Long noncoding RNA X-inactive specific transcript regulates NLR family pyrin domain containing 3/caspase-1-mediated pyroptosis in diabetic nephropathy

BACKGROUND NLRP3-mediated pyroptosis is recognized as an essential modulator of renal disease pathology.Long noncoding RNAs(lncRNAs)are active participators of diabetic nephropathy(DN).X inactive specific transcript(XIST)expression has been reported to be elevated in the serum of DN patients.AIM To evaluate the mechanism of lncRNA XIST in renal tubular epithelial cell(RTEC)pyroptosis in DN.METHODS A DN rat model was established through streptozotocin injection,and XIST was knocked down by tail vein injection of the lentivirus LV sh-XIST.Renal metabolic and biochemical indices were detected,and pathological changes in the renal tissue were assessed.The expression of indicators related to inflammation and pyroptosis was also detected.High glucose(HG)was used to treat HK2 cells,and cell viability and lactate dehydrogenase(LDH)activity were detected after silencing XIST.The subcellular localization and downstream mechanism of XIST were investigated.Finally,a rescue experiment was carried out to verify that XIST regulates NLR family pyrin domain containing 3(NLRP3)/caspase-1-mediated RTEC pyroptosis through the microRNA-15-5p(miR-15b-5p)/Toll-like receptor 4(TLR4)axis.RESULTS XIST was highly expressed in the DN models.XIST silencing improved renal metabolism and biochemical indices and mitigated renal injury.The expression of inflammation and pyroptosis indicators was significantly increased in DN rats and HG-treated HK2 cells;cell viability was decreased and LDH activity was increased after HGtreatment. Silencing XIST inhibited RTEC pyroptosis by inhibiting NLRP3/caspase-1. Mechanistically,XIST sponged miR-15b-5p to regulate TLR4. Silencing XIST inhibited TLR4 by promotingmiR-15b-5p. miR-15b-5p inhibition or TLR4 overexpression averted the inhibitory effect ofsilencing XIST on HG-induced RTEC pyroptosis.CONCLUSIONSilencing XIST inhibits TLR4 by upregulating miR-15b-5p and ultimately inhibits renal injury inDN by inhibiting NLRP3/caspase-1-mediated RTEC pyroptosis.

Effect and mechanism of reactive oxygen species-mediated NOD-like receptor family pyrin domain-containing 3 inflammasome activation in hepatic alveolar echinococcosis

BACKGROUND The NOD-like receptor family pyrin domain-containing 3(NLRP3)inflammasome is a significant component of the innate immune system that plays a vital role in the development of various parasitic diseases.However,its role in hepatic alveolar echinococcosis(HAE)remains unclear.AIM To investigate the NLRP3 inflammasome and its mechanism of activation in HAE.METHODS We assessed the expression of NLRP3,caspase-1,interleukin(IL)-1β,and IL-18 in the marginal zone and corresponding normal liver of 60 patients with HAE.A rat model of HAE was employed to investigate the role of the NLRP3 inflammasome in the marginal zone of HAE.Transwell experiments were conducted to investigate the effect of Echinococcus multilocularis(E.multilocularis)in stimulating Kupffer cells and hepatocytes.Furthermore,immunohistochemistry,Western blotting,and enzyme-linked immunosorbent assay were used to evaluate NLRP3,caspase-1,IL-1β,and IL-18 expression;flow cytometry was used to detect apoptosis and reactive oxygen species(ROS).RESULTS NLRP3 inflammasome activation was significantly associated with ROS.Inhibition of ROS production decreased NLRP3-caspase-1-IL-1βpathway activation and mitigated hepatocyte damage and inflammation.CONCLUSION E.multilocularis induces hepatocyte damage and inflammation by activating the ROS-mediated NLRP3-caspase-1-IL-1βpathway in Kupffer cells,indicating that ROS may serve as a potential target for the treatment of HAE.

