钟青芮,黄虹凯,兰悦嘉,王欢,曾勇,吴嘉思
ZHONG Qing-rui,HUANG Hong-kai,LAN Yue-jia,WANG Huan,ZENG Yong,WU Jia-si

• 1:成都中医药大学药学院/现代中药产业学院
• 2:成都中医药大学针灸推拿学院

摘要(Abstract)：

该研究旨在探究黄连-黄芩对CpG1826诱导小鼠细胞因子风暴继发性肺损伤(CSSLI)的治疗作用，揭示黄连-黄芩主要效应成分黄连碱和汉黄芩素抑制线粒体膜通透性转换孔(mPTP)/核苷酸结合寡聚结构域样受体蛋白3(NLRP3)炎症小体细胞焦亡路径，并减轻CSSLI的潜在分子机制。体内实验通过CpG1826诱导复制CSSLI小鼠模型，结合肺组织湿干比(W/D)评价肺肿胀程度，苏木素-伊红(HE)染色评价小鼠肺损伤程度，透射电镜(TEM)观察肺组织超微结构，酶联免疫吸附测定(ELISA)检测肺泡灌洗液中白细胞介素(IL)-1β、高迁移率族蛋白B1(HMGB1)、IL-18、IL-1α水平，发现黄连-黄芩水煎液较单味药显著降低肺水肿、肺损伤程度和肺泡灌洗液中相关细胞因子浓度，从而改善CSSLI。体外实验通过小鼠肺泡巨噬细胞(MH-S)建立CpG1826诱导的CSSLI模型，通过钙黄绿素淬灭筛选黄连-黄芩中抑制mPTP通道开启效果最优单体成分，发现黄连碱(5、10、20μmol·L~(-1))和汉黄芩素(10、20、40μmol·L~(-1))明显抑制mPTP通道的开启，作用效果最优且呈剂量依赖性；且黄连碱与汉黄芩素通过抑制mPTP通道开放，减少线粒体DNA(mtDNA)的释放和活性氧(ROS)累积，有效逆转CpG1826导致的线粒体膜电位(MMP)降低。进一步研究发现，2个单体成分能抑制细胞焦亡过程，下调NLRP3/半胱天冬蛋白酶-1(Caspase-1)/消皮素D(GSDMD)通路相关蛋白表达。综上所述，黄连-黄芩能改善CpG1826诱导的小鼠CSSLI,与其抑制mPTP/NLRP3细胞焦亡路径有关，为该药的临床应用及开发提供了科学依据。
This study aims to investigate the therapeutic effects of the Coptidis Rhizoma-Scutellariae Radix on cytokine storm secondary lung injury(CSSLI) induced by CpG1826 in mice, and to elucidate the potential molecular mechanisms by which its major active components, i.e., coptisine and wogonin, alleviate CSSLI by inhibiting the mitochondrial permeability transition pore(mPTP)/nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3) inflammasome pyroptosis pathway. In vivo, a mouse model of CSSLI was established by CpG1826 induction. Pulmonary edema was assessed by lung wet-to-dry weight ratio(W/D), lung injury was evaluated by hematoxylin-eosin(HE) staining, and ultrastructural changes in lung tissue were observed by transmission electron microscopy(TEM). The levels of interleukin(IL)-1β, high mobility group box 1 protein(HMGB1), IL-18, and IL-1α in bronchoalveolar lavage fluid were measured by enzyme-linked immunosorbent assay(ELISA). The results showed that the decoction of the Coptidis Rhizoma-Scutellariae Radix significantly reduced pulmonary edema, alleviated lung injury, and decreased the concentrations of related cytokines in BALF more effectively than either single herb alone, thereby improving CSSLI. In vitro, a CpG1826-induced CSSLI model was established in mouse alveolar macrophage MH-S cells. Calcein-AM quenching was used to screen for the most effective monomer components from the herb pair in inhibiting mPTP opening. Coptisine(5, 10, 20 μmol·L~(-1)) and wogonin(10, 20, 40 μmol·L~(-1)) markedly inhibited mPTP opening, with optimal effects and a clear dose-dependent pattern. These components suppressed mPTP opening, thereby reducing the release of mitochondrial DNA(mtDNA) and the accumulation of reactive oxygen species(ROS), effectively reversing the CpG1826-induced decrease in mitochondrial membrane potential(MMP). Further studies revealed that both coptisine and wogonin inhibited pyroptosis and downregulated the expression of key proteins in the NLRP3/Caspase-1/gasdermin D(GSDMD) pathway. In conclusion, the Coptidis Rhizoma-Scutellariae Radix improves CpG1826-induced CSSLI in mice, and this effect is associated with the inhibition of the mPTP/NLRP3 pyroptosis pathway, providing scientific evidence for its clinical application and further development.

关键词(KeyWords)： 黄连-黄芩;CpG1826;细胞因子风暴继发性肺损伤;线粒体膜通透性转换孔(mPTP);细胞焦亡
Coptidis Rhizoma-Scutellariae Radix;CpG1826;cytokine storm secondary lung injury;mitochondrial permeability transition pore(mPTP);pyroptosis

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金青年科学基金项目(82104491);; 四川省自然科学基金面上项目(2023NSFSC0674)

作者(Author): 钟青芮,黄虹凯,兰悦嘉,王欢,曾勇,吴嘉思
ZHONG Qing-rui,HUANG Hong-kai,LAN Yue-jia,WANG Huan,ZENG Yong,WU Jia-si

DOI: 10.19540/j.cnki.cjcmm.20250428.503

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