姜黄素介导的抗菌光动力疗法在口腔念珠菌病小鼠模型上的应用
Summary
该方案描述了抗菌光动力疗法（aPDT）在口腔念珠菌病小鼠模型中的应用。使用姜黄素和蓝色LED灯的水溶性混合物进行aPDT。
Abstract
抗菌光动力疗法 （aPDT） 已 在体外进行了广泛的研究，感染的临床前动物模型适用于在临床试验之前评估替代疗法。本研究描述了 aPDT 在口腔念珠菌病小鼠模型中的疗效。40只小鼠通过皮下注射泼尼松龙进行免疫抑制，并使用先前浸泡在 白色念珠菌 细胞悬浮液中的口腔拭子接种它们的舌头。在实验过程中 ，四 环素通过饮用水给药。真菌接种5天后，将小鼠随机分为8组;第九组未经治疗的未感染小鼠作为阴性对照（n = 5）。用蓝色LED灯（89.2 mW /cm 2;~455 nm）和无光（分别为C + + 和 C + L- 组）测试姜黄素混合物的三种浓度（20 μM、40 μM 和 80 μM）。仅光照 （C-L+）、不治疗 （C-L-） 和无感染动物被评估为对照。使用 Welch 方差分析和 Games-Howell 检验 （α = 0.05） 分析数据。口腔念珠菌病在所有受感染的动物中都已确定，并通过舌背上的特征性白斑或假膜在宏观上可视化。组织病理学切片证实，在C-L-组中，酵母和细丝的存在仅限于上皮的角化层，并且在从使用40μM或80μM姜黄素进行aPDT的小鼠获得的图像中，真菌细胞的存在在视觉上减少。与C-L-组相比，由80μM姜黄素介导的aPDT促进了2.47 log10 的菌落计数减少（p = 0.008）。所有其他组的菌落数量均无统计学意义减少，包括光敏剂（C+L-）或光敏（C-L+）组。姜黄素介导的aPDT减少了小鼠舌头的真菌负荷。
Introduction
口腔念珠菌病（OC）是口腔的主要真菌感染;它是由 念珠菌 属的过度生长引起的。OC 的诱发因素包括内分泌功能障碍、使用广谱抗生素、放疗和化疗、营养缺乏、口干症（唾液流量低）、使用假牙、卫生条件差，尤其是免疫抑制1。在 念珠菌 物种中， 白色念珠菌 是最普遍和最致命的一种;它被发现为人体内的共生物种和机会性病原体。 白色念珠菌 有能力将其形态从共生酵母（胚孔）转变为致病丝（菌丝和假菌丝）2。丝状形式，尤其是菌丝，可以通过内吞作用或主动渗透侵入宿主上皮，引起感染3. 白色念珠 菌的其他毒力因子包括粘附、生物膜形成以及脂解和水解酶和毒素的分泌，例如脂肪酶、磷脂酶、蛋白酶和念珠菌溶血素4。
OC治疗包括使用抗真菌药物，特别是局部多烯和唑类（制霉菌素和咪康唑）5。然而，它们仅显示出短期疗效，并且复发频繁。此外，抗真菌药物的过度使用引发了抗真菌药物耐药性发展和传播的问题6。因此，需要替代疗法，例如抗菌光动力疗法 （aPDT），它在氧气存在下将光敏剂 （PS） 和适当波长（与 PS 吸收相同）的光结合在一起。PS与细胞结合或被细胞吸收，当被光激活时，会产生对致敏细胞有毒的活性氧（ROS）7。
在aPDT中，使用的光敏剂（PS）之一是姜黄素（CUR），这是一种从姜黄植物（Curcuma longa L.）的根茎中提取的天然化合物。姜黄素具有多种治疗特性，包括抗炎、抗氧化、抗癌和抗菌能力 8,9。先前的一项研究发现，利用 CUR 的 aPDT 有效地减少了口腔念珠菌病小鼠模型中的白色念珠菌，而不会对宿主的组织造成任何伤害10。CUR是从姜黄中提取的主要姜黄素，但其他多酚，如脱甲氧基姜黄素和双脱甲氧基姜黄素，也存在于这种植物中。姜黄素介导的 aPDT 对导管中生长的金黄色葡萄球菌的生物膜具有抗菌活性11。然而，据我们所知，其对白色念珠菌的抗真菌活性仍不清楚。因此，在这项研究中，我们评估了在 OC 小鼠模型中由姜黄素盐介导的针对白色念珠菌的 aPDT。
Protocol
使用小鼠的研究方案已获得UNESP阿拉拉夸拉牙科学院的动物使用伦理委员会（案例编号05/2008和09/2020）的批准。 白色念珠菌 （ATCC 90028）作为参比菌株。本研究使用体重范围为 20-30 g 的六周龄雌性瑞士小鼠 （n = 45）。这些动物由圣保罗州立大学、UNESP、Botucatu提供。 1. PS的制备和aPDT光源的选择 根据要测试的物质的规格准备PS。注：在这项研究中，?…
Representative Results
OC的小鼠模型显示所有感染小鼠的舌头上有典型的白斑和假膜（图4A）。从C-L-动物中回收 的白色念珠菌 证实了该微生物的组织定植（值范围为1.62 x 104 至4.80 x 105 CFU/mL）。正如预期的那样，来自NCtr组的动物在取样后没有表现出任何组织改变或菌落生长（图4B）。 当在80μM下使用姜黄素进行光敏化时，aPDT降低?…
Discussion
白色念珠菌与免疫功能低下、糖尿病、长期使用抗生素和口腔卫生不良的个体的口腔和食管感染有关 1,3。人类传染病的研究需要进行体外和体内研究，然后才能安全准确地设计临床试验。本研究描述了一种建立OC小鼠模型的方法，可用于评估白色念珠菌口腔感染的发病机制和抗真菌方法的疗效15,16,17,18,19。<su…
Acknowledgements
作者感谢FAPESP（圣保罗研究基金会，过程编号FAPESP #2013/07276-1（CePID CePOF）和2008/00601-6的财政支持。我们还要感谢 Ana Paula Silva 博士提供有关 CUR 基水溶性盐的信息。
Materials
| C. albicans | ATCC (Rockville, Md, USA) | 90028 | Used to prepare the Candida inoculum |
| Centrifuge | Eppendorf Centrifuge 5804/5804R,B. Braun, Melsungen, Hesse, Germany | 022628146 (NA) | Used to prepare the Candida inoculum |
| Chlorpromazine chloride 2 mg/mL | Compounding pharmacy, Araraquara, SP, Brazil | - | Used to sedate animals during candida inoculation |
