the lungs, as well as the expression of NLRP3, caspase-1 (p20), and pro-IL-1β proteins. These results demonstrated the promising anti-inflammatory effects of EAE in endotoxin-poisoned mice. Furthermore, EAE could alleviate the lung injury of endotoxin-poisoned mice by antagonizing the activation of the NLRP3 inflammasome.     

白藜芦醇对急性肝损伤小鼠NLRP3炎性体表达的影响

目的观察白藜芦醇对急性肝损伤(acute liver injury,ALI)小鼠Nod样受体家族3(Nod-like receptor 3,NLRP3)炎性体表达的影响,探讨白藜芦醇对ALI的保护作用及其机制。方法本实验采用四氯化碳制作ALI小鼠模型。将雄性ICR小鼠随机分成正常对照组、模型组、白藜芦醇低、中、高剂量组及阳性对照组,每组7只。白藜芦醇低、中、高剂量组及阳性对照组于造模前24 h及1 h分别腹腔注射剂量为10、20及30 mg/kg的白藜芦醇或剂量为100 mg/kg的乙酰半胱氨酸,对照组及模型组在相应时间点腹腔注射等量生理盐水,造模时模型组及各药物干预组采用腹腔注射5%四氯化碳,对照组腹腔注射等量橄榄油。采用蛋白质印迹(Western blot,WB)法测定小鼠肝组织NLRP3、凋亡相关微粒蛋白(apoptosis-associated speck-like protein contain,ASC)、炎性半胱天冬酶-1(caspase-1)蛋白,酶联免疫吸附测定(enzyme linked immunosorbent assay,ELISA)法检测炎症因子白介素(interleukin,IL)-1β及IL-18,全自动生化分析仪测定小鼠肝功能,病理组织学观察肝脏损伤情况及其程度。结果模型组小鼠谷丙转氨酶(alanine aminotransferase,ALT)及谷草转氨酶(aspartate aminotransferase,AST)水平高于正常对照组(P<0.01);白藜芦醇各剂量组及阳性对照组小鼠ALT及AST水平均低于模型组(P<0.01)。模型组小鼠肝脏炎症积分及损伤面积均高于正常对照组(P<0.01);白藜芦醇各剂量组及阳性对照组小鼠肝脏炎症积分及损伤面积均低于模型组(P<0.05或P<0.01)。模型组小鼠NLRP3、ASC、caspase-1、IL-1β及IL-18表达高于正常对照组(P<0.01);白藜芦醇各剂量组及阳性对照组小鼠NLRP3、ASC、caspase-1、IL-1β及IL-18表达均低于模型组(P<0.05或P<0.01)。病理组织切片显示,模型组小鼠肝细胞结构表现为胞浆疏松,小叶内坏死灶较多,坏死灶中可见中性粒细胞浸润;白藜芦醇各剂量组及阳性对照组小叶内坏死灶及中性粒细胞浸润等改变较模型组减少,肝细胞的受损面积较小。结论白藜芦醇可以显著减轻四氯化碳诱导的ALI,其机制可能与抑制NLRP3炎性体活化及其下游炎症级联反应有关。     

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。     

Involvement of endoplasmic reticulum stress in angiotensin II-induced NLRP3 inflammasome activation in human renal proximal tubular cells in vitro

Aim:

NLRP3 inflammasome plays an important role in renal injury and may be a therapeutic target in the treatment of patients with progressive chronic kidney disease. In this study we investigated whether angiotensin II (Ang II)-induced NLRP3 inflammasome activation was linked to endoplasmic reticulum stress (ERS) in human renal proximal tubular cells in vitro.

Methods:

Human kidney proximal epithelial cells (HK-2) were pretreated with telmisartan or 4-PBA, and then treated with Ang II. The expression levels of mRNAs and proteins related to NLRP3 inflammasomes and ERS was examined by real-time PCR, Western blot and immunofluorescence.

Results:

Treatment with Ang II (10, 100, and 1000 nmol/L) increased the expression of the inflammasome markers NLRP3 and ASC, as well as caspase-1, IL-1β, and IL-18 in dose- and time-dependent manners with peak levels detected at 100 nmol/L and 12 h. Ang II-induced increases in the expression of NLRP3, ASC, caspase-1, IL-1β, and IL-18 were significantly reduced by pretreatment with telmisartan (1 μmol/L). Immunofluorescence studies showed that Ang II increased the expression of NLRP3 and ASC, which was inhibited by telmisartan. Furthermore, Ang II treatment increased the expression of ERS markers GRP78 and p-eIF2α in dose- and time-dependent manners, which was significantly reduced by telmisartan. Moreover, Ang II-induced increases in the expression of NLRP3, ASC, caspase-1, IL-1β, and IL-18 were significantly inhibited by pretreatment with the ERS inhibitor 4-PBA (5 mmol/L).

Conclusion:

Ang II treatment induces NLRP3 inflammasome activation in HK-2 cells in vitro and ER stress is involved in this process, which may represent a new mechanism for the renal rennin-angiotensin system to induce tubulointerstitial inflammation.     

Asian sand dust enhances murine lung inflammation caused by Klebsiella pneumoniae

Inhaling concomitants from Asian sand dust (ASD) may result in exacerbation of pneumonia by the pathogen. The exacerbating effect of ASD on pneumonia induced by Klebsiella pneumoniae (KP) was investigated in ICR mice. The organic substances adsorbed onto ASD collected from the atmosphere of Iki-island in Japan were excluded by heat treatment at 360 °C for 30 min. ICR mice were instilled intratracheally with ASD at doses of 0.05 mg or 0.2 mg/mouse four times at 2-week intervals (total dose of 0.2 mg or 0.8 mg/mouse) and were administrated with ASD in the presence or absence of KP at the last intratracheal instillation. Pathologically, ASD caused exacerbation of pneumonia by KP as shown by increased inflammatory cells within the bronchiolar and the alveolar compartments. ASD enhanced the neutrophil number dose dependently as well as the expression of cytokines (IL-1β, IL-6, IL-12, IFN-γ, TNF-α) and chemokines (KC, MCP-1, MIP-1α) related to KP in BALF. In an in vitro study using RAW264.7 cells, combined treatment of ASD and KP increased gene expression of IL-1β, IL-6, IFN-β, KC, MCP-1, and MIP-1α. The same treatment tended to increase the protein level of IL-1β, TNF-α and MCP-1 in a culture medium compared to each treatment alone. The combined treatment tended to increase the gene expression of Toll-like receptor 2 (TLR2), and NALP3, ASC and caspase-1 compared with KP alone. These results suggest that the exacerbation of pneumonia by ASD + KP was due to the enhanced production of pro-inflammatory mediators via activation of TLR2 and NALP3 inflammasome pathways in alveolar macrophages.