微生物学报

Gene	Primer sequences (5′→3′)	Product length (bp)	Function of gene product	References
arcA	F: ATGCAGACCCCGCACATTC R: TTAATCTTCCAGATCACCGCAGA	717	Anoxic redox control, mediating the metabolic transition from aerobiosis to anaerobiosis	[34-37]
fnr	F: TCAGGCAACGTTACGCGTATG R: ATGATCCCGGAAAAGCGAA	753	Global transcription factor, managing the distribution of RNA polymerase in response to oxygen starvation	[38-39]
fdnH	F: ATGGCTATGGAAACGCAGGA R: TTACTCATGATGATCCTCCTCGTC	885	Functions in the formate-nitrate respiratory chain, rich of Fe-S cluster	[40-41]
soxS	F: ATGTCCCATCAGAAAATTATTCAGG R: TTACAGGCGGTGGCGATAAT	324	Activator of superoxide stress	[42-43]
sucB	F: ATGAGTAGCGTAGATATTCTGGTCCC R: CTACACGTCCAGCAGCAGACG	1 218	Alpha-ketoglutarate dehydrogenase, TCA cycle enzyme	[44]
gapA	F: ATGACTATCAAAGTAGGTATCAACGGTT R: TTATTTGGAGATGTGAGCGATCA	996	GADPH, universal reference sequences	[45]

Gene	Primer sequences (5′→3′)	Product length (bp)	Function of gene product	References
arcA	F: ATGCAGACCCCGCACATTC R: TTAATCTTCCAGATCACCGCAGA	717	Anoxic redox control, mediating the metabolic transition from aerobiosis to anaerobiosis	[34-37]
fnr	F: TCAGGCAACGTTACGCGTATG R: ATGATCCCGGAAAAGCGAA	753	Global transcription factor, managing the distribution of RNA polymerase in response to oxygen starvation	[38-39]
fdnH	F: ATGGCTATGGAAACGCAGGA R: TTACTCATGATGATCCTCCTCGTC	885	Functions in the formate-nitrate respiratory chain, rich of Fe-S cluster	[40-41]
soxS	F: ATGTCCCATCAGAAAATTATTCAGG R: TTACAGGCGGTGGCGATAAT	324	Activator of superoxide stress	[42-43]
sucB	F: ATGAGTAGCGTAGATATTCTGGTCCC R: CTACACGTCCAGCAGCAGACG	1 218	Alpha-ketoglutarate dehydrogenase, TCA cycle enzyme	[44]
gapA	F: ATGACTATCAAAGTAGGTATCAACGGTT R: TTATTTGGAGATGTGAGCGATCA	996	GADPH, universal reference sequences	[45]

Strains	Silver nanoparticles	MIC (μg/mL)	MBC (μg/mL)	MBC/MIC
Escherichia coli BW25113	PVP-AgNPs	12.50	25.0	2.0
PVP-AgNPs@AKG	6.25	12.5	2.0
E.coli 8099	PVP-AgNPs	12.50	25.0	2.0
PVP-AgNPs@AKG	6.25	12.5	2.0
Staphylococcus aureus ATCC6538	PVP-AgNPs	25.00	50.0	2.0
PVP-AgNPs@AKG	12.50	25.0	2.0
Pseudomonas aeruginosa 1118	PVP-AgNPs	50.00	＞100.0	＞2.0
PVP-AgNPs@AKG	25.00	50.0	2.0
Acinetobacter baumnnii TY-19	PVP-AgNPs	50.00	100.0	2.0
PVP-AgNPs@AKG	25.00	100.0	4.0

Strains	Silver nanoparticles	MIC (μg/mL)	MBC (μg/mL)	MBC/MIC
Escherichia coli BW25113	PVP-AgNPs	12.50	25.0	2.0
PVP-AgNPs@AKG	6.25	12.5	2.0
E.coli 8099	PVP-AgNPs	12.50	25.0	2.0
PVP-AgNPs@AKG	6.25	12.5	2.0
Staphylococcus aureus ATCC6538	PVP-AgNPs	25.00	50.0	2.0
PVP-AgNPs@AKG	12.50	25.0	2.0
Pseudomonas aeruginosa 1118	PVP-AgNPs	50.00	＞100.0	＞2.0
PVP-AgNPs@AKG	25.00	50.0	2.0
Acinetobacter baumnnii TY-19	PVP-AgNPs	50.00	100.0	2.0
PVP-AgNPs@AKG	25.00	100.0	4.0

Silver nanoparticles	Concentration (μg/mL)	The amount of silver^* (μg)		The ratio of silver (%)
^：计算为每10⁶ CFUE.coli中的总银量 ^: Calculated as the total amount of silver per 10⁶ CFUEscherichia coli.
PVP-AgNPs	6.25	2.70±0.01	2.92±0.02		15.37	16.60
12.50	2.85±0.02	3.29±0.02		8.11	9.35
PVP-AgNPs@AKG	6.25	2.63±0.01	2.76±0.03		14.93	15.67
12.50	2.81±0.02	2.92±0.02		8.00	8.31

Silver nanoparticles	Concentration (μg/mL)	The amount of silver^* (μg)		The ratio of silver (%)
^：计算为每10⁶ CFUE.coli中的总银量 ^: Calculated as the total amount of silver per 10⁶ CFUEscherichia coli.
PVP-AgNPs	6.25	2.70±0.01	2.92±0.02		15.37	16.60
12.50	2.85±0.02	3.29±0.02		8.11	9.35
PVP-AgNPs@AKG	6.25	2.63±0.01	2.76±0.03		14.93	15.67
12.50	2.81±0.02	2.92±0.02		8.00	8.31

靶向α-酮戊二酸脱氢酶增强纳米银的抗菌作用

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何雨婧 , 杜华茂 ^*

微生物学报 | 研究报告 2024,64(7): 2277-2294

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微生物学报 | 研究报告 2024, 64(7): 2277-2294