| Curcumin-based water-soluble salt | PDTPharma, Cravinhos, Brazil | - | Consisting of 53.4% of natural curcumin, and 46.6% of other curcuminoids (demethoxycurcumin and bis-demethoxycurcumin). Prepared in water and N-MethylD-Glucamine (final average molecular weight of 730.32 g.mol−1) |
| Digital colony counter | CP 600 Plus, Phoenix Ind Com Equipamentos Científicos Ltda, Araraquara, SP, Brazil | - | Used to count colonies on agar plates |
| Extruded mouse chow | Benelab food, Industry Qualy Animal Nutrition and Commerce Ltda., Lindóia, São Paulo State, Brazil. | - | Used for the feeding of the mice |
| Ketamine Hydrochloride 10% | Ketamina Agener, União Química Farmacêutica Nacional S/A, Embu-Guaçu, SP, Brazil | - | Used to anesthetize animals before treatments and for euthanasia |
| Light-emitting diode handpiece (prototype) | Instituto de Física de São Carlos, University of São Paulo, São Carlos, SP, Brazil | - | Fabricated with LXHL-PR09, Luxeon III Emitter, Lumileds Lighting, San Jose, California, USA |
| Methylprednisolone acetate 40 mg | DEPO-MEDROL, Pfizer, New York | - | Used as an immunosuppressant |
| Microtome | Leica Microsystems, Bannockburn, IL, USA | SM2500 | Used to cut the serial sections of the tongues |
| Propylene boxes (cages housing) H13 x L20 x D30 cm | Bonther Equipaments, Ribeirão Preto, SP, Brazil | - | Used to keep the animals throughout the experimental period |
| Sabouraud Dextrose Agar with Chloramphenicol | HiMedia, Mumbai, India | MM1067-500G | Culture medium for yeast growth (agar) |
| Spectrophotometer | Spectrophotometer Kasvi K37-VIS , São José dos Pinhais, PR, Brazil | K37-VIS | Used to standardize the inoculum concentration |
| Tetracycline hydrochloride | Compounding pharmacy, Araraquara, SP, Brazil | - | Antibiotic given to induce oral dysbiosis |
| Wood shavings | J.R. Wood Shavings, Comerce of Sawdust Ltda., Conchal, São Paulo State, Brazil | - | Used for floor covering inside the housing boxes |
| Xylazine 2% | Calmiun, União Química Farmacêutica Nacional S/A, Embu-Guaçu, SP, Brazil | - | Used in combination with ketamine for anesthesia |
| Yeast Nitrogen Broth | Difco, InterLab, Detroit, MI, USA | DF0919-07-3 | Culture medium for yeast growth (broth) |
| Yeast Peptone Dextrose Broth | NutriSelect Basic, Sigma Aldrich | Y1375 | Culture medium for maintaining the strains at -80°C and grow |
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Cite This Article
Mima, E. G. d. O., Pavarina, A. C., Jordão, C. C., Vieira, S. M., Dovigo, L. N. Curcuminoid-Mediated Antimicrobial Photodynamic Therapy on a Murine Model of Oral Candidiasis. J. Vis. Exp. (200), e65903, doi:10.3791/65903 (2023).