| 杜仲抗菌肽EuCHIT1对白色念珠菌的抑菌作用及机制研究 |
| 投稿时间：2019-12-28 点此下载全文
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| 引用本文：赵丹,张湘燕,罗振华,董旋,赵德刚.杜仲抗菌肽EuCHIT1对白色念珠菌的抑菌作用及机制研究[J].中国现代中药,2020,22(12):1991-1995 |
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| 基金项目:国家自然科学基金项目（31360272）；贵州省中医药管理局中医药、民族医药科学技术研究项目（QZYY2017-025）；贵州省特色植物种质资源利用与创新人才基地项目（RCJD2018-14） |
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| 中文摘要:目的：研究杜仲抗菌肽EuCHIT1对白色念珠菌的抗菌活性及机制。方法：通过聚合酶链式反应（PCR）扩增EuCHIT1蛋白的编码区，构建原核表达质粒并转化到大肠杆菌Escherichia coli BL21（DE3），经异丙基硫代半乳糖苷（IPTG）诱导，采用镍亲和色谱法纯化得到可溶性的EuCHIT1；采用二倍最小抑菌浓度（MIC）法稀释EuCHIT1并测定其对白色念珠菌的抑菌圈直径；采用荧光分光光度计和酶标仪分别检测EuCHIT1对白色念珠菌细胞膜的去极化作用及对细胞膜通透性的影响；通过鲎试验动态浊度法和高效液相色谱法分别测定白色念珠菌中（1，3）-β-D-葡聚糖和麦角甾醇含量；酶联免疫吸附法（ELISA）检测EuCHIT1对白色念珠菌感染的SD大鼠肺上皮WTRL1细胞中白细胞介素-1β（IL-1β）、IL-6及肿瘤坏死因子-α（TNF-α）表达的影响。结果：纯化得到的杜仲抗菌肽EuCHIT1相对分子质量为77 210，对白色念珠菌MIC值为32 μg·mL-1，EuCHIT1质量浓度与抑菌活性之间具有量效关系；EuCHIT1可增加白色念珠菌细胞壁的去极化及通透性，降低白色念珠菌细胞壁（1，3）-β-D-葡聚糖及麦角甾醇的含量，同时EuCHIT1可降低白色念珠菌感染的WTRL1细胞中IL-6及TNF-α水平（P<0.05）。结论：EuCHIT1对白色念珠菌具有抑菌作用，主要机制可能是破坏细胞壁，导致细胞膜去极化、通透性增加，并降低白色念珠菌感染的肺上皮细胞IL-6及TNF-α水平。 |
| 中文关键词:杜仲 抗菌肽EuCHIT1 白色念珠菌 抑菌 机制 |
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| Antibacterial Effect and Mechanism of Antimicrobial Peptides EuCHIT1 from Eucommia ulmoides on Candida albicans |
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| Abstract:Objective：To study the antibacterial activity and mechanism of Eucommia ulmoides EuCHIT1 against Candida albicans. Methods：The coding region of EuCHIT1 was amplified by polymerase chain reaction （PCR），the prokaryotic expression plasmid was constructed and transformed into Escherichia coli BL21 （DE3）. The soluble EuCHIT1 protein was purified by nickel affinity chromatography after isopropyl thiogalactoside （IPTG） induction. EuCHIT1 was diluted with double minimum inhibitory concentration （MIC）. The inhibition zone diameter of EuCHIT1 at different concentration against C.albicans was determined. The depolarization effect of EuCHIT1 on the cell membrane of C.albicans and its influence on the membrane permeability were determined by fluorescence spectrophotometer and microplate reader respectively. The contents of （1，3）-β-D-glucan and ergosterol in C.albicans were determined by limulus limulus kinetic turbidimetry and high performance liquid chromatography respectively. The effects of EuCHIT1 on the expression of interleukin-1β （IL-1β），IL-6 and tumor necrosis factor-α （TNF-α） in WTRL1 cells of SD rats infected with C.albicans were detected by Enzyme-linked immunosorbent assay （ELISA）. Results：The molecular weight of purification of E.ulmoides antimicrobial peptides EuCHIT1 was 77 210，the MIC value against C.albicans was 32 μg·mL-1. There was a dose-activity relationship between EuCHIT1 concentration and antibacterial activity. E.ulmoides antimicrobial peptides EuCHIT1 increased depolarization and permeability of C.albicans cell walls，and decreased contents of （1，3）-β-D-glucan and ergosterol in C.albicans cell walls，and decreased the levels of IL-6 and TNF-α in lung epithelial cells of SD rats infected with C.albicans （P<0.05）. Conclusion：EuCHIT1 has antibacterial effect on C.albicans. The main mechanism may be the destruction of cell wall，resulting in depolarization and increase permeability of cell membrane，and the reduction of IL-6 and TNF-α levels in SD rat lung epithelial cells. |
| keywords:Eucommia ulmoides Oliv. antimicrobial peptides EuCHIT1 Candida albicans bacteriostatic mechanism |
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