1、第 29 卷第 5 期2023年 3 月Vol.29,No.5Mar.,2023中国实验方剂学杂志Chinese Journal of Experimental Traditional Medical Formulae八宝丹通过抑制 NLRP3/Caspase-1通路减轻对乙酰氨基酚诱导的小鼠急性肝损伤赵若玮1,张庆1,朱铭星2,刘跃洋3,程再兴1,黄鸣清1,郑燕芳1*,林彦翔1*(1.福建中医药大学 药学院,福州 350122;2.福建中医药大学 中医学院,福州 350122;3.福建中医药大学 针灸学院,福州 350122)摘要 目的:研究八宝丹(BBD)对对乙酰氨基酚(APAP)诱导的急
2、性肝损伤小鼠 NOD 样受体热蛋白结构域相关蛋白 3/胱天蛋白酶-1(NLRP3/Caspase-1)通路蛋白的影响。方法:将 C57BL/6 小鼠随机分组,BBD(75、150、300 mg kg-1)灌胃给药3 d,2次/d,于末次给药后 2 h,腹腔注射 APAP(400 mg kg-1),14 h后摘眼球取血,随后脱颈椎处死取材。苏木素-伊红(HE)染色法观察肝组织细胞形态学变化;生化法检测各组小鼠血清中丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、超氧化物歧化酶(SOD)、丙二醛(MDA)和髓过氧化物酶(MPO)的活性;实时荧光定量聚合酶链式反应(Real-time P
3、CR)检测小鼠肝脏中肿瘤坏死因子-(TNF-)、白细胞介素-1(IL-1)及白细胞介素-6(IL-6)mRNA 表达水平;蛋白免疫印迹法(Western blot)检测小鼠肝脏中环氧化酶-2(COX-2)、诱导型一氧化氮合酶(iNOS)、NLRP3、Caspase-1、白细胞介素-18(IL-18)的蛋白表达水平。结果:与正常组比较,模型组小鼠肝脏组织小叶结构部分破坏、肝窦扩张,血清中 ALT和 AST水平,肝组织中 NLRP3、Caspase-1、iNOS、IL-18、COX-2蛋白表达水平和 IL-1、IL-6、TNF-mRNA 水平均明显升高(P0.05,P0.01)。与模型组比较,各给
4、药组小鼠肝细胞破裂现象有所好转,肝血窦挤压情况有所缓解,血清中 ALT 和 AST 水平,肝组织中 NLRP3、Caspase-1、iNOS、IL-18、COX-2 蛋白表达水平和 IL-1、IL-6、TNF-mRNA 水平均明显降低(P0.05,P0.01),并呈剂量依赖性。结论:BBD 可减轻 APAP 诱导的小鼠急性肝损伤,其机制可能与抗氧化应激,抑制 NLRP3/Caspase-1 通路,进而降低 IL-1、IL-18、TNF-、IL-6表达水平有关。关键词 八宝丹;急性肝损伤;炎症因子;NOD样受体热蛋白结构域相关蛋白 3(MRP3);胱天蛋白酶-1(Caspase-1)中图分类号
5、R2-0;R22;R285.5;R289;R33 文献标识码 A 文章编号 1005-9903(2023)05-0122-07doi 10.13422/ki.syfjx.20222001 网络出版地址 https:/ 2022-11-02 10:54:42Babaodan Alleviates APAP-induced Acute Liver Injury in Mice by Inhibiting NLRP3/Caspase-1 PathwayZHAO Ruowei1,ZHANG Qing1,ZHU Mingxing2,LIU Yueyang3,CHENG Zaixing1,HUANG Mi
6、ngqing1,ZHENG Yanfang1*,LIN Yanxiang1*(1.College of Pharmacy,Fujian University of Traditional Chinese Medicine(TCM),Fuzhou 350122,China;2.College of Traditional Chinese Medicine,Fujian University of TCM,Fuzhou 350122,China;3.College of Acupuncture and Moxibustion,Fujian University of TCM,Fuzhou 3501
7、22,China)Abstract Objective:To explore the effect of Babaodan(BBD)on the NOD-like receptor pyrin domain containing 3/cysteine aspartate-specific protease-3(NLRP3/Caspase-1)pathway proteins in mice with acetaminophen(APAP)-induced acute liver injury.Method:C57BL/6 mice were randomly grouped,and BBD 收
8、稿日期 2022-08-05基金项目 国家自然科学基金项目(81973437)第一作者 赵若玮,在读硕士,从事中药药理研究,E-mail:通信作者 *郑燕芳,博士,副教授,从事中药药理研究,Tel:0591-22861335,E-mail:;*林彦翔,博士,副教授,从事药用植物与分子生药研究,Tel:0591-22861283,E-mail:122第 29 卷第 5 期2023年 3 月中国实验方剂学杂志Chinese Journal of Experimental Traditional Medical FormulaeVol.29,No.5Mar.,2023(75,150,300 mg k
