石菖蒲挥发油抑制NLRP3炎症体介导的小胶质细胞焦亡实验研究  

Essential oil of Acori Tatarinowii Rhizoma inhibits pyroptosis of microglia by regulating NLRP3 inflammasomes

作　　者：徐志伟 周小杰[3] 黄筠皓 姬丽婷 李昌煜 XU Zhi-wei;ZHOU Xiao-jie;HUANG Jun-hao;JI Li-ting;LI Chang-yu(the Second School of Clinical Medicine,Zhejiang Chinese Medical University,Hangzhou 310053,China;Jinhua Academy,Zhejiang Chinese Medical University,Jinhua 321000,China;Academy of Chinese Medical Sciences,Zhejiang Chinese Medical University,Hangzhou 310053,China;School of Pharmaceutical Sciences,Zhejiang Chinese Medical University,Hangzhou 310053,China)

机构地区：[1]浙江中医药大学第二临床医学院,浙江杭州310053 [2]浙江中医药大学金华研究院,浙江金华321000 [3]浙江中医药大学中医药科学院,浙江杭州310053 [4]浙江中医药大学药学院,浙江杭州310053

出　　处：《中国中药杂志》2024年第18期4986-4995,共10页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(82204918)。

摘　　要：探讨石菖蒲挥发油对小胶质细胞焦亡的影响及其作用机制。采用1μg·mL^(-1)脂多糖和10μmol·L^(-1)尼日利亚菌素钠盐制备BV-2细胞焦亡模型,给予不同剂量的石菖蒲挥发油干预,CCK-8法检测细胞存活率;流式细胞术检测细胞凋亡情况;ELISA法检测炎症因子肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)和白细胞介素-18(IL-18)含量;qRT-PCR检测NOD样受体热蛋白结构域相关蛋白3(NLRP3)、凋亡相关斑点样蛋白(ASC)和半胱氨酸蛋白酶-1(caspase-1)mRNA水平;免疫荧光法检测离子钙结合衔接分子1(Iba-1)、NLRP3蛋白表达;Western blot检测B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、Iba-1、κB抑制因子激酶β(IKKβ)、p-IKKβ(Y188)、核转录因子-κB p65(NF-κB p65)、p-NF-κB(S536)、NLRP3、ASC、caspase-1、cleaved caspase-1、焦孔素D(GSDMD)和GSDMD-N蛋白表达。结果显示,8、16、32μg·mL^(-1)石菖蒲挥发油能明显改善脂多糖和尼日利亚菌素钠盐诱导的细胞存活率降低现象,增加Bcl-2/Bax比值,改善细胞凋亡情况;降低Iba-1蛋白表达;减少TNF-α、IL-1β和IL-18炎症因子含量;降低NLRP3、ASC和caspase-1 mRNA水平以及NLRP3、ASC、caspase-1、p-IKKβ(Y188)/IKKβ、p-NF-κB(S536)/NF-κB p65、cleaved caspase-1和GSDMD-N蛋白表达。结果表明,石菖蒲挥发油能够抑制NLRP3炎症体介导的小胶质细胞焦亡。This study aims to investigate the effect of essential oil of Acori Tatarinowii Rhizoma on microglial pyroptosis and decipher the underlying mechanism.BV-2 cells were treated with 1μg·mL^(-1)lipopolysaccharide(LPS)and 10μmol·L^(-1)nigericin sodium salt for the modeling of pyroptosis.The cells were treated with different doses of essential oil of Acori Tatarinowii Rhizoma,and then the cell viability and apoptosis were examined by the CCK-8 assay and flow cytometry,respectively.The levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),and interleukin-18(IL-18)were measured by ELISA.The mRNA levels of nucleotide-binding domain,leucine-rich repeat,and pyrin domain-containing protein 3(NLRP3),apoptosis-associated speck-like protein containing CARD(ASC),and cysteine proteinase-1(caspase-1)were determined by qRT-PCR.The immunofluorescence assay was used to detect the expression of ionized calcium-binding adapter molecule 1(Iba-1)and NLRP3.Western blot was employed to determine the protein levels of B lymphocytoma-2(Bcl-2),Bcl-2 associated X protein(Bax),Iba-1,κB-inhibitor kinase beta(IKKβ),p-IKKβ(Y188),nuclear factor-κB p65(NF-κB p65),p-NF-κB(S536),NLRP3,ASC,caspase-1,cleaved caspase-1,gasdermin D(GSDMD),and GSDMD-N.The results showed that 8,16,and 32μg·mL^(-1)essential oil of Acori Tatarinowii Rhizoma increased the viability,increased the expression of Bcl-2/Bax,and reduced the apoptosis of the cells exposed to LPS and nigericin sodium salt.In addition,the essential oil inhibited the expression of Iba-1,lowered the levels of TNF-α,IL-1β,and IL-18,and down-regulated the mRNA levels of NLRP3,ASC,and caspase-1 as well as the protein levels of NLRP3,ASC,caspase-1,p-IKKβ(Y188)/IKKβ,p-NF-κB(S536)/NF-κB p65,cleaved caspase-1,and GSDMD-N.The results indicated that the essential oil of Acori Tatarinowii Rhizoma inhibited the NLRP3 inflammasome-mediated pyroptosis of microglia.

关 键 词：石菖蒲挥发油 小胶质细胞 神经炎症 细胞焦亡 NLRP3 

分 类 号：R285[医药卫生—中药学]