【作者】 梁蓉梅；

【导师】 姜远英； 曹永兵；

【作者基本信息】 第二军医大学 ， 药理学， 2010， 博士

【摘要】 白念珠菌感染由于在艾滋病患者,器官移植患者以及其他免疫缺陷患者中具有高发病率和高死亡率的特点,已成为一个严重的医学问题。尽管抗真菌药物品种不断增加,但仍然满足不了病情复杂患者对控制感染越来越高的需求。本课题的研究目的是探讨具有新型化学结构的抗真菌化合物2-氨基壬烷-6-甲氧基四氢萘盐酸盐(10b)的体外抗真菌活性和作用机制,为降低真菌相关感染提供有用的信息和策略。微量液基稀释法结果显示10b对氟康唑敏感白念珠菌以及氟康唑耐药白念珠菌均有良好的抗真菌活性,MIC80值的范围从0.031μg/ml到8μg/ml,活性强与氟康唑或酮康唑相当或者更强；对氟康唑耐药白念珠菌的抗真菌活性强大,其作用普遍强于酮康唑,尤其对氟康唑高度耐药的实验室诱导白念珠菌SC5314R(对氟康唑和伊曲康唑都高度耐药,MIC80值均为256μg/ml)作用更加显著,MIC80值为0.125μg/ml。琼脂平皿纸片扩散实验进一步证实10b对氟康唑耐药白念珠菌的抗真菌活性强于氟康唑和酮康唑并且表现出一定的杀菌作用。生长曲线实验,半数抑菌浓度(IC50)测定以及最低杀菌浓度(MFC)测定等多种实验手段和方法均证实10b具有强大的体外抗真菌活性。结晶紫染色实验以及XTT还原分析实验考察了10b对白念珠菌生物被膜的影响,结果显示10b对白念珠菌生物被膜的形成以及成熟(48 h)白念珠菌生物被膜细胞代谢活性均有强大的抑制作用,并且抑制能力强于法尼醇。激光共聚焦显微镜观察也进一步证实了10b强大的抑制白念珠菌生物被膜的作用：正常白念珠菌生物被膜表现为主要由假菌丝和真菌丝组成的典型三维空间结构；当白念珠菌细胞在0h与10μM法尼醇共孵育48 h后,被膜的形成没有受到抑制,仍然主要由真菌丝和假菌丝组成；与0.1μM的10b共孵育48 h后,尽管真菌丝和假菌丝仍然能够观察到,但被膜典型的三维空间结构(相互缠绕的丝状结构和芽生孢子基底层)受到了破坏,被膜主要由酵母态细胞组成；当10b的浓度增加10倍(1μM),粘附细胞从酵母态向菌丝态的转变被彻底抑制,导致了被膜的消失或者仅能生成少量的全部由酵母态细胞组成的被膜。为了探讨10b对白念珠菌的作用机制,我们首先采用薄层色谱分析(TLC)和气相色谱-质谱联用(GC/MS)分析考察10b在麦角甾醇生物合成通路上的作用靶点,通过透射电镜观察10b处理后对白念珠菌超微结构的影响,然后采用cDNA芯片分析和realtime RT-PCR分析从分子水平上考察10b处理酵母态白念珠菌以及被膜态白念珠菌基因表达谱的变化,最后采用MTS/PMS还原分析法评价了10b的体外细胞毒性。TLC分析结果显示10b处理后的薄层色谱图上羊毛甾醇及其类似物的斑点与酮康唑处理后完全不同,前者斑点直径明显变小颜色变淡,甚至观察不到而后者与空白对照相比斑点直径显著增加,颜色变深,这说明10b可能抑制了麦角甾醇生物合成通路上羊毛甾醇C-14去甲基酶之外的其他酶,导致羊毛甾醇向其他中间甾醇的转化。GC/MS分析结果显示10b处理组在气相色谱图上出现了与酮康唑处理组完全不同的色谱峰,前者所产生的甾醇成分与erg24菌株所产生的甾醇成分完全相同,均以ergosta-8,14-dienol(ignosterol)作为主要甾醇同时伴随大量ergosta-8,14,22-trienol的产生而后者则以24-methylene-lanost-8-en-3-ol作为主要甾醇。因此,10b在麦角甾醇生物合成通路上的真正作用靶点可能是ERG24基因编码的甾醇C-14还原酶而不是ERG11基因编码的羊毛甾醇C-14去甲基酶。为了证实上述观点,我们比较了10b和甾醇C-14还原酶抑制剂丁苯吗啉(Fm)处理野生型白念珠菌(BWP17)以及erg24菌株(NJ51-2)所产生的甾醇成分种类及含量变化情况。