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.
Zhongguo Zhong Yao Za Zhi. 2024 Sep;49(18):4986-4995. doi: 10.19540/j.cnki.cjcmm.20240603.401.
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 1 μg·mL(-1) lipopolysaccharide(LPS) and 10 μmol·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 32 μg·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.
本研究旨在探讨石菖蒲挥发油对小胶质细胞焦亡的影响，并阐明其潜在机制。用1μg·mL⁻¹脂多糖（LPS）和10μmol·L⁻¹尼日利亚菌素钠盐处理BV-2细胞以建立焦亡模型。用不同剂量的石菖蒲挥发油处理细胞，然后分别通过CCK-8法和流式细胞术检测细胞活力和凋亡情况。通过酶联免疫吸附测定（ELISA）检测肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）和白细胞介素-18（IL-18）的水平。通过实时定量聚合酶链反应（qRT-PCR）测定含核苷酸结合结构域、富含亮氨酸重复序列和吡啉结构域的蛋白3（NLRP3）、含半胱天冬酶激活和招募结构域的凋亡相关斑点样蛋白（ASC）以及半胱氨酸蛋白酶-1（caspase-1）的mRNA水平。采用免疫荧光法检测离子钙结合衔接分子1（Iba-1）和NLRP3的表达。采用蛋白质免疫印迹法检测B淋巴细胞瘤-2（Bcl-2）、Bcl-2相关X蛋白（Bax）、Iba-1、κB抑制激酶β（IKKβ）、磷酸化IKKβ（Y188）、核因子-κB p65（NF-κB p65）、磷酸化NF-κB（S536）、NLRP3、ASC、caspase-1、裂解的caspase-1、gasdermin D（GSDMD）和GSDMD-N的蛋白水平。结果显示，8、16和32μg·mL⁻¹的石菖蒲挥发油可提高细胞活力，增加Bcl-2/Bax的表达，并减少暴露于LPS和尼日利亚菌素钠盐的细胞凋亡。此外，该挥发油抑制Iba-1的表达，降低TNF-α、IL-1β和IL-18的水平，并下调NLRP3、ASC和caspase-1的mRNA水平以及NLRP3、ASC、caspase-1、磷酸化IKKβ（Y188）/IKKβ、磷酸化NF-κB（S536）/NF-κB p65、裂解的caspase-1和GSDMD-N的蛋白水平。结果表明，石菖蒲挥发油可抑制NLRP3炎性小体介导的小胶质细胞焦亡。