神经胶质瘤患者术前血清NLRP3、ILs表达水平对预后的影响研究

目的研究术前NLR家族Pyrin域蛋白3(NLR family pyrin domain-containing protein 3,NLRP3)、白细胞介素(Interleukin,ILs)表达谱特征对神经胶质瘤患者手术预后的影响。方法以2019年1月-2022年12月新疆医科大学第二附属医院神经外科收治的86例神经胶质瘤患者为研究对象,根据患者术后1年内随访情况将患者划分为手术预后良好组(n=52)和手术预后不良组(n=34)。对比两组患者一般临床资料及术前NLRP3、ILs(IL-1β、IL-4、IL-12、IL-17、IL-33)表达谱的表达水平差异。采用Spearman相关性分析,经单因素及多因素Logistic回归分析筛选神经胶质瘤患者手术预后不良的危险因素,通过受试者工作特征(Receiver operating characteristic,ROC)曲线评价各危险因素对患者手术预后的影响。结果与手术预后良好组比较,手术预后不良组患者肿瘤大小、肿瘤周围水肿患者比例、合并癫痫发作史患者比例、血清NLRP3、IL-1β、IL-4、IL-33水平均升高,术前卡氏功能状态评分、血清IL-12水平均降低,差异有统计学意义(P<0.05);Spearman相关性分析结果表明,肿瘤大小、肿瘤周围水肿、有癫痫发作史、血清中NLRP3、IL-1β、IL-4、IL-33水平均与手术预后不良呈正相关性,卡氏功能状态评分、IL-12与手术预后不良呈负相关性(P<0.05);单因素及多因素Logistic回归分析表明血清中NLRP3、IL-1β、IL-4水平均是神经胶质瘤患者手术预后不良的重要危险因素,IL-12是神经胶质瘤患者手术预后不良的重要保护因素(P<0.05);ROC曲线分析表明NLRP3、IL-1β、IL-4、IL-12独立及联合对于神经胶质瘤患者手术预后不良均具有较高预测效能(P<0.05)。结论神经胶质瘤患者NLRP3、ILs水平较高均是手术预后不良的重要危险因素,通过早期干预减轻神经胶质瘤患者炎症水平可能有利于改善手术治疗效果。

术前血清Lp-PLA2与NLRP3水平对IABP辅助PCI治疗的高危冠心病患者发生MACE的预测价值

【目的】探讨术前血清脂蛋白相关磷脂酶A2(Lp-PLA2)与核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)水平对主动脉内球囊反搏(IABP)辅助经皮冠状动脉介入治疗(PCI)高危冠心病患者发生心血管不良事件(MACE)的预测价值。【方法】选取本院收治的120例高危冠心病患者,所有患者均行IABP辅助PCI治疗。统计术后6个月内MACE发生情况,分为非MACE组和MACE组,比较两组术前血清Lp-PLA2、NLRP3水平,分析术前血清Lp-PLA2、NLRP3水平与冠脉狭窄程度的相关性,采用受试者工作特征(ROC)曲线分析术前血清Lp-PLA2、NLRP3水平预测术后发生MACE的价值。【结果】120例IABP辅助PCI术后高危冠心病患者MACE发生率为34.17%(41/120);MACE组术前血清Lp-PLA2、NLRP3水平均高于非MACE组(P<0.05)。Spearman相关性分析显示,术前血清Lp-PLA2、NLRP3水平与冠脉狭窄程度呈正相关(r_(s)=0.750、0.815,均P<0.05)。重度患者术前血清Lp-PLA2、NLRP3水平高于中度、轻度患者,中度患者高于轻度患者(P<0.05)。ROC曲线分析显示,术前血清Lp-PLA2、NLRP3水平单独预测的曲线下面积分别为0.770、0.844,二者联合预测AUC为0.909(P<0.05)。【结论】术前血清Lp-PLA2、NLRP3水平与高危冠心病患者冠脉狭窄程度呈正相关,二者联合检测可为临床预测高危冠心病患者IABP辅助PCI手术治疗后发生MACE提供一定参考依据。

血清FOXO3a、NLRP3、sICAM-1水平在冠心病合并高尿酸血症患者中的意义

目的探讨冠心病合并高尿酸血症(HUA)患者叉头转录因子O亚家族成员3a(FOXO3a)、核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)、可溶性细胞间黏附分子1(sICAM-1)水平变化的意义。方法选取河南科技大学第一附属医院2021年1月至2022年8月收治的156例冠心病患者为研究对象,根据尿酸水平分为HUA组(62例)和UA正常组(94例)。比较两组一般临床资料及FOXO3a、NLRP3、sICAM-1水平,分析FOXO3a、NLRP3、sICAM-1水平与临床资料中有统计学差异指标及UA水平相关性,采用logistic回归分析影响冠心病合并HUA的危险因素,以受试者工作特征(ROC)曲线分析FOXO3a、NLRP3、sICAM-1水平联合检测对冠心病合并HUA的诊断价值。结果HUA组总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)水平高于UA正常组(P<0.05);HUA组FOXO3a、NLRP3、sICAM-1水平高于UA正常组(P<0.05);相关性分析发现,FOXO3a、NLRP3、sICAM-1水平与TC、TG、LDL-C、UA水平均呈正相关(P<0.05);logistic回归分析发现,TC(>5.04 mmol·L^(-1))、TG(>1.88 mmol·L^(-1))、LDL-C(>2.53 mmol·L^(-1))、FOXO3a(>4.65μg·L^(-1))、sICAM-1(>195.73μg·L^(-1))、NLRP3(>1322.12 ng·L^(-1))水平是冠心病患者合并HUA的危险因素(P<0.05);ROC分析发现,FOXO3a、sICAM-1、NLRP3水平联合诊断冠心病合并HUA的曲线下面积(AUC)为0.811,敏感度、特异度分别为95.16%、67.02%,优于各指标单一指标诊断,具有较高诊断效能(P<0.05)。结论FOXO3a、NLRP3、sICAM-1参与冠心病合并HUA发生过程,且在临床诊断冠心病合并HUA方面具有较高效能。