靶向α-酮戊二酸脱氢酶增强纳米银的抗菌作用

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何雨婧, 杜华茂^*

作者信息

西南大学蚕桑纺织与生物质科学学院, 重庆 400715

Targeting alpha-ketoglutarate dehydrogenase enhances antibacterial activity of silver nanoparticles

Yujing HE, Huamao DU^*

Affiliations

College of Sericulture, Textile and Biomass Sciences, Southwest University, Chongqing 400715, China

出版时间: 2024-07-04 doi: 10.13343/j.cnki.wsxb.20230644

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摘要

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【目的】纳米银(silver nanoparticles, AgNPs)的生物安全性一直受业界诟病，扩大纳米银的治疗窗将为治疗人和动物多耐药性细菌感染提供有效的备选药物。本研究拟用三羧酸循环的重要成员α-酮戊二酸(alpha-ketoglutaric acid, AKG)对纳米银进行表面修饰以提高其抗菌的生物安全性。【方法】芦丁在常温下合成纳米银，用全波长分光光度计、粒度仪及透射电镜进行表征。加1 mmol/L聚乙烯吡咯烷酮(polyvinylpyrrolidone, PVP)作为稳定剂(PVP-AgNPs)，另加10 mmol/L AKG作为封端剂(PVP-AgNPs@AKG)，比较2种纳米银的抗菌性及对人正常宫颈上皮细胞(human cervical epithelial cells, HCerEpic)的毒性作用，再分析2种纳米银对大肠杆菌(Escherichia coli) BW25113能量代谢、抗氧化应激和无氧呼吸相关基因表达等的影响。【结果】PVP-AgNPs@AKG对多株革兰阳性细菌和革兰阴性细菌的最小抑菌浓度(minimal inhibit concentration, MIC)和最低杀菌浓度(minimum bactericidal concentration, MBC)均比PVP-AgNPs低50%或50%以上，而对HCerEpic细胞的毒性无显著差异。与PVP-AgNPs相比，PVP-AgNPs@AKG在MIC浓度下对E.coli α-酮戊二酸脱氢酶活性的抑制作用增强，AKG蓄积，ATP水平显著降低，同时活性氧(reactive oxygen species, ROS)的水平显著升高，soxS表达上调，但是，厌氧呼吸相关的arcA、fnr、fdnH基因表达上调的程度显著降低。【结论】AKG修饰纳米银能通过靶向α-酮戊二酸脱氢酶抑制细菌的能量代谢，使其对氧化损伤更敏感，从而获得更强的抗菌能力，是一种扩大纳米银治疗窗的有效手段。

关键词

纳米银 / α-酮戊二酸 / 抗菌作用 / 治疗窗

Abstract

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[Objective] The biosafety of silver nanoparticles (AgNPs) has been a subject of concern due to the narrow therapeutic window. Expanding the therapeutic window could facilitate the application of AgNPs in the treatment of multi-drug resistant bacterial infections in humans and animals. This study aimed to enhance the biosafety of AgNPs by modifying their surface with alpha-ketoglutaric acid (AKG), a crucial component of the tricarboxylic acid cycle. [Methods] Silver ion was reduced to AgNPs by rutin at room temperature, and then AgNPs were stabilized with 1 mmol/L polyvinylpyrrolidone (PVP) solution to generate PVP-AgNPs. AKG (10 mmol/L) was added to generate PVP-AgNPs@AKG. The prepared AgNPs were characterized by a full-wavelength spectrophotometer, a particle size analyzer, and a transmission electron microscope. The antibacterial activities of PVP-AgNPs and PVP-AgNPs@AKG were evaluated based on minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), time-kill curve, and post-antibiotic effect. The cytotoxicity of the prepared AgNPs to human cervical epithelial cells (HCerEpic) was examined by the MTT assay and flow cytometry. Furthermore, the effects of the prepared AgNPs on the energy metabolism, oxidative stress, and expression of genes involved in anaerobic respiration ofEscherichia coli BW25113 were studied. [Results] The MIC and MBC of PVP-AgNPs@AKG against Gram-positive and Gram-negative bacteria were 50% or above 50% lower than those of PVP-AgNPs. PVP-AgNPs@AKG and PVP-AgNPs showed no significant difference in the cytotoxicity to HCerEpic cells. Compared with PVP-AgNPs, PVP-AgNPs@AKG at the MIC showed significantly enhanced inhibitory effect on the α-ketoglutarate dehydrogenase inEscherichia coli, increased accumulation of AKG, lowered ATP level, and elevated reactive oxygen species level. Moreover, PVP-AgNPs@AKG significantly up-regulated the expression ofsoxS and down-regulated the expression of genes involved in anaerobic respiration, such asarcA,fnr, andfdnH. [Conclusion] The findings suggested that PVP-AgNPs@AKG disrupted the energy metabolism by targeting α-ketoglutarate dehydrogenase, rending bacteria more vulnerable to oxidative damage. Modifying with AKG would be a potential method to expand the therapeutic window of AgNPs.

