基于NLRP3/Caspase-1/GSDMD通路探讨木犀草素对LPS/ATP诱导的细胞焦亡的保护机制  

Exploring protective mechanism of luteolin against LPS/ATP-induced pyroptosis based on NLRP3/Caspase-1/GSDMD pathway

作　　者：刘双慧 张衍坤 赵毅[1,2] 贺常亮[2,3] LIU Shuanghui;ZHANG Yankun;ZHAO Yi;HE Changliang(Department of Basic Veterinary,College of Veterinary Medicine,Sichuan Agricultural University,Chengdu 611134,China;Natural Medicine Research Center,College of Veterinary Medicine,Sichuan Agricultural University,Chengdu 611134,China;Department of Clinical Veterinary,College of Veterinary Medicine,Sichuan Agricultural University,Chengdu 611134,China)

机构地区：[1]四川农业大学动物医学院基础兽医系,四川成都611134 [2]四川农业大学动物医学院天然药物研究中心,四川成都611134 [3]四川农业大学动物医学院临床兽医系,四川成都611134

出　　处：《中草药》2025年第6期1989-1998,共10页Chinese Traditional and Herbal Drugs

基　　金：国家自然科学基金面上项目(32273047)。

摘　　要：目的基于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)/半胱氨酸天冬氨酸蛋白酶-1(cystein-asparate protease-1,Caspase-1)/消皮素D(gasdermin D,GSDMD)通路研究木犀草素对脂多糖(lipopolysaccharide,LPS)联合三磷腺苷(adenosine triphosphate,ATP)诱导的小鼠单核巨噬细胞J774A.1焦亡的作用及其机制。方法利用分子对接技术预测木犀草素与焦亡相关蛋白结合的可能性。将对数生长期的小鼠单核巨噬细胞J774A.1细胞分为对照组、LPS+ATP组、木犀草素(8μmol/L)组和木犀草素低、中、高(2、4、8μmol/L)+LPS+ATP组。采用LPS+ATP诱导J774A.1细胞焦亡,采用木犀草素预防治疗12 h。通过碘化丙啶(propidium iodide,PI)染色、乳酸脱氢酶(lactate dehydrogenase,LDH)释放和细胞计数(cell counting kit-8,CCK-8)法检测细胞活力和细胞膜损伤,利用ELISA法测定白细胞介素-1β(interleukin-1β,IL-1β)和IL-18的水平。采用Western blotting检测细胞中NLRP3、凋亡相关颗粒样蛋白(apoptosis-associated speck-like protein containing a CARD,ASC)、Caspase-1、GSDMD的蛋白表达。同时,将ICR雄性小鼠随机分为对照组、LPS(50 mg/kg)组、木犀草素(80 mg/kg)组和木犀草素低、中、高剂量(40、60、80 mg/kg)+LPS(50 mg/kg)组,进行木犀草素干预LPS诱导小鼠脾脏细胞焦亡的体内实验,检测血清中IL-1β、IL-18的水平和脾脏中NLRP3、ASC、Caspase-1、GSDMD的表达。结果分子对接结果表明,木犀草素与细胞焦亡相关蛋白Caspase-1、NLRP3、ASC、GSDMD的结合良好,结合能均小于−5 kcal/mol。体外实验表明,经过木犀草素预处理(2~8μmol/L)12 h能显著提高LPS+ATP诱导的J774A.1细胞活力(P<0.05),显著减少PI染色阳性率和LDH释放(P<0.05),显著降低IL-1β和IL-18水平(P<0.01),并显著下调NLRP3、Caspase-1 p10及GSDMD p30的蛋白表达(P<0.01)。体内实验表明,木犀草素可以显著降低小鼠血清中IL-1β和IL-18水平(P<0.01),并�Objective To clarify the protective effect of Guilingji on the liver of mild cognitive impairment(MCI)rats,and reveal its potential mechanism based on metabolomics technology.Methods Sixty male SD rats were randomly divided into control group,model group,Guilingji high-dose group(150 mg/kg),Guilingji low-dose group(75 mg/kg),Ginkgo leaf tablets group(7.2 mg/kg)and donepezil group(0.625 mg/kg).The MCI rats was established by D-galactose combined with semi-high fat diet.Liver index,the level of glutamic-pyruvic transaminase(ALT),aspartate aminotransferase(AST)and alkaline phosphatase(ALP)were detected in each group,and hematoxylin eosin staining(HE)were used to observe the pathological changes of liver tissue.Based on metabolomics,differential metabolites were screened and metabolic pathways were analyzed.Results Compared with the rats in the control group,the rats in the model group had pathological liver injury,accompanied by a significant decrease in liver index,and the level of liver function indicators including ALT and AST were increased significantly(P<0.05,0.01),the level of ALP was decreased significantly(P<0.05);Compared with the model group,the high-dose Guilingji group and the Ginkgo leaf tablets group showed significant reductions in AST and ALT levels in rats(P<0.05,0.01),and the low-dose Guilingji group exhibited a significant decrease in ALT levels(P<0.01),donepezil had no significant effect on the liver function indexes.The liver metabolomics identified 23 differential metabolites,all of which could be significantly reversed by Guilingji.The main metabolic pathways included purine metabolism,Dglutamine and D-glutamate metabolism,arginine biosynthesis,alanine,aspartate and glutamate metabolism,phenylalanine,tyrosine and tryptophan biosynthesis.Conclusion Guilingji has a significant protective effect on the liver of MCI rats,and the effect is better than that of Ginkgo leaf tablets and donepezil.The mechanism is closely related to the regulation of amino acid metabolism and lipid metabolism.

关 键 词：细胞焦亡 木犀草素 NOD样受体热蛋白结构域相关蛋白3 半胱氨酸天冬氨酸蛋白酶-1 消皮素D 

分 类 号：R285.5[医药卫生—中药学]