大黄素提高新生隐球菌琥珀酸脱氢酶活性增强氧化作用的研究

doi:10.3877/cma.j.issn.1674-0785.2023.03.014

目的

探索大黄素提高新生隐球菌（cryptococcus neoformans，C. neoformans）琥珀酸脱氢酶（succinate dehydrogenase，SDH）活性，增强氧化作用，阐述大黄素新的生物学功能，提高以SDH为靶点的抗菌药物的敏感性，发挥协同抗菌作用，缓解临床耐药的严重问题。

方法

用微量肉汤稀释法和MTT法检测大黄素对C. neoformans的最小抑菌浓度（minimum inhibitory concentration，MIC），并检测大黄素诱导后C. neoformans的活性氧（reactive oxygen species，ROS）和线粒体膜电位的变化，qPCR检测SDH mRNA的表达，用表面等离子共振方法检测大黄素与SDH的相互作用。

结果

大黄素无明显的抑制C. neoformans作用，MIC＞256 μg/ml，但1~4 μg/ml的大黄素可以促进C. neoformans的SDH活性升高，1 μg/ml时ROS升高和SDH的mRNA表达也明显增强，大黄素与SDH可发生特异性结合。

结论

大黄素具有促进C. neoformans的SDH活性升高、增强ROS作用的新生物学功能。

关键词: 大黄素, 新生隐球菌, 琥珀酸脱氢酶, 氧化作用

Objective

To explore the effects of emodin on succinate dehydrogenase (SDH) activity and oxidation in Cryptococcus neoformans (C. neoformans) and elaborate the new biological function of emodin, so as to improve the sensitivity of antibacterial drugs targeting SDH and alleviate the serious problem of drug resistance.

Methods

The minimal inhibitory concentration of emodin against C. neoformans was detected by broth microdilution and MTT assay, and the changes of reactive oxygen species (ROS) and mitochondrial membrane potential of C. neoformans treated with emodin were detected. The expression of SDH mRNA was detected by qPCR, and the interaction between emodin and SDH was detected by surface plasmon resonance.

Results

Emodin had no obvious inhibitory effects on C. neoformans, with an MIC＞256 μg/ml, but emodin at a concentration of 1~4 μg/ml was able to promote the SDH activity of C. neoformans. At 1 μg/ml, emodin significantly increased the ROS and SDH mRNA expression. Emodin can bind with SDH specifically.

Conclusion

Emodin can promote the SDH activity and enhance ROS production in C. neoformans, which represents the new biological function of emodin.

Key words: Emodin, Cryptococcus neoformans, Succinate dehydrogenase, Oxidation

图1 大黄素的分子结构式

表1 引物列表

引物名称	序列（5’ to 3’）
GADPH-F	TGAGAAGGACCCTGCCAACA
GADPH-R	ACTCCGGCTTGTAGGCATCAA
CNI03270-F	GCACGAACGATTGCCTGGTA
CNI03270-R	TGAGGACTTCGCCGGTGTAT

表1 引物列表

引物名称	序列（5’ to 3’）
GADPH-F	TGAGAAGGACCCTGCCAACA
GADPH-R	ACTCCGGCTTGTAGGCATCAA
CNI03270-F	GCACGAACGATTGCCTGGTA
CNI03270-R	TGAGGACTTCGCCGGTGTAT

表2 大黄素对C. neoformans的MIC

菌株名称	MIC（μg/ml）
ATCC^® MYA4906	＞256
ATCC^® MYA4907	＞256
BHKS388	＞256
BHKS390	＞256
BHKS480	＞256
BHKS481	＞256
BHKS482	＞256

表2 大黄素对C. neoformans的MIC

菌株名称	MIC（μg/ml）
ATCC^® MYA4906	＞256
ATCC^® MYA4907	＞256
BHKS388	＞256
BHKS390	＞256
BHKS480	＞256
BHKS481	＞256
BHKS482	＞256

图2 大黄素对C. neoformans的活力。MA为50 mM丙二酸，丙二酸作为底物竞争抑制剂抑制SDH活性。（*P＜0.05，**P＜0.01）

图3 大黄素处理4 h后C. neoformans的SDH活性（*为P＜0.05，****为P＜0.001）

图4 大黄素处理后C. neoformans ROS和线粒体膜电位检测，图a为ROS检测荧光显微镜图片；图b为线粒体膜电位检测荧光显微镜图片；图c为ROS相对荧光强度；图d为线粒体膜电位相对荧光强度。（*P＜0.05，***P＜0.001）

图5 SDH mRNA的表达

图6 大黄素与SDH分子互作

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Emodin enhances succinate dehydrogenase activity and oxidation in Cryptococcus neoformans

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Emodin enhances succinate dehydrogenase activity and oxidation in Cryptococcus neoformans