Key words

silver nanoparticles / alpha-ketoglutaric acid / antibacterial effect / therapeutic window

引用本文

何雨婧, 杜华茂. 靶向α-酮戊二酸脱氢酶增强纳米银的抗菌作用. 微生物学报, 2024 , 64 (7) : 2277 -2294 . DOI: 10.13343/j.cnki.wsxb.20230644

Yujing HE, Huamao DU. Targeting alpha-ketoglutarate dehydrogenase enhances antibacterial activity of silver nanoparticles[J]. Acta Microbiologica Sinica, 2024 , 64 (7) : 2277 -2294 . DOI: 10.13343/j.cnki.wsxb.20230644

正文

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由于多耐药性细菌日益泛滥，含银制剂又重新受到业界和学界的重视，特别是纳米银(silver nanoparticles, AgNPs)因其强效广谱的抗菌特性和可修饰性得到更深入的研究^[1]。Appapalam等报道AgNPs对多重抗生素耐药性糖尿病足溃疡衍生的细菌分离株具有良好的抗菌效果^[2]，Ismail等报道用AgNPs治愈了多耐药菌引起的皮肤癣^[3]。纳米银水凝胶已应用于烧伤和妇科的感染疾病，对多耐药的肺炎克雷伯菌(Klebsiella pneumoniae)^[4]、铜绿假单胞菌(Pseudomonas aeruginosa)^[5]、单核细胞增生李斯特菌(Listeria monocytogenes)^[6]、白色念珠菌(Candida albicans)^[7]等有明显治疗效果。刺激产生活性氧(reactive oxygen, ROS)是金属纳米粒子和某些抗生素实现抗菌作用的重要机制之一^[8]，除此以外，AgNPs还能与含巯基的酶结合并使之失活，但由于微生物本身已有较为完备的胁迫应对机制，如外排系统^[9]、抗氧化系统^[10]、特异性吸附蛋白^[11]、生物膜^[12]等，在某些条件下仍能对重金属纳米粒子产生耐性。大肠杆菌中的cusABC基因簇编码产物形成外排泵，将进入胞内的Ag⁺、Cu²⁺排出胞外而表现出耐受性^[13]；因Ag⁺产生的抗性也能诱导大肠杆菌对AgNPs产生耐受性^[13-14]。Panáček等研究发现大肠杆菌经亚致死剂量纳米银处理13代后，纳米银对其的最小抑菌浓度(minimum inhibitory concentration, MIC)值从3.38 mg/L升高至108.00 mg/L，对铜绿假单胞菌的MIC也从1.69 mg/L提升到了54.00 mg/L，二者均上升30倍以上^[15]。Gliga等研究发现亚致死水平的Ag⁺和AgNPs暴露改变了细胞膜结构(饱和脂肪酸的顺式/反式异构化)，增强了恶臭假单胞菌对AgNPs的抗性^[16]。Kaweeteerawat等发现细菌暴露于亚致死剂量的AgNPs中，5 d后对AgNPs的耐受性增加，其中大肠杆菌的半数最大抑制浓度(half-maximal inhibitory concentration, IC₅₀)值从11.89 mg/L增加到17.59 mg/L，金黄色葡萄球菌的IC₅₀值从6.98 mg/L增加到18.09 mg/L^[17]。此外，细菌代谢方式发生改变也可能导致其耐受AgNPs，如∆narH单基因缺失株能在含100 μg/mL纳米银(8 MIC值)琼脂平板上生长^[18]。这些发现使纳米银的临床应用不得不慎重考虑。

纳米银的细胞毒性、遗传毒性和神经毒性与其形状、粒径、表面电荷、表面修饰相关，也与细胞的种类、细胞亚类有关，这些数据经常因实验的方案不同而异，但毒副作用的剂量依赖性是一致的^[19-21]，这种毒性作用主要表现为DNA损伤、细胞滞留于G2/M期^[22]。Ćurlin等对小鼠连续28 d复用纳米银(0.1 mg/kg-b.w.)，亚急性毒性试验虽未导致小鼠死亡，但研究发现纳米银在雌鼠脏器的蓄积高于雄鼠，雌鼠的氧化应激反应高于雄鼠^[23]。纳米银的大量生产与广泛应用必然导致大量排放，产生基于蓄积量的环境毒性。纳米银在上述生物安全性方面的问题很大程度上源于治疗窗窄^[24]，如何增强纳米银的抗菌、抗病毒、抗癌作用成为研究热点。

对纳米银的尺寸、形状、表面修饰物或封端剂等进行改造可以增强其抗菌作用。例如Ajitha等利用pH变化控制AgNPs粒径能增强其抗菌活性^[25]，Kumari等通过控制各项物理参数，用生物控制剂绿色木霉合成了不同形状的AgNPs，这些AgNPs展现出了不同的抗菌活性^[26]；用寡核苷酸修饰的AgNPs可加速伤口愈合、抵抗细菌感染^[27]；各类表面修饰能影响AgNPs的生物学活性，但对其影响机制的研究并不明确。

α-酮戊二酸(alpha-ketoglutaric acid, AKG)与柠檬酸同为三羧酸循环(tricarboxylic acid cycle, TCA)的成员，柠檬酸是生成纳米银的还原剂和良好的稳定剂^[28-30]，但并未见其增强纳米银抗菌性能的报道。AKG不仅是连接生物体内碳循环和氮循环的关键物质，同时还能作为α-酮戊二酸依赖性双加氧酶家族的限速底物，是一种具有多效活性的代谢物，参与细菌的多个抗氧化反应过程^[31]。我们推测用AKG修饰纳米银可引导其对AKG参与代谢的重要酶促反应产生干扰或抑制作用。本研究用α-酮戊二酸修饰纳米银以增强其抗菌作用，通过测定时间-杀菌曲线、检测细菌对纳米银的摄取、对几种关键代谢物浓度进行测定，并结合基因表达检测探究了其增强机理，同时为确定其生物安全性，用人正常宫颈上皮细胞(human cervical epithelial cells, HCerEpic)进行了细胞毒性检测。