虽然10b与吗啉类都属于甾醇C-14还原酶抑制剂,但两者在麦角甾醇生物合成通路上的作用机制仍然存在差异。吗啉类除抑制甾醇C-14还原酶外,还抑制由ERG2基因编码的甾醇C-8异构酶,因此丁苯吗啉处理的野生型白念珠菌(BWP17)除以ergosta-8.14-dienol(ignosterol)作为主要甾醇外,还出现ergosta-8-en-3-ol的堆积。由于甾醇C-8异构酶位于甾醇C-14还原酶下游,因此丁苯吗啉处理的erg24菌株(NJ51-2)所产生的甾醇种类及比例均与未处理的erg24菌株完全相同。10b处理的erg24菌株(NJ51-2)所产生的甾醇成分与未处理的erg24菌株(NJ51-2)所产生的甾醇成分完全相同,但两者ergosta-8,14,22-trienol与ignosterol的比例却不同。10b处理组所产生的ergosta-8,14,22-trienol量明显高于未处理组,这表明10b在麦角甾醇合成通路上除抑制甾醇C-14还原酶活性外,可能还存在另外一个靶点,并且这个靶点应该位于甾醇C-14还原酶的上游。通过ergosta-8,14,22-trienol与ignosterol比例变化规律,我们推测它可能是与甾醇C-5去饱和酶相关的一个酶。另外,我们发现ignosterol的量随着10b剂量的增加而增加,这表明10b在低浓度时甾醇C-5去饱和酶相关酶是主要的作用靶点；随着10b剂量增加,10b与甾醇C-14还原酶的结和力增强,最终使甾醇C-14还原酶成为占主导作用的靶点。尽管ERG24基因编码的C-14还原酶是酿酒酵母有氧生长所必须的,但白念珠菌erg24突变体却能够在正常有氧条件下在标准限定滋养培养基中生长。电镜观察结果也显示10b处理后细胞膜结构完整,光滑,边界清晰,无胞内物质外流进入胞膜和胞壁之间。这表明10b虽然能够抑制麦角甾醇生物合成通路上的甾醇C-14还原酶和甾醇C-5去饱和酶相关酶的活性,导致麦角甾醇含量急剧降低,但由于中间甾醇的替代作用并不能破坏细胞膜结构的完整性,也不能完全抑制白念珠菌细胞的生长。因此,除了抑制麦角甾醇合成通路上的作用靶点之外,10b强大的抗白念珠菌活性必然还存在更为重要的作用机制。酵母态白念珠菌芯片分析和Real-time RT-PCR分析结果显示10b处理后,能量代谢相关基因,包括糖酵解相关基因(PFK1,CDC19和HXK2),生醇发酵相关基因(PDC11,ALD5和ADH1)以及线粒体呼吸链复合物相关基因(CBP3,COR1和QCR8)的表达水平显著降低。实时荧光定量分析结果显示10b处理白念珠菌后能够显著增加内源性活性氧(ROS)的产生,并且能力强于咪康唑。线粒体功能分析结果表明10b处理后能够降低线粒体膜电位(ΔΨm),泛醌-细胞色素C还原酶(复合物Ⅲ)活性以及细胞内ATP的含量。另外,加入抗氧化剂抗坏血酸(ascorbic acid,AA)能显著降低10b的抗真菌活性。这说明线粒体有氧呼吸链的转换与内源性ROS产生的增加可能在10b抑制酵母态白念珠菌活性过程中起了重要作用。被膜态白念珠菌芯片分析和Real-time RT-PCR分析结果显示10b处理后菌丝特异性基因ECE1表达急剧降低,编码转录抑制因子Nrg1p的基因NRG1表达水平显著增加,这与10b抑制白念珠菌被膜形成的作用直接相关。另外,10b处理后还能引起能量代谢相关基因表达降低,包括糖酵解相关基因(HXK2和PFK1),生醇发酵相关基因(ADH1)以及内源性ROS清除相关基因(SOD5)表达水平显著降低。功能分析结果显示加入抗坏血酸能显著降低10b对成熟白念珠菌生物被膜细胞代谢活性的抑制效率。上述结果表明10b对白念珠菌被膜形成的抑制作用可能主要依赖于改变被膜形成相关基