9、g-1,ig)was administered twice a day for three days.After 2 hours of the last administration,the mice were treated with APAP(400 mg kg-1,ip),and the eyeballs were removed to collect blood after 14 hours.Then they were sacrificed by cervical dislocation for sample collection.Hematoxylin-eosin(HE)stain
10、ing was used to observe the morphological changes of liver tissue cells,and biochemical methods were used to detect the activities of alanine aminotransferase(ALT),aspartate aminotransferase(AST),superoxide dismutase(SOD),malondialdehyde(MDA)and myeloperoxidase(MPO)in serum of mice in each group.Rea
11、l-time fluorescence quantitative polymerase chain reaction(Real-time PCR)was performed to determine the mRNA expression of tumor necrosis factor-(TNF-),interleukin-1(IL-1)and IL-6,and Western blot was performed to determine the protein expression of cyclooxygenase-2(COX-2),inducible nitric oxide syn
12、thase(iNOS),NLRP3,Caspase-1 and IL-18 in the liver of mice.Result:Compared with the conditions in normal group,the hepatic lobule structure of mice in the model group was partially destroyed,and the hepatic sinusoids were dilated.And the expression levels of ALT and AST in serum,the protein levels o
13、f NLRP3,Caspase-1,iNOS,IL-18 and COX-2 and the mRNA levels of IL-1,IL-6 and TNF-were increased(P0.05,P0.01).Compared with the model group,the administration groups had improvement in liver cell rupture and hepatic sinusoidal compression,and a dose-dependent decrease in the levels of ALT and AST in s
14、erum as well as the protein levels of NLRP3,Caspase-1,iNOS,IL-18 and COX-2 and the the mRNA levels of IL-1,IL-6 and TNF-in liver tissue(P0.05,P0.01).Conclusion:BBD can reduce APAP-induced acute liver injury in mice.The mechanism may be related to anti-oxidative stress,inhibition of NLRP3/Caspase-1 p
15、athway,and decreased expression levels of IL-1,IL-18,TNF-and IL-6.Keywords Babaodan;acute liver injury;inflammatory factors;NOD-like receptor pyrin domain containing 3(NLRP3);cysteine aspartate-specific protease-3(Caspase-1)急性肝损伤(ALI)是指由药物中毒、酒精摄入过多等各种不同诱因引起的,肝功能短期突发异常的疾病,急性肝损伤在我国的发病率呈逐年上升的趋势。严重的急性肝损
16、伤还会转变为急性肝衰竭(ALF),导致肝细胞大量死亡,肝功能迅速恶化,病患死亡率极高。对乙酰氨基酚(APAP)别称扑热息痛,可以减少前列腺素 E1(PGE1)的合成和释放,进而通过扩张外周血管来起到解热的效果,临床上常用于解热镇痛。然而,APAP 存在于多种非处方的复方药物中,容易导致过量服用,如果日剂量超过4 g就可致急性肝损伤,更大剂量会导致急性肝衰竭甚至死亡。APAP 进入体内,小部分由细胞色素P4502E1(CYP2E1)代谢,形成活性毒性代谢产物N-乙酰基-对苯醌亚胺(NAPQI),NAPQI 优先与肝脏中谷胱甘肽(GSH)结合形成无毒半胱氨酸和酸醚氨酸衍生物,从而解除毒性。过量服用 APAP 会产生大量 NAPQI,使肝脏中谷胱甘肽耗竭,再与肝细胞中蛋白质结合,导致肝细胞死亡。APAP 过量服用还会刺激致炎因子和炎症介质的产生,从而进一步加重肝脏损伤。因而 APAP成为诱发药物性急性肝损伤的主要原因之一1-2。同时,临床上唯一使用的 APAP 解毒剂 N-乙酰-L-半胱氨酸(NAC)具有局限性,其治疗窗窄,对于 APAP中毒超过 24 h的患者,NAC 已经失去了解毒功能;