1 材料与方法

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1.1 主要试剂

芦丁购自生工生物工程(上海)股份有限公司；硝酸银和聚乙烯吡咯烷酮(polyvinylpyrrolidone, PVP)均购自成都市科龙化工试剂厂；α-酮戊二酸购自上海毕得医药科技股份有限公司；3-(4, 5-二甲基噻唑-2)-2, 5-二苯基四氮唑溴盐(3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide, MTT)细胞增殖及细胞毒性检测试剂盒购自上海贝博生物科技有限公司；Annexin V-FITC/PI凋亡检测试剂盒购自北京索莱宝科技有限公司；高纯总RNA快速提取试剂盒购自北京百泰克生物技术有限公司；α-酮戊二酸脱氢酶购自Sigma-Aldrich公司；ATP含量检测试剂盒以及还原型谷胱甘肽含量检测试剂盒均购自金克隆(北京)生物技术有限公司；微生物活性氧簇检测试剂盒购自江苏酶免实业有限公司；细菌α-酮戊二酸脱氢酶活性试剂盒、细菌α-酮戊二酸检测试剂盒以及细菌谷氨酸检测试剂盒均购自上海优选生物科技有限公司。

1.2 菌株和细胞

大肠杆菌(Escherichia coli) K-12 BW25113购自CGSC (the Coli Genetic Stock Center)；E.coli 8099和金黄色葡萄球菌(Staphylococcus aureus) ATCC6538购自上海鲁微科技有限公司；铜绿假单胞菌(Pseudomonas aeruginosa) 1118和鲍曼不动杆菌(Acinetobacter baumannii) TY-19由山西省中医院王晓玲博士惠赠，该2株菌均为临床分离株，对β-内酰胺类、氨基糖苷类、喹若酮类、磺胺类等多种类型的抗生素均不敏感。人正常宫颈上皮细胞(HCerEpic)购自青旗(上海)生物技术发展有限公司。

1.3 纳米银的合成与表征

用芦丁作为还原剂在pH 11.0条件下制备纳米银^[32]，经2次离心去除大颗粒和银离子，加入终浓度为1 mmol/L PVP作为稳定剂。在200 μg/mL PVP-AgNPs胶体溶液中加入20 mmol/L AKG，于37 ℃避光过夜，用去离子水洗涤后得到PVP-AgNPs@AKG。采用超微量分光光度计(ThermoFisher Scientific公司)、透射电子显微镜(FEI公司)、纳米粒度电位仪(Malvern公司)、酸度计[赛多利斯科学仪器(北京)有限公司]对2种纳米银进行表征。

1.4 纳米银的抗菌作用

按文献[33]的方法在96孔板中检测PVP-AgNPs和PVP-AgNPs@AKG对E.coli BW25113、E.coli 8099、S.aureus ATCC6538、P.aeruginosa 1118和A.baumannii TY-19这5株细菌的最小抑菌浓度(MIC)和最低杀菌浓度(minimum bactericidal concentration, MBC)，以及对E.coli BW25113和S.aureus ATCC6538的时间-杀菌曲线和抗生素后效应，其中细菌终浓度均为1×10⁶ CFU/mL，MIC和MBC值的检测中纳米银终浓度依次为100、50、25、12.5、6.25、3.13、1.56、0.78 μg/mL，时间-杀菌曲线和抗生素后效应中对E.coli使用的纳米银浓度统一为6.25 μg/mL，对S.aureus使用的纳米银浓度统一为12.50 μg/mL。每次实验设3个平行重复，实验重复3次。

1.5 纳米银的细胞毒性

将HCerEpic细胞培养至对数生长期(按细胞铺满瓶底的80%计)，PBS洗涤后用DMEM基础培养基重悬细胞，转至96孔细胞培养板，约8 000个细胞/孔(每孔100 μL)，于37 ℃、5% CO₂培养箱中培养18 h，用浓度分别为20、40、80、120 μg/mL的PVP-AgNPs和PVP-AgNPs@AKG处理2 h，弃去含纳米银的培养液并用PBS洗涤1次。按MTT法测定细胞增殖及细胞毒性。

将HCerEpic细胞按1×10⁶个细胞/孔铺板，培养24 h后分别用12.5 μg/mL和50.0 μg/mL的PVP-AgNPs和PVP-AgNPs@AKG处理12 h，按说明书用Annexin V-FITC/PI试剂盒对细胞进行染色，染色完成后在流式细胞仪(BD Biosciences公司)上检测细胞坏死、凋亡情况。

1.6 纳米银释放银离子

将浓度为200、100、50、25、12.5、6.25、3.125、1.563 μg/mL的PVP-AgNPs@AKG溶液加入截流值为10 kDa的超滤管中，4 ℃、3 000×g离心15 min后，按1.4的方法在96孔板中测定超滤前后的PVP-AgNPs@AKGE.coli BW25113的MIC和MBC值。同时测定浓度为200、100、50、25、12.5、6.25、3.125、1.563 μg/mL AgNO₃溶液和浓度为20、15、10、5、2.5 mmol/L的AKG溶液对E.coli BW25113的MIC和MBC值作为对照。

1.7 检测细菌的纳米银摄入量

将4×10⁵ CFU/mLE.coli BW25113用纳米银处理15 min和30 min后，5 000 r/min离心10 min收集菌体，PBS洗涤3次后用1 mL 65%浓硝酸于80 ℃处理2 h，完全消解后用ddH₂O定容至5 mL，经0.22 μm滤膜过滤后使用电感耦合等离子体质谱仪(PerkinElmer公司)检测溶液中的银含量，并换算为纳米银的质量，即为细菌的摄入量。

纳米银进入细菌内部的质量百分比计算公式：百分比(%/×10⁶ CFU)=(测得进入细菌内的纳米银含量×细菌消解液体积)/(纳米银浓度×纳米银体积×菌液浓度×菌液体积)×100。

