木犀草素抑制丙酮酸铁氧还蛋白氧化还原酶及其抗艰难梭菌作用研究  

Study on the Inhibitory of Pyruvate-Ferredoxin Oxidoreductase by Luteolin and Its Anti-Clostridium Difficile Effect

作　　者：肖丽君 赵文静 齐晓怡 吕沐瀚 梁思成 XIAO Lijun;ZHAO Wenjing;QI Xiaoyi;LYU Muhan;LIANG Sicheng(Department of Gastroenterology,The Affiliated Hospital of Southwest Medical University,Luzhou 646000 Sichuan,China;Department of Dermatology,The Affiliated Hospital of Southwest Medical University,Luzhou 646000 Sichuan,China.)

机构地区：[1]西南医科大学附属医院消化内科,四川泸州646000 [2]西南医科大学附属医院皮肤科,四川泸州646000

出　　处：《中药新药与临床药理》2024年第2期230-236,共7页Traditional Chinese Drug Research and Clinical Pharmacology

基　　金：国家自然科学基金项目(81672458,82003850);四川省科技规划项目(2021JDTD0003,2022NSFSC0576)。

摘　　要：目的考察木犀草素对丙酮酸铁氧还蛋白氧化还原酶(PFOR酶)的抑制作用及其抗艰难梭菌活性。方法将艰难梭菌PFOR编码序列克隆至表达载体pET-2a,转染至感受态大肠杆菌,异丙基-β-D-硫代吡喃半乳糖苷(Isopropylβ-D-Thiogalactoside,IPTG)诱导表达后进行粗酶液制备。40μmol·L^(-1)待测化合物与PFOR酶在厌氧25℃条件反应8 h后,测定待测化合物对PFOR酶的抑制率。通过对菌液OD_(600)的考察,测定PFOR酶强抑制剂对艰难梭菌细菌(ATCC BAA 1382和ATCC BAA 1870)的最小抑菌浓度(MIC)。借助分子对接技术考察PFOR-抑制剂间可能的相互作用机制。结果在所测化合物中,黄酮木犀草素对PFOR的抑制活性最强,单点抑制率约为33%,与阳性抑制剂硝唑尼特的抑制率(40%)相当。分子对接发现木犀草素可与PFOR结构域中的Asp428、Val431、Gly429、Asp456、Lys458、Lys459等形成氢键。木犀草素对艰难梭菌的MIC约为32μg·mL^(-1)。结论木犀草素具有较好的抗艰难梭菌活性,PFOR酶可能是其抗菌作用靶点。Objective To investigate the inhibitory effects of pyruvate-ferredoxin oxidoreductase(PFOR)by luteolin and its anti-Clostridium difficile effect.Methods The PFOR encoding sequence of Clostridium difficile was cloned into the expression vector pET-2a and transformed into competent Escherichia coli.The crude enzyme was prepared after induction with IPTG(Isopropylβ-D-Thiogalactoside).The inhibitory rate of the test compounds on PFOR was determined after an 8-hour anaerobic reaction between PFOR and 40μmol·L^(-1)of test compounds at 25℃.The minimum inhibitory concentration(MIC)of PFOR inhibitors against C.difficile strains(ATCC BAA 1382 and ATCC BAA 1870)was determined by monitoring the OD_(600)of the bacterial culture.Molecular docking was performed to investigate the possible interaction mechanisms between PFOR and inhibitors.Results Among the tested compounds,the luteolin showed the strongest inhibitory activity against PFOR,with a single-point inhibition rate of approximately 33%,which is comparable to that observed with the positive inhibitor nitazoxanide(40%).Molecular docking revealed that luteolin could form hydrogen bonds with Asp428,Val431,Gly429,Asp456,Lys458,Lys459,and other residues in the PFOR domain.The MIC of luteolin against C.difficile was approximately 32μg·mL^(-1).Conclusion Luteolin exhibits good activity against C.difficile,and PFOR may be a target for its antibacterial action.

关 键 词：木犀草素 丙酮酸铁氧还蛋白氧化还原酶 艰难梭菌 PFOR酶 

分 类 号：R285.5[医药卫生—中药学]