益气升清方调节HIF-1α/NLRP3信号通路对缺血性脑卒中大鼠神经元焦亡的影响

目的 探究益气升清方调节缺氧诱导因子-1α(HIF-1α)/NOD样受体热蛋白结构域相关蛋白3(NLRP3)信号通路对缺血性脑卒中大鼠神经元焦亡的影响。方法 将SD大鼠随机分为假手术组(S组),模型组(M组),益气升清方低、中、高剂量组(3.465、6.930、13.860 g/kg)及益气升清方高剂量+HIF-1α激活剂DMOG组(13.860 g/kg益气升清方+40 mg/kg DMOG),每组15只。采用线栓法构建脑卒中模型。造模成功后进行神经功能缺陷评估;TTC染色评估脑梗死体积;ELISA法检测血清白细胞介素(IL)-1β、IL-18水平;HE染色检测缺血皮质区病理变化;TUNEL染色检测神经元凋亡;Western blot检测焦亡及HIF-1α/NLRP3通路相关蛋白表达。结果 与S组比较,M组神经功能缺陷评分、梗死体积、IL-1β含量、IL-18含量、神经细胞凋亡率、胞膜穿孔蛋白D-N端(GSDMD-N)、胱天蛋白酶1(Caspase-1)、HIF-1α、NLRP3蛋白水平均上升(P<0.05);与M组比较,低、中、高剂量益气升清方组神经功能缺陷评分、梗死体积、IL-1β含量、IL-18含量、神经细胞凋亡率、GSDMD-N、Caspase-1、HIF-1α、NLRP3蛋白水平均下调(P<0.05);DMOG减弱了高剂量益气升清方对缺血性脑卒中大鼠神经元焦亡的改善作用。结论 益气升清方可能通过下调HIF-1α/NLRP3信号通路减轻缺血性脑卒中大鼠神经元焦亡。

NLRP3基因敲减对高脂高糖饮食诱导的非酒精性脂肪性肝炎小鼠模型的影响

目的探讨NOD样受体热蛋白结构域相关蛋白3(NLRP3)基因敲减对高脂高糖诱导的非酒精性脂肪性肝炎(NASH)小鼠模型的影响。方法将44只小鼠随机分为正常饮食组(CON)20只,高脂高糖造模组(HFHC)24只。造模14周末,随机选取4只HFHC组小鼠进行腺相关病毒9(AAV9)尾静脉注射预实验,4周后验证NLRP3敲减模型是否成功。18周末确认敲减成功后,对剩余40只小鼠进行AAV9一次性尾静脉注射,分为CON+NLRP3敲减阴性对照组(CON+NLRP3-NC)、CON+NLRP3敲减组(CON+NLRP3-KD)、HFHC+NLRP3-NC及HHFHC+NLRP3-KD组,每组10只,继续造模6周。24周末取材观察炎症小体活化效应,检测小鼠体质量、肝质量、肝指数及糖代谢(空腹血糖、空腹胰岛素及HOMA-IR指数)指标;检测小鼠肝脂质含量(肝组织TG及油红O染色)、肝脏炎症(血清ALT活性、HE染色及炎症相关基因)及肝纤维化(天狼星红染色及纤维化相关基因)指标。计量资料多组间比较使用单因素方差分析,进一步两两比较采用LSD-t检验。结果与CON+NLRP3-NC组相比,Western Blot结果提示,HFHC+NLRP3-NC组的NLRP3、pro-Caspase1、Caspase1、ASC及IL-1β蛋白水平均升高,HFHC+NLRP3-KD组均降低(P值均<0.05);HFHC+NLRP3-NC组小鼠体质量、肝质量、肝指数及糖代谢指标均有不同程度升高,HFHC+NLRP3-KD组均显著改善(P值均<0.05);在肝脂肪沉积方面,与CON+NLRP3-NC组相比,HFHC+NLRP3-NC组肝脏TG明显增高,油红O染色显示大量红色脂滴,HFHC+NLRP3-KD组肝脏TG及肝脂滴数量显著减少(P值均<0.01);在肝脏炎症方面,HFHC+NLRP3-NC组血清ALT,非酒精性脂肪性肝病活动度(NAS)评分及炎症相关基因均较CON+NLRP3-NC组明显升高,HFHC+NLRP3-KD组均明显降低(P值均<0.01);在肝纤维化方面,HFHC+NLRP3-NC组肝胶原纤维面积以及纤维化相关基因均较CON+NLRP3-NC组明显升高,HFHC+NLRP3-KD组纤维化相关基因均明显降低(P值均<0.05),胶原纤维面积虽有降低趋势但差异无统计学意义(P>0.05)。结论NLRP3基因敲减可显著改善高脂高糖饮食诱导的NASH小鼠模型肝脂肪沉积及炎症。