1.8 代谢物浓度的测定

检测纳米银处理后E.coli BW25113胞内ATP、ROS、AKG以及谷氨酸(Glu)的含量及浓度变化。用6.25 μg/mL和12.50 μg/mL PVP-AgNPs、PVP-AgNPs@AKG于37 ℃处理1 mL 4×10⁵ CFU/mLE.coli BW25113 15 min和30 min后，5 000 r/min离心5 min收集菌体并用PBS洗涤，随后用2 mL超声缓冲液重悬菌体，将样品管置于冰水浴中进行超声破碎，条件为功率100 W，工作1.5 s，停2.5 s，共处理10 min。分别用肌酸激酶法测定细菌内ATP含量，用ELISA法检测细菌内ROS含量，具体测定步骤按试剂盒说明书进行。用相同方法处理4×10⁵ CFU/mLE.coli BW25113 12 h和24 h，取10 μL菌液进行平板活菌计数，收集其余菌体，加入200 μL放射免疫沉淀法(radio immunoprecipitation assay, RIPA)强裂解液，混匀后冰上静置2 h。4 ℃、3 000×g离心10 min，取25 μL上清液用酶联免疫吸附试验(enzyme-linked immuno sorbent assay, ELISA)法测定AKG和Glu的浓度。每个处理设3个细胞孔，实验重复3次。根据标准品绘制标准曲线，计算样品中的各代谢物浓度。

1.9 纳米银抑制α-酮戊二酸脱氢酶活性

用ddH₂O稀释α-酮戊二酸脱氢酶(alpha-ketoglutarate dehydrogenase, α-KGDC)至约400 IU/L，分别配制浓度为64、32、16、8、4、2、1、0.5、0.25 μg/mL的纳米银溶胶。将二者等体积混合，在37 ℃培养箱中避光孵育1 h。孵育完成后立即置于冰上，用α-KGDC活性ELISA试剂盒按说明书方法检测酶活。

将1 mL 4×10⁵ CFU/mLE.coli BW25113菌液用MIC浓度纳米银处理15 min、30 min、12 h和24 h，取少量菌液进行平板活菌计数，收集其余菌体加入200 μL RIPA强裂解液，混匀后冰上静置2 h。4 ℃、3 000×g离心10 min后，取25 μL上清液用ELISA法测定细菌内α-KGDC活性。

1.10 检测细菌内关键基因的表达水平

用实时荧光定量PCR检测2种纳米银处理E.coli后arcA^[34-37]、fnr^[38-39]、fdnH^[40-41]、soxS^[42-43]和sucB^[44] 5个基因的转录水平变化，以三磷酸甘油醛脱氢酶的编码基因gapA作为内参，所用引物序列见表1 (引物由Primer 5软件设计)。E.coli BW25113经12.50 μg/mL纳米银处理15 min和30 min。

用总RNA快速提取试剂盒提取RNA，每组3个平行重复，总RNA重复提取3次。反转录反应体系(20 μL)：5×TRUEscript RT MasterMix 4 μL，gDNA Remover 1 μL，RNA (10 ng/μL) 15 μL。混合各管中液体后置于PCR仪，设置反应程序：25 ℃ 10 min，42 ℃ 15 min，85 ℃ 5 s。所得的cDNA产物用NanoDrop检测浓度和纯度后分装，保存于−20 ℃。在96孔PCR板中依次加入ddH₂O 8 μL，UltraSYBR Mixture 10 μL，上、下游引物各50 μmol/L，cDNA 80 ng。反应程序：95 ℃ 10 min；95 ℃ 15 s，60 ℃ 60 s，共40个循环；72 ℃ 10 min。最后进行熔解曲线分析。

1.11 数据分析

所有数据采用SPSS v21.0进行单因素方差分析(one-way ANOVA)，进行组间比较：若Leven’s检验方差齐，采用最小显著性差异法(least significant difference, LSD)检验进行两两比较；若方差不齐，改用Dunner’s T3检验。用^*表示差异显著(P＜0.05)，^**表示差异非常显著(P＜0.01)，^***表示差异极其显著(P＜0.001)。图形绘制由Origin软件完成。流式细胞仪检测数据采用FlowJo v10.6.2进行分析。

2 结果与分析

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2.1 PVP-AgNPs@AKG的合成与表征

芦丁在碱性条件下将Ag⁺还原为单质银，溶液从无色逐渐变为淡黄色、黄色、棕色，全波长扫描可见404 nm处有单一吸收峰，说明有纳米银生成^[46-47] (图1A)。透射电镜观察可见纳米银呈球形，粒径分布在6−22 nm，平均粒径13.2 nm (图1B、1D)；平均ζ电位为−17.1 mV，水合半径为39.23 nm。加入20 mmol/L AKG作封端剂后纳米银形貌未发生改变，粒径分布在4−16 nm，平均粒径10.6 nm (图1C、1E)；平均ζ电位−3.58 mV，水合粒径为89.61 nm。PVP-AgNPs和PVP-AgNPs@AKG胶体溶液的pH值分别为5.5和2.3。

2.2 AKG增强纳米银的抗菌作用

PVP-AgNPs和PVP-AgNPs@AKG对E.coli BW25113、E.coli 8099、S.aureus ATCC6538、P.aeruginosa 1118和A.baumnnii TY-19的MIC和MBC值见表2，结果表明AKG封端后对5株细菌的MIC值均降低50%；除A.baumnnii TY-19的MBC值保持不变以外，其余菌株MBC值均降低50%或50%以上。

用6.25 μg/mL PVP-AgNPs@AKG处理E.coli BW25113 9 h后活菌数量下降3个数量级(图2A)，用12.50 μg/mL浓度处理S.aureus ATCC6538能下降1个数量级(图2C)，而未封端纳米银在相同浓度下对2株细菌仅表现为抑制作用。抗生素后效应结果显示，即使在1 MIC浓度下，AKG封端纳米银处理后BW25113株恢复生长延迟0.79 h，ATCC6538株的恢复生长延迟0.07 h (图2B、2D)。总体来看，AKG修饰纳米银后抗菌性增强。

