基于多途径联合效应的甘草防治脓毒血症活性成分筛选及作用机制研究  

Screening and mechanism study of active ingredients of Glycyrrhizae Radix et Rhizoma in prevention and treatment of sepsis based on combined effects of multiple pathways

作　　者：梁龙鑫 任璐彤 刘婷婷 高源 徐广 肖小河 柏兆方 LIANG Long-xin;REN Lu-tong;LIU Ting-ting;GAO Yuan;XU Guang;XIAO Xiao-he;BAI Zhao-fang(School of Pharmacy,Jiangxi University of Traditional Chinese Medicine,Nanchang 330004,China;Department of Liver Medicine,Fifth Medical Center,PLA General Hospital,Beijing 100039,China;Department of Pharmacy,People’s Hospital of Inner Mongolia Autonomous Region,Hohhot 010010,China;Third Affiliated Hospital of Zunyi Medical University(Zunyi First People’s Hospital),Zunyi 563002,China;College of Traditional Chinese Medicine,Capital Medical University,Beijing 100069,China)

机构地区：[1]江西中医药大学药学院,江西南昌330004 [2]解放军总医院第五医学中心肝病医学部,北京100039 [3]内蒙古自治区人民医院药学处,内蒙古呼和浩特010010 [4]遵义医科大学第三附属医院(遵义市第一人民医院),贵州遵义563002 [5]首都医科大学中医药学院,北京100069

出　　处：《中草药》2023年第11期3524-3533,共10页Chinese Traditional and Herbal Drugs

基　　金：国家中医药传承创新团队项目(ZYYCXTD-C-202005);国家自然科学基金重点项目(81930110);国家自然科学基金青年科学基金项目(81903891);国家自然科学基金创新研究群体项目(81721002)。

摘　　要：目的研究甘草中有效组分对脓毒血症的防治作用及机制。方法采用小鼠骨髓来源巨噬细胞(bone marrow-derived macrophages,BMDM)构建体外NOD样受体热蛋白结构域相关蛋白3(NOD-like receptor thermal protein domain associated protein 3,NLRP3)炎症小体激活模型及H2O2诱导的氧化应激模型筛选出甘草抗炎、抗氧化应激的有效组分,采用ip脂多糖(lipopolysaccharide,LPS,20 mg/kg)诱导C57BL/6小鼠脓毒血症致死模型以及脓毒血症模型,评价致死模型中小鼠的生存率及脓毒血症模型小鼠腹腔灌洗液中巨噬细胞和中性粒细胞在渗出细胞中的占比以及外周血和腹腔灌洗液中白细胞介素-1β(interleukin-1β,IL-1β)及肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)水平。结果基于BMDM细胞NLRP3炎症小体激活模型筛选出甘草中有效组分刺甘草查耳酮,可显著抑制半胱氨酸天冬氨酸蛋白酶-1(cystein-asparate protease-1,Caspase-1)p20的蛋白表达及IL-1β的释放(P<0.05、0.001)。基于H2O2诱导的BMDM细胞氧化应激模型筛选出有效组分甘草酸,其可改善H2O2诱导的细胞氧化损伤,显著升高细胞活力和超氧化物歧化酶(superoxide dismutase,SOD)活性(P<0.01、0.001),降低活性氧(reactive oxygen species,ROS)产生(P<0.001)。基于LPS诱导的脓毒血症致死模型发现,甘草酸高剂量组及刺甘草查耳酮低、高剂量组和联合组均可显著升高小鼠生存率(P<0.05、0.001),并且联合用药组的效果优于甘草酸组和刺甘草查耳酮低剂量组。在脓毒血症小鼠模型中,刺甘草查耳酮和甘草酸均可降低巨噬细胞和中性粒细胞在渗出细胞中的占比(P<0.001),降低外周血及腹腔灌洗液中IL-1β和TNF-α的水平(P<0.01、0.001),同时联合组与单药组相比体现出更佳的作用效果(P<0.05、0.01、0.001)。结论甘草活性成分刺甘草查耳酮与甘草酸联用可抑制BMDM细胞NLRP3炎症小体激活和氧化应激,从而多途径地发挥较强的防治脓毒血Objective To explore the effect and mechanism of effective components in Gancao(Glycyrrhizae Radix et Rhizoma)on the prevention and treatment of sepsis.Methods The effective components of anti-inflammation and anti-oxidant stress in Glycyrrhizae Radix et Rhizoma were screened by NOD-like receptor thermal protein domain associated protein 3(NLRP3)inflammasome activation model and in vitro oxidative stress model induced by H2O2 in bone marrow-derived macrophages(BMDM).The mortality model of C57BL/6 mice induced by lipopolysaccharide(LPS,20 mg/kg)and sepsis model were used to evaluate the survival rate of mice in mortality model,the proportions of macrophages and neutrophils in exudative cells in peritoneal lavage fluid,interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α)levels in peripheral blood and peritoneal lavage fluid of sepsis model mice.Results Based on NLRP3 inflammasome activation model in BMDM cells,the effective component(echinatin)of Glycyrrhizae Radix et Rhizoma was screened out,which significantly inhibited the protein expression of cysteine-aspartate protease-1(Caspase-1)p20 and the release of IL-1β(P<0.05,0.001).Based on H2O2-induced oxidative stress model of BMDM cells,glycyrrhizic acid was screened out,which improved H2O2-induced oxidative damage of cells,significantly increased cell viability and superoxide dismutase(SOD)activity(P<0.01,0.001),and reduced the production of reactive oxygen species(ROS)(P<0.001).Based on the death model of sepsis induced by LPS,it was found that glycyrrhizic acid high-dose group,echinatin low-,high-dose groups and the combined group significantly improved the survival rate of mice(P<0.05,0.001),and the effect of the combined drug group was better than that of glycyrrhizic acid group and echinatin low-dose group.In the mouse model of sepsis,both echinatin and glycyrrhizic acid reduced the proportions of macrophages and neutrophils in exudative cells(P<0.001),reduced the levels of IL-1βand TNF-αin peripheral blood and peritoneal lavage fluid(P<0.01,0.001).At

关 键 词：甘草 组分中药 联合增效 刺甘草查耳酮 甘草酸 脓毒血症 

分 类 号：R285.5[医药卫生—中药学]