NLRP3炎症小体在人骨关节炎软骨中的表达及作用

目的探究NOD样受体热蛋白结构域相关蛋白3(NLRP3)炎症小体在人骨关节炎(OA)软骨中的表达及作用。方法收集OA病人软骨30例和正常软骨10例,采用逆转录实时定量聚合酶链反应(RT-qPCR)法检测NLRP3炎症小体组分NLRP3、凋亡相关斑点样蛋白(ASC)、半胱氨酸天冬氨酸蛋白水解酶1(caspase-1)及炎症因子白细胞介素1β(IL-1β)mRNA的表达,采用蛋白质印迹法(Western blot)检测NLRP3、ASC、caspase-1蛋白的表达,采用酶联免疫吸附测定(ELISA)法检测IL-1β蛋白的表达。结果OA组软骨组织内NLRP3、ASC、caspase-1在mRNA水平和蛋白水平上均较正常软骨组显著升高,差异具有统计学意义(t=4.042~6.704,P<0.001)。OA组软骨组织中IL-1β的mRNA和蛋白水平均较正常软骨组显著升高,差异具有统计学意义(t=4.826、6.144,P<0.001)。结论OA软骨中NLRP3炎症小体组分表达升高以及IL-1β含量增加,参与了OA关节软骨炎症环境的形成。

连翘苷调节NLRP3炎性通路对急性胸膜炎大鼠肺损伤的影响

目的探讨连翘苷通过调节NOD样受体热蛋白结构域相关蛋白3(NLRP3)炎性通路对急性胸膜炎大鼠渗出液和肺损伤的影响。方法将90只大鼠采用随机数字表法均分为对照组、模型组、连翘苷低剂量(PH-L,5 mg/kg)组、连翘苷中剂量(PH-M,10 mg/kg)组、连翘苷高剂量(PH-H,20 mg/kg)组及NLRP3通路抑制剂(PJ34,10 mg/kg)组。肺功能分析仪检测大鼠用力肺活量(FVC)、第0.1秒用力呼气量(FEV 0.1)、第0.3秒用力呼气量(FEV 0.3);电子天平称量胸腔渗出物质量;瑞氏染色检测渗出物白细胞数;酶联免疫吸附试验检测渗出液中前列腺素E2(PGE2)、单核细胞趋化蛋白-1(MCP-1)、白细胞介素(IL)-6、肿瘤坏死因子-α(TNF-α)含量;全自动血气分析仪检测大鼠动脉CO_(2)分压[p(CO_(2))]、动脉氧分压[p(O_(2))];HE染色观察肺组织病理学变化;免疫组织化学染色检测NOD样受体热蛋白结构域相关蛋白3(NLRP3)、胱天蛋白酶1(Caspase-1)蛋白表达;Western blot检测NLRP3通路蛋白表达。结果与对照组比较,模型组大鼠胸腔渗出物质量和白细胞数、渗出液PGE2、MCP-1、IL-6、TNF-α含量、p(CO_(2))、NLRP3通路蛋白表达增加,FVC、FEV 0.1、FEV 0.3、p(O_(2))降低,肺组织出现明显的病理损伤(P<0.05);与模型组比较,PH各组和PJ34组大鼠胸腔渗出物质量、白细胞数、渗出液PGE2、MCP-1、IL-6、TNF-α含量、p(CO_(2))、NLRP3通路蛋白表达降低,FVC、FEV 0.1、FEV 0.3、p(O_(2))增加,肺组织病理损伤好转(P<0.05);与PH-H组比较,PJ34组上述指标差异无统计学意义(P>0.05)。结论PH可通过抑制NLRP3通路激活,抑制炎症反应,改善急性胸膜炎引起的肺损伤。

NOD样受体蛋白3(NLRP3)炎性小体在肝细胞癌发生发展中的作用

近年来,关于NOD样受体蛋白3(NLRP3)炎性小体在肿瘤中的研究已成为热点话题,尤其是在黑色素瘤、结直肠癌、肺癌、乳腺癌等肿瘤中,越来越多的证据表明炎症在促进肿瘤的发生发展、血管生成和肿瘤侵袭中具有重要的作用。肝细胞癌(HCC)是原发性肝癌中最常见的类型,而关于NLRP3炎性小体在HCC发生发展中的作用仍争议不断。因此,本文就NLRP3炎性小体在HCC进展过程中的潜在影响以及在抗癌治疗中的作用机制作一综述,认为NLRP3炎性小体可以作为HCC患者的有效治疗靶点。

