川藏香茶菜丙素抑制NLRP3炎症小体活化并缓解小鼠脓毒性休克  

Isodopharicin C inhibits NLRP3 inflammasome activation and alleviates septic shock in mice

作　　者：曹海若 张玮[2,3] 李明远 杨燕青 李玉云 CAO Hairuo;ZHANG Wei;LI Mingyuan;YANG Yanqing;LI Yuyun(Anhui Provincial Key Laboratory of Cancer Research and Clinical Laboratory Diagnosis,school of laboratory Medicine,Bengbu Medical College,Bengbu 233030,China;Clinical Laboratory,First Affiliated Hospital of Bengbu Medical College,Bengbu 233004,China;Anhui Provincial Key Laboratory of Immunology in Chronic Disease,Bengbu Medical College,Bengbu 233030,China)

机构地区：[1]蚌埠医学院肿瘤基础研究与临床检验诊断重点实验室,安徽蚌埠233030 [2]蚌埠医学院第一附属医院检验科,安徽蚌埠233004 [3]蚌埠医学院慢性疾病免疫学基础与临床安徽省重点实验室,安徽蚌埠233030

出　　处：《南方医科大学学报》2023年第9期1476-1484,共9页Journal of Southern Medical University

基　　金：国家自然科学基金(82071775);蚌埠医学院2022年度研究生科研创新计划项目(Byycx22044);安徽省科研编制计划优秀青年科研项目(2022AH030140);安徽省重点科研平台开放课题基金项目(KLICD-2022-D1)。

摘　　要：目的探究中草药提取物川藏香茶菜丙素(Iso C)对NLRP3炎症小体活化的影响以及对脂多糖(LPS)诱导的小鼠脓毒性休克是否具有缓解作用。方法体外实验:利用LPS预刺激小鼠骨髓来源巨噬细胞(BMDM)以及人急性单核细胞白血病(THP-1)细胞系。通过加入多种NLRP3炎症小体激动剂活化经典NLRP3炎症小体。利用胞内转染LPS活化非经典NLRP3炎症小体。利用转染聚脱氧腺苷酸(poly A:T)活化AIM2炎症小体。通过蛋白质印迹法(Western blot)检测NLRP3炎症小体活化产物caspase-1的剪切,酶联免疫吸附测定法(ELISA)检测上清中NLRP3依赖与非依赖的多种促炎细胞因子分泌情况。利用电感耦合等离子体发射光谱仪检测细胞内钾离子含量。体内实验:挑选SPF级C57BL/6J小鼠随机分为空白对照组(Control组),脓毒性休克组(LPS组),Iso C治疗组(LPS+Iso C组)。ELISA分析小鼠血清和腹腔灌洗液中促炎细胞因子白细胞介素1β(1L-1β)、肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)的分泌情况,并观察注射LPS后小鼠48 h内生存时间,绘制小鼠生存曲线。结果体外实验表明,在BMDM细胞中Iso C以剂量依赖的方式抑制多种激动剂引起的经典NLRP3炎症小体活化以及胞内转染LPS诱导非经典NLRP3炎症小体的活化(P<0.05),Iso C对NLRP3炎症小体非依赖的促炎细胞因子TNF-α和IL-6的分泌无显著影响(P>0.05)。Iso C对AIM2炎症小体的活化无显著影响(P>0.05)。Iso C不影响NLRP3炎症小体活化上游信号钾离子外流(P>0.05)。在人THP-1细胞中Iso C抑制NLRP3炎症小体活化(P<0.05)。体内实验结果表明,与脓毒性休克组相比,Iso C治疗组小鼠血清和腹腔灌洗液中IL-1β的水平显著下降(P<0.05)且生存时间更长(P<0.05)。结论中草药来源的Iso C可以特异性抑制NLRP3炎症小体的活化从而缓解小鼠脓毒性休克,是潜在的治疗炎症性疾病的小分子化合物。Objective To investigate the effect of Isodopharicin C(Iso C),a traditional Chinese herbal medicine extract,on NLRP3 inflammasome activation and lipopolysaccharide(LPS)-induced septic shock in mice.Methods Murine bone marrowderived macrophages(BMDM)and human monocytic THP-1 cells were stimulated with LPS before treatment with different NLRP3 inflammasome agonists to activate canonical NLRP3 inflammasomes.The non-canonical NLRP3 inflammasomes were activated by intracellular LPS transfection,and AIM2 inflammasomes were activated with poly A:T.The cleavage of caspase-1 induced by NLRP3 activation was measured using Western blotting.The levels of NLRP3-dependent and-independent proinflammatory cytokines in the cell culture supernatant were detected using ELISA,and the intracellular potassium ion concentration was measured using ICP-OES.In the animal experiment,C57BL/6J mouse models of septic shock(induced by intraperitoneal LPS injection)were treated with Iso C,and the levels of IL-1β,TNF-αand IL-6 in the serum and peritoneal lavage fluid were detected using ELISA.The survival time of the mice was observed within 48 h after LPS injection and a survival curve was plotted.Results In BMDM cells,Iso C dose-dependently inhibited the activation of canonical NLRP3 inflammasomes and non-canonical NLRP3 inflammasomes(P<0.05)without obviously affecting the secretion levels of TNF-αand IL-6(P>0.05),the activation of AIM2 inflammasomes(P>0.05),or K+efflux,the upstream signaling of NLRP3 activation(P>0.05).Iso C inhibited the activation of canonical NLRP3 inflammasomes in human THP-1 cells.In septic C57BL/6J mice,Iso C treatment significantly reduced IL-1βlevels in the serum and peritoneal lavage fluid,and prolonged the survival time of the mice(P<0.05).Conclusion Iso C specifically inhibits NLRP3 inflammasome activation and alleviates septic shock in mice,and can serve as a potential small molecule compound for treatment of inflammatory diseases.

关 键 词：川藏香茶菜丙素 NLRP3炎症小体 脓毒性休克 川藏香茶菜 

分 类 号：R285.5[医药卫生—中药学]