2.3 AKG未增强对正常细胞的毒性作用

用MTT法检测纳米银处理2 h的细胞毒性，结果显示2种纳米银的细胞毒性均表现出剂量的依赖性，AKG封端前后对人正常细胞HCerEpic的毒性无显著改变，IC₅₀值分别为104.39 μg/mL和100.82 μg/mL (图3A)，远大于上文检测的细菌MIC值。经流式细胞仪检测2种纳米银处理12 h后细胞凋亡和坏死情况，结果表明AKG封端前后相同浓度纳米银处理组间无显著差异，均主要诱导细胞凋亡(12.5 μg/mL: 65.74%vs. 62.40%; 50.0 μg/mL: 50.24%vs. 51.12%)，少数细胞发生坏死(12.5 μg/mL: 0.23%vs. 0.14%; 50.0 μg/mL: 0.31%vs. 0.15%) (图3B−3I)。

2.4 AKG未提升Ag⁺的释放

PVP-AgNPs@AKG溶胶的pH值为2.3，可能促进银离子的释放从而增强抗菌作用。本研究将系列浓度PVP-AgNPs@AKG溶液用截流值为10 kDa的超滤管过滤分离出银离子，仅200 μg/mL浓度下所得滤过液对E.coli有抑制作用，这相当于1.563 μg/mL AgNO₃的抑菌效果，说明在PVP-AgNPs@AKG胶体溶液中银离子的释放量低于1%。研究还发现AKG对E.coli的MIC值为15 mmol/L，而本研究中即使所有AKG分子结合纳米银(100 μg/mL PVP-AgNPs溶液中加入10 mmol/L AKG)，当纳米银稀释到E.coli的MIC值时，AKG浓度仅0.625 mmol/L。这些结果说明AKG封端后纳米银抗菌作用增强与酸性条件促进银离子释放无关。

2.5 AKG未提高细菌纳米银摄入量

为检测AKG是否促进细菌摄入纳米银，用ICP-MS测定了处理不同时间后菌体内的总银量(表3)。用2种纳米银处理E.coli 15 min和30 min后，菌体内(按10⁶ CFU计)的银含量分别增加3.91%和15.44%，总银占比提升幅度分别为0.31%和1.24%，12.50 μg/mL PVP-AgNPs处理组增加量最多，但差异无统计学意义。PVP-AgNPs@AKG处理组在不同剂量和不同时间点上，菌体内的总银量均低于PVP-AgNPs，说明AKG未促进菌体摄入更多纳米银。

2.6 对三羧酸循环的抑制作用

ATP耗竭是纳米银抗菌的一个显著特征^[48]，本研究结果显示，6.25 μg/mL PVP-AgNPs处理15 min后E.coli ATP含量下降15.32%，而相同浓度PVP-AgNPs@AKG导致E.coli ATP下降45.97%；在12.50 μg/mL浓度下，2种纳米银分别导致ATP含量下降30.65%和61.30%。12.50 μg/mL PVP-AgNPs处理30 min也仅下降51.94%，PVP-AgNPs@AKG组无显著变化(图4A)。考虑到AKG未增强银离子的释放，也不增强细菌摄入更多纳米银，而细菌的ATP亏耗更严重，我们推测PVP-AgNPs@AKG可能靶向于AKG相关的酶，比如三羧酸循环的α-KGDC，它是TCA的限速酶。体外试验显示该酶对纳米银极其敏感，1 μg/mL PVP-AgNPs@AKG使酶活降低39.43%，相同浓度PVP-AgNPs使其活性降低28.89%，说明AKG封端的纳米银对α-KGDC的抑制作用更强，这种抑制程度在低浓度时表现得更加明显，在高浓度时逐渐趋于饱和状态(图4B)。PVP-AgNPs@AKG对菌体内α-KGDC也表现出更强的抑制作用：经MIC浓度处理大肠杆菌12 h后，AKG封端纳米银处理组的酶活为PVP-AgNPs组的84.04% (0.569 IU/CFUvs. 0.677 IU/CFU)，到24 h时仅为52.17% (0.276 IU/CFUvs. 0.529 IU/CFU) (图4C)。经荧光定量PCR检测发现，纳米银处理大肠杆菌15−30 min后，sucB基因转录水平降低，PVP-AgNPs@AKG处理组的下降程度显著程度高于PVP-AgNPs处理组(图4D)，暗示这种抑制作用是多水平的。为进一步验证对菌体α-酮戊二酸脱氢酶的抑制作用，我们还分析了AKG的蓄积与Glu的生成情况，结果显示在MIC浓度下，PVP-AgNPs@AKG处理12 h后AKG的浓度显著高于PVP-AgNPs处理组，24 h后差异更为显著(图4E)。24 h后，PVP-AgNPs@AKG组存活的细菌仅为PVP-AgNPs组的10% (1.0×10⁴ CFU/mLvs. 1.5×10⁵ CFU/mL)，相应地，菌体内Glu的浓度在两个时间点也显著高于PVP-AgNPs处理组(图4F)。实验结果表明AKG封端纳米银严重抑制TCA效率，影响细菌的能量代谢。

2.7 AKG封端阻碍大肠杆菌向厌氧代谢转变

我们前期研究发现大肠杆菌受AgNPs刺激后的适应性反应是从有氧呼吸转变为厌氧呼吸，缺失arcA基因将提高大肠杆菌对纳米银的敏感性^[18]。本研究采用12.50 μg/mL纳米银处理E.coli BW25113，荧光定量PCR结果显示PVP-AgNPs处理后arcA基因显著上调，而PVP-AgNPs@AKG组arcA基因表达水平仅略有上升(图5A)。受此影响，2处理组的fdnH基因在30 min后显著上调，PVP-AgNP@AKG组的上调幅度显著低于PVP-AgNPs组(图5C)。fnr基因表现为先下调再上调，并且PVP-AgNP@AKG组在第30 min上调幅度更显著(图5B)。