含NOD样受体家族Pyrin域蛋白3/白介素-1β信号通路在脓毒症相关肾损伤中的研究进展

脓毒症相关急性肾损伤(SA-AKI)发病机制复杂,有研究显示,在SA-AKI的发病过程中,NOD样受体家族的NLRP3(NLRP3)被激活,进而增加白细胞介素1β (IL-1β)的产生,介导炎症反应的发展。因此,针对近年NLRP3/IL-1β信号通路在SA-AKI中的研究进展进行了综述,以期为阐述SA-AKI的发病机制提供新的思路,同时也为SA-AKI的诊断和治疗提供了新的策略。

苦豆碱通过抑制TLR4/NF-κB/NLRP3通路改善香烟烟雾诱导的人支气管上皮细胞损伤

目的探究苦豆碱(Alo)对香烟烟雾诱导的人支气管上皮细胞损伤的作用及其可能的作用机制。方法16HBE人支气管上皮细胞经100 mL/L香烟烟雾提取物(CSE)和(50、100、200)μmol/L Alo共处理后,CCK-8法检测细胞活力,试剂盒检测乳酸脱氢酶(LDH)活性;原位末端转移酶标记技术(TUNEL)、Western blot法检测细胞凋亡,ELISA检测炎性因子水平;2′,7′-二氯二氢荧光素二乙酸酯(DCFH-DA)荧光探针和相关试剂盒检测氧化应激水平;Western blot法检测Toll样受体4(TLR4)/核因子κB(NF-κB)/含pyrin结构域核苷酸结合寡聚结构域样受体家族蛋白3(NLRP3)通路相关蛋白表达水平。16HBE细胞经100 mL/L CSE和200μmol/L Alo共处理后,采用上述方法检测过表达TLR4对TLR4/NF-κB/NLRP3通路、细胞LDH活性、凋亡、炎症反应及氧化应激的影响。结果CSE暴露可降低16HBE细胞活力,增加LDH释放和细胞凋亡,增强炎症反应和氧化应激水平,且激活TLR4/NF-κB/NLRP3通路;经Alo处理后,细胞活性升高,LDH释放减少、凋亡降低、炎症减轻、氧化应激水平下降,且TLR4/NF-κB/NLRP3通路失活;TLR4过表达可逆转Alo处理对CSE诱导的16HBE细胞损伤的保护作用。结论Alo可通过抑制TLR4/NF-κB/NLRP3通路减轻CSE诱导的人支气管上皮细胞损伤。

内质网应激和NLRP3炎症小体在急性肾损伤中的作用及其机制

急性肾损伤(acute kidney injury,AKI)是临床常见的危急重症,主要临床症状为肾功能短时间内急剧下降。AKI的发病机制复杂,目前尚未完全阐明。近年来研究发现,内质网应激(endoplasmic reticulum stress,ERS)和Nod样受体蛋白3(Nod-like receptor family pyrin domain containing 3,NLRP3)炎症小体的激活均与AKI的发生密切相关。肾脏受损时,肾细胞内环境稳态被破坏,ERS被激活,过度的ERS可引起肾细胞凋亡,导致AKI的发生。另外,NLRP3炎症小体可以介导宿主识别内源性和外源性危险信号分子,继而激活caspase-1、IL-1β和IL-18等,诱导炎症反应,促使肾细胞凋亡。在AKI的动物模型中,ERS标志物的表达水平升高会伴随NLRP3炎症小体相关蛋白表达水平的升高,表明ERS可以调控NLRP3炎症小体的活化过程。阐明ERS和NLRP3炎症小体在AKI中的作用及其机制,有望为AKI的防治提供新的思路。

枸杞多糖通过调控NLRP3/Caspase-1通路在抑制高糖诱导的HRMEC损伤中的作用

目的观察枸杞多糖(LBP)是否可以通过调控核苷酸寡聚化结构域样受体家族3/半胱天冬酶-1(NLRP3/Caspase-1)细胞焦亡途径抑制高糖诱导的人视网膜微血管内皮细胞(HRMEC)损伤。方法将体外培养的HRMEC随机分组,即正常组(给予5.5 mmol·L^(-1)葡萄糖)、高糖组(给予55.5 mmol·L^(-1)葡萄糖)、LBP低浓度组(给予55.5 mmol·L^(-1)葡萄糖+100 mg·L^(-1) LBP)、LBP中浓度组(给予55.5mmol·L^(-1)葡萄糖+500 mg·L^(-1) LBP)、LBP高浓度组(给予55.5 mmol·L^(-1)葡萄糖+1000 mg·L^(-1) LBP)、si-NC组(转染20μmol·L^(-1) si-NC后给予55.5 mmol·L^(-1)葡萄糖)和si-NLRP3组(转染20μmol·L^(-1) si-NLRP3后给予55.5 mmol·L^(-1)葡萄糖);利用CCK-8法检测各组HRMEC细胞增殖情况,流式细胞术检测各组HRMEC细胞焦亡情况,RT-PCR法检测各组HRMEC中NLRP3、Caspase-1、核因子(NF)-κB、Gasdermin-D(GSDMD)、血管内皮生长因子(VEGF)的mRNA相对表达水平,Western blot法检测各组HRMEC焦亡相关NLRP3、Caspase-1、NF-κB、GSDMD、VEGF的蛋白相对表达水平,ELISA检测各组HRMEC细胞上清液中细胞焦亡下游白细胞介素(IL)-1β和IL-18的表达水平。结果与正常组相比,高糖组HRMEC细胞增殖率降低,细胞焦亡率升高,NLRP3、Caspase-1、NF-κB、GSDMD、VEGF的mRNA和蛋白相对表达水平均升高,IL-1β和IL-18表达水平均升高(均为P<0.05);与高糖组相比,si-NLRP3组中HRMEC细胞增殖率升高,细胞焦亡率降低,NLRP3、Caspase-1、NF-κB、GSDMD、VEGF的mRNA和蛋白相对表达水平均降低,IL-1β和IL-18表达水平均降低(均为P<0.05);与高糖组相比,si-NC组的细胞增殖率,细胞焦亡率,NLRP3、Caspase-1、NF-κB、GSDMD、VEGF蛋白和mRNA以及IL-1β、IL-18的表达水平差异均无统计学意义(均为P>0.05);与高糖组相比,LBP中、高浓度组中HRMEC细胞增殖率升高,细胞焦亡率降低,NLRP3、Caspase-1、NF-κB、GSDMD、VEGF的mRNA和蛋白相对表达水平均降低,IL-1β和IL-18表达水平均降低(均为P<0.05);与高糖组相比,除LBP低浓度组HRMEC细胞增殖率、各蛋白相对表达水平差异均无统计学意义外(均为P>0.05),其余指标表现和LBP中、高浓度组一致。结论LBP对高糖诱导的HRMEC损伤具有保护作用,能够促进细胞增殖,抑制细胞焦亡,其作用机制与抑制NLRP3/Caspase-1信号通路的激活,降低相关炎症因子的表达有关。

Tranylcypromine upregulates Sestrin 2 expression to ameliorate NLRP3-related noise-induced hearing loss

Noise-induced hearing loss is the primary non-genetic factor contributing to auditory dysfunction.However,there are currently no effective pharmacological interventions for patients with noise-induced hearing loss.Here,we present evidence suggesting that the lysine-specific demethylase 1 inhibitor–tranylcypromine is an otoprotective agent that could be used to treat noise-induced hearing loss,and elucidate its underlying regulatory mechanisms.We established a mouse model of permanent threshold shift hearing loss by exposing the mice to white broadband noise at a sound pressure level of 120 d B for 4 hours.We found that tranylcypromine treatment led to the upregulation of Sestrin2(SESN2)and activation of the autophagy markers light chain 3B and lysosome-associated membrane glycoprotein 1 in the cochleae of mice treated with tranylcypromine.The noise exposure group treated with tranylcypromine showed significantly lower average auditory brainstem response hearing thresholds at click,4,8,and 16 k Hz frequencies compared with the noise exposure group treated with saline.These findings indicate that tranylcypromine treatment resulted in increased SESN2,light chain 3B,and lysosome-associated membrane glycoprotein 1 expression after noise exposure,leading to a reduction in levels of 4-hydroxynonenal and cleaved caspase-3,thereby reducing noise-induced hair cell loss.Additionally,immunoblot analysis demonstrated that treatment with tranylcypromine upregulated SESN2 expression via the autophagy pathway.Tranylcypromine treatment also reduced the production of NOD-like receptor family pyrin domaincontaining 3(NLRP3)production.In conclusion,our results showed that tranylcypromine treatment ameliorated cochlear inflammation by promoting the expression of SESN2,which induced autophagy,thereby restricting NLRP3-related inflammasome signaling,alleviating cochlear hair cell loss,and protecting hearing function.These findings suggest that inhibiting lysine-specific demethylase 1 is a potential therapeutic strategy for preventing hair cell loss and noise-induced hearing loss.