2.8 氧化应激反应

氧化应激是纳米银抗菌的另一个机制，刺激产生的ROS打破菌体的氧化还原平衡，导致生物大分子功能受损^[49]。用6.25 μg/mL纳米银处理30 min内，E.coli产生ROS的量呈上升趋势，但PVP-AgNPs@AKG与PVP-AgNPs处理组差异不大(120.41%vs. 121.14%)。在12.50 μg/mL浓度下，纳米银刺激E.coli产生ROS的情况表现为时间依赖性，30 min时AKG封端前后具有显著差异(126.24%vs. 178.72%,P＜0.001)；实时荧光定量PCR检测显示，2种纳米银处理组的soxS基因表达水平呈上调趋势，其中30 min后PVP-AgNPs组基因出现显著上调(P＜0.05)，而AKG封端后该基因上调程度的显著性更高(P＜0.01) (图6)。总之，这些结果说明AKG封端后能促进纳米银诱导细菌产生更多ROS，引起更强烈的氧化应激反应。

3 讨论与结论

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纳米银因其粒径微小而形成的较大比表面积能使它们能够更好地与微生物接触，因此即使在低浓度下也具有良好的抗菌能力，并且能对鲍曼不动杆菌、铜绿假单胞菌、肺炎克雷伯菌、结核分枝杆菌、耐万古霉素肠球菌、耐甲氧西林金黄色葡萄球菌等多耐药细菌具有抗菌活性^[50]。然而，随着研究的深入，近年来发现了细菌对纳米银的耐受问题，筛选具有高生物相容性的小分子物质对纳米银进行各类表面修饰，改变其物理化学特性，进而获得更加优良的生物学活性或将成为主要的解决途径之一^[51-54]。本研究首次使用了具有多效活性的α-酮戊二酸作为纳米银封端剂，合成的PVP-AgNPs@AKG具有更加优良的抗菌效果，同时又不改变其细胞毒性大小。PVP-AgNPs对多耐药菌株P.aeruginosa 1118和A.baumannii TY-19的MIC值为50.00 μg/mL，为PVP-AgNPs@AKG对它们的MIC值的2倍(表2)，该浓度下PVP-AgNPs@AKG处理组活细胞仅比对照组降低4.00% (图3B、3F)，扩大了纳米银的治疗窗。

通常情况下酸性条件会促进纳米银释放Ag^+[55-56]，但本研究发现PVP-AgNPs@AKG胶体溶液中银离子的释放量低于1%，其抗菌性的增强与Ag⁺释放无关。本研究还发现AKG封端是通过促使纳米银靶向α-酮戊二酸脱氢酶来增强纳米银的抗菌活性，并使大肠杆菌向厌氧代谢转变的过程受阻，诱导更多ROS的产生。

本研究提出了PVP-AgNPs@AKG的抗菌作用增强机制(图7)。ROS一直以来被认为是纳米银介导的细菌和细胞毒性的主要效应分子，Grzelak等的研究证明AgNPs对线粒体呼吸链的破坏会增加ROS的生成，阻断ATP的合成，从而导致DNA损伤^[57]；AgNPs释放的Ag⁺也可诱导ROS的产生^[58-59]。线粒体的存在是真核细胞和原核细胞的重要差异之一，真核细胞中有氧呼吸发生在线粒体中。因此，我们推测PVP-AgNPs虽然在细菌中能低浓度下抑制其TCA循环中的α-酮戊二酸脱氢酶，并进而引发ROS水平上升、呼吸方式转变；但在正常细胞中，由于线粒体膜的阻隔，相同浓度下其对该酶的抑制程度远不及在细菌中，这也是纳米银对细胞IC₅₀值远大于对细菌的MIC值的重要原因之一。AKG的封端使得其对细菌中该酶的抑制程度更加显著，而在细胞中却远远未达到进入线粒体后产生破坏的浓度阈值。同时，真核细胞中更为完善的抗氧化系统也使得ROS的破坏作用并不如细菌中那样显著。

此外，在生物体中，AKG除作为连接碳代谢与氮代谢的关键节点外^[60-61]，还参与多种信号通路。生物体对环境压力进行响应时，TCA代谢产物水平和代谢通路发生的改变可以解释癌细胞在变化环境中的适应性和可塑性，AKG及其结构类似物(琥珀酸盐、富马酸盐等)参与调控促进致癌的信号通路^[62]。例如，研究表明AKG作为羟化酶的底物，影响脯氨酸/天冬氨酸/赖氨酸羟化，进而调节低氧诱导因子-1 (hypoxic inducible factor-1, HIF-1)的稳定性和胶原合成，而HIF-1是癌症发生和发展中的重要转录因子^[63-64]。以AKG为重要底物的铁(Ⅱ)/α-酮戊二酸依赖的双加氧酶(alpha-ketoglutarate-dependent dioxygenase, α-OGDDs)是含铁的非血红素加氧酶超家族中最大的亚家族，在许多生物过程中发挥着不同的作用，包括调节缺氧适应，细胞外基质形成，基因转录的表观遗传调控和细胞代谢的重编程。其中，铁(Ⅱ)/α-酮戊二酸依赖性双加氧酶的AlkB家族是一类普遍存在的DNA修复酶，能通过氧化脱烷基作用去除碱基上的烷基加合物^[65]。因此，由于AKG参与的代谢反应众多，未来我们还需要对更多这些与AKG有关的酶类进行研究，深入挖掘PVP-AgNPs@AKG更加优良的作用以及进一步合成和修饰纳米银的改进方向。

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2024年第64卷第7期

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doi: 10.13343/j.cnki.wsxb.20230644