The emerging role of mesenchymal stem cell-derived extracellular vesicles to ameliorate hippocampal NLRP3 inflammation induced by binge-like ethanol treatment in adolescence

Our previous studies have reported that activation of the NLRP3(NOD-,LRR-and pyrin domain-containing protein 3)-inflammasome complex in ethanol-treated astrocytes and chronic alcohol-fed mice could be associated with neuroinflammation and brain damage.Mesenchymal stem cell-derived extracellular vesicles(MSC-EVs)have been shown to restore the neuroinflammatory response,along with myelin and synaptic structural alterations in the prefrontal cortex,and alleviate cognitive and memory dysfunctions induced by binge-like ethanol treatment in adolescent mice.Considering the therapeutic role of the molecules contained in mesenchymal stem cell-derived extracellular vesicles,the present study analyzed whether the administration of mesenchymal stem cell-derived extracellular vesicles isolated from adipose tissue,which inhibited the activation of the NLRP3 inflammasome,was capable of reducing hippocampal neuroinflammation in adolescent mice treated with binge drinking.We demonstrated that the administration of mesenchymal stem cell-derived extracellular vesicles ameliorated the activation of the hippocampal NLRP3 inflammasome complex and other NLRs inflammasomes(e.g.,pyrin domain-containing 1,caspase recruitment domain-containing 4,and absent in melanoma 2,as well as the alterations in inflammatory genes(interleukin-1β,interleukin-18,inducible nitric oxide synthase,nuclear factor-kappa B,monocyte chemoattractant protein-1,and C–X3–C motif chemokine ligand 1)and miRNAs(miR-21a-5p,miR-146a-5p,and miR-141-5p)induced by binge-like ethanol treatment in adolescent mice.Bioinformatic analysis further revealed the involvement of miR-21a-5p and miR-146a-5p with inflammatory target genes and NOD-like receptor signaling pathways.Taken together,these findings provide novel evidence of the therapeutic potential of MSC-derived EVs to ameliorate the hippocampal neuroinflammatory response associated with NLRP3 inflammasome activation induced by binge drinking in adolescence.

Yemazhui(Herba Eupatorii Lindleyani)ameliorates lipopolysaccharide-induced acute lung injury via modulation of the toll-like receptor 4/nuclear factor kappa-B/nod-like receptor family pyrin domain-containing 3 protein signaling pathway and intestinal flor

OBJECTIVE:To investigate the impact of Yemazhui(Herba Eupatorii Lindleyani,HEL)against lipopolysaccharide(LPS)-induced acute lung injury(ALI)and explore its underlying mechanism in vivo.METHODS:The chemical constituents of HEL were analyzed by ultra-high performance liquid chromatographyquadrupole time-of-flight mass spectrometry method.Then,HEL was found to suppress LPS-induced ALI in vivo.Six-week-old male Sprague-Dawley rats were randomly divided into 6 groups:control,LPS,Dexamethasone(Dex),HEL low dose 6 g/kg(HEL-L),HEL medium dose 18 g/kg(HEL-M)and HEL high dose 54 g/kg(HEL-H)groups.The model rats were intratracheally injected with 3 mg/kg LPS to establish an ALI model.Leukocyte counts,lung wet/dry weight ratio,as well as myeloperoxidase(MPO)activity were determined followed by the detection with hematoxylin and eosin staining,enzyme linked immunosorbent assay,quantitative real time polymerase chain reaction,western blotting,immunohistochemistry,and immunofluorescence.Besides,to explore the effect of HEL on ALI-mediated intestinal flora,we performed 16s rRNA sequencing analysis of intestinal contents.RESULTS:HEL attenuated LPS-induced inflammation in lung tissue and intestinal flora disturbance.Mechanism study indicated that HEL suppressed the lung coefficient and wet/dry weight ratio of LPS-induced ALI in rats,inhibited leukocytes exudation and MPO activity,and improved the pathological injury of lung tissue.In addition,HEL reduced the expression of tumor necrosis factoralpha,interleukin-1beta(IL-1β)and interleukin-6(IL-6)in bronchoalveolar lavage fluid and serum,and inhibited nuclear displacement of nuclear factor kappa-B p65(NF-κBp65).And 18 g/kg HEL also reduced the expression levels of toll-like receptor 4(TLR4),myeloid differentiation factor 88,NF-κBp65,phosphorylated inhibitor kappa B alpha(phospho-IκBα),nod-like receptor family pyrin domain-containing 3 protein(NLRP3),IL-1β,and interleukin-18(IL-18)in lung tissue,and regulated intestinal flora disturbance.CONCLUSIONS:In summary,our findings revealed that HEL has a protective effect on LPS-induced ALI in rats,and its mechanism may be related to inhibiting TLR4/NF-κB/NLRP3 signaling pathway and improving intestinal flora disturbance.

NLRP3炎症小体在自身免疫性肝炎中的作用机制

自身免疫性肝炎(AIH)是由自身免疫系统攻击肝细胞所致的慢性肝炎,目前关于AIH的发病机制尚不十分明确。炎症小体是先天免疫的重要组成部分,参与多种病理生理学过程。研究表明核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)炎性小体相关的炎性反应在AIH的发病机制中起重要作用,其主要介导促炎因子的释放和细胞焦亡,进而参与AIH的病理生理过程。因此,可以通过抑制NLRP3炎性小体的激活来延缓AIH发生发展,从而为AIH的防治提供新思路。

漆黄素抑制小胶质细胞NLRP3炎症小体活化缓解脓毒症后认知功能损害的研究

目的探讨漆黄素抑制小胶质细胞NOD样受体家族蛋白3(NLRP3)炎症小体活化缓解脓毒症后认知功能损害的机制。方法选用C57BL/6小鼠,用盲肠结扎穿孔法构建脓毒症模型。分组包括:假手术组、脓毒症组、脓毒症+胱天蛋白酶(caspase)-1敲除组(脓毒症+Cas-1-/-组)、脓毒症+漆黄素组。Morris水迷宫评估小鼠的认知功能。Western blot和免疫荧光双染检测脑组织和小胶质细胞NLRP3炎症小体相关蛋白caspase-1、GSDMD蛋白N端片段(GSDMD-N)、白细胞介素(IL)-1β和IL-18,线粒体自噬相关蛋白Pink1、Parkin和微管相关蛋白轻链3(LC3)-Ⅱ的表达。用伊文思蓝检测血脑屏障的通透性。结果与假手术组相比,脓毒症组caspase-1、GSDMD-N、IL-1β和IL-18的表达水平显著升高(P<0.05);脓毒症+Cas-1-/-组caspase-1、GSDMD-N、IL-1β和IL-18的表达水平明显低于脓毒症组(P<0.05)。脓毒症+漆黄素组Pink1、Parkin和LC3-Ⅱ的表达水平明显高于脓毒症组(P<0.05),caspase-1、GSDMD-N、IL-1β和IL-18的表达水平明显低于脓毒症组(P<0.05)。与脓毒症组相比,脓毒症+漆黄素组伊文思蓝渗漏减少、逃逸潜伏期缩短和穿越平台次数增加(均P<0.05)。结论漆黄素可能通过Pink1/Parkin通路激活线粒体自噬,抑制小胶质细胞NLRP3炎症小体活化,进而缓解脓毒症后中枢炎症反应和认知功能损害。

敲低IL-33下调炎症因子和NLRP3表达抑制哮喘小鼠炎症反应

目的探讨白细胞介素33(IL-33)对哮喘小鼠炎症反应的调控机制。方法利用10μg/mL脂多糖(LPS)构建小鼠骨髓间充质干细胞(BMMSC)的体外细胞炎症模型,根据是否转染IL-33的小干扰RNA(si-IL-33)质粒和过表达(IL-33-OE)质粒,将细胞分成si-IL-33组,IL-33-OE组和模型组。实时荧光定量PCR检测IL-33、含pyrin结构域核苷酸结合寡聚结构域样受体家族蛋白3(NLRP3)、IL-1β和IL-6的信使RNA(mRNA)表达水平。钙离子荧光探针检测试剂盒(Fluo-3 AM)检测钙离子含量,JC-1线粒体膜电位检测试剂盒检测线粒体膜电位变化。利用腹腔注射致敏剂和激发剂的方法建立小鼠哮喘模型,根据处理方案的不同,将哮喘小鼠分成si-IL-33组、IL-33-OE组和模型组,每组5只。ELISA试剂盒检测小鼠血清IL-1β、IL-6和NLRP3含量,HE染色和Masson染色检测肺组织的病变情况。结果与模型组相比,si-IL-33组的IL-33、NLRP3、IL-1β和IL-6的mRNA表达量均降低,IL-33-OE组的IL-33、NLRP3、IL-1β和IL-6的mRNA表达量增加。BMMSC中钙离子荧光降低,而IL-33-OE组中钙离子荧光升高。si-IL-33组细胞线粒体中JC-1以聚合物形式存在,呈明亮的红色荧光,细胞中的绿色荧光非常弱;说明si-IL-33组细胞线粒体稳定,线粒体功能尚在。使用IL-33-OE质粒处理使线粒体膜电位下降后,JC-1不能以聚合物形式存在于线粒体基质中,此时线粒体内红色荧光强度显著降低,而细胞质中的绿色荧光显著增强。si-IL-33组小鼠血清中IL-1β、IL-6、NLRP3的含量明显降低,而IL-33-OE组小鼠血清中IL-1β、IL-6、NLRP3的含量明显升高。si-IL-33组几乎未见炎细胞浸润,炎症缓解,上皮细胞正常。si-IL-33组肺组织的纤维增生部分趋于正常。与模型组相比,si-IL-33组的支气管壁总面积(WAt)/支气管基底膜周径(Pbm)和气管壁平滑肌面积(WAm)/Pbm降低,IL-33-OE组的WAt/Pbm和WAm/Pbm增加。结论敲低IL-33通过下调NLRP3、IL-1β、IL-6表达抑制哮喘小鼠的炎症反应。