接收时间：2023-10-22
首发时间：2026-03-19
出版时间：2024-07-04

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西南大学蚕桑纺织与生物质科学学院, 重庆 400715

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Gene	Primer sequences (5′→3′)	Product length (bp)	Function of gene product	References
arcA	F: ATGCAGACCCCGCACATTC R: TTAATCTTCCAGATCACCGCAGA	717	Anoxic redox control, mediating the metabolic transition from aerobiosis to anaerobiosis	[34-37]
fnr	F: TCAGGCAACGTTACGCGTATG R: ATGATCCCGGAAAAGCGAA	753	Global transcription factor, managing the distribution of RNA polymerase in response to oxygen starvation	[38-39]
fdnH	F: ATGGCTATGGAAACGCAGGA R: TTACTCATGATGATCCTCCTCGTC	885	Functions in the formate-nitrate respiratory chain, rich of Fe-S cluster	[40-41]
soxS	F: ATGTCCCATCAGAAAATTATTCAGG R: TTACAGGCGGTGGCGATAAT	324	Activator of superoxide stress	[42-43]
sucB	F: ATGAGTAGCGTAGATATTCTGGTCCC R: CTACACGTCCAGCAGCAGACG	1 218	Alpha-ketoglutarate dehydrogenase, TCA cycle enzyme	[44]
gapA	F: ATGACTATCAAAGTAGGTATCAACGGTT R: TTATTTGGAGATGTGAGCGATCA	996	GADPH, universal reference sequences	[45]

Gene

Primer sequences (5′→3′)

Product length (bp)

Function of gene product

References

arcA

F: ATGCAGACCCCGCACATTC
R: TTAATCTTCCAGATCACCGCAGA

717

Anoxic redox control, mediating the metabolic transition from aerobiosis to anaerobiosis

[34-37]

fnr

F: TCAGGCAACGTTACGCGTATG
R: ATGATCCCGGAAAAGCGAA

753

Global transcription factor, managing the distribution of RNA polymerase in response to oxygen starvation

[38-39]

fdnH

F: ATGGCTATGGAAACGCAGGA
R: TTACTCATGATGATCCTCCTCGTC

885

Functions in the formate-nitrate respiratory chain, rich of Fe-S cluster

[40-41]

soxS

F: ATGTCCCATCAGAAAATTATTCAGG
R: TTACAGGCGGTGGCGATAAT

324

Activator of superoxide stress

[42-43]

sucB

F: ATGAGTAGCGTAGATATTCTGGTCCC
R: CTACACGTCCAGCAGCAGACG

1 218

Alpha-ketoglutarate dehydrogenase, TCA cycle enzyme

[44]

gapA

F: ATGACTATCAAAGTAGGTATCAACGGTT
R: TTATTTGGAGATGTGAGCGATCA

996

GADPH, universal reference sequences

[45]

Strains	Silver nanoparticles	MIC (μg/mL)	MBC (μg/mL)	MBC/MIC
Escherichia coli BW25113	PVP-AgNPs	12.50	25.0	2.0
PVP-AgNPs@AKG	6.25	12.5	2.0
E.coli 8099	PVP-AgNPs	12.50	25.0	2.0
PVP-AgNPs@AKG	6.25	12.5	2.0
Staphylococcus aureus ATCC6538	PVP-AgNPs	25.00	50.0	2.0
PVP-AgNPs@AKG	12.50	25.0	2.0
Pseudomonas aeruginosa 1118	PVP-AgNPs	50.00	＞100.0	＞2.0
PVP-AgNPs@AKG	25.00	50.0	2.0
Acinetobacter baumnnii TY-19	PVP-AgNPs	50.00	100.0	2.0
PVP-AgNPs@AKG	25.00	100.0	4.0

Strains

Silver nanoparticles

MIC (μg/mL)

MBC (μg/mL)

MBC/MIC

Escherichia coli BW25113

PVP-AgNPs

12.50

25.0

2.0

PVP-AgNPs@AKG

6.25

12.5

2.0

E.coli 8099

PVP-AgNPs

12.50

25.0

2.0

PVP-AgNPs@AKG

6.25

12.5

2.0

Staphylococcus aureus ATCC6538

PVP-AgNPs

25.00

50.0

2.0

PVP-AgNPs@AKG

12.50

25.0

2.0

Pseudomonas aeruginosa 1118

PVP-AgNPs

50.00

＞100.0

＞2.0

PVP-AgNPs@AKG

25.00

50.0

2.0

Acinetobacter baumnnii TY-19

PVP-AgNPs

50.00

100.0

2.0

PVP-AgNPs@AKG

25.00

100.0

4.0

Silver nanoparticles	Concentration (μg/mL)	The amount of silver^* (μg)		The ratio of silver (%)
^：计算为每10⁶ CFUE.coli中的总银量 ^: Calculated as the total amount of silver per 10⁶ CFUEscherichia coli.
PVP-AgNPs	6.25	2.70±0.01	2.92±0.02		15.37	16.60
12.50	2.85±0.02	3.29±0.02		8.11	9.35
PVP-AgNPs@AKG	6.25	2.63±0.01	2.76±0.03		14.93	15.67
12.50	2.81±0.02	2.92±0.02		8.00	8.31

Silver nanoparticles

Concentration (μg/mL)

The amount of silver^* (μg)

The ratio of silver (%)

15 min

30 min

15 min

30 min

^*：计算为每10⁶ CFUE.coli中的总银量
^*: Calculated as the total amount of silver per 10⁶ CFUEscherichia coli.

PVP-AgNPs

6.25

2.70±0.01

2.92±0.02

15.37

16.60

12.50

2.85±0.02

3.29±0.02

8.11

9.35

PVP-AgNPs@AKG

6.25

2.63±0.01

2.76±0.03

14.93

15.67

12.50

2.81±0.02

2.92±0.02

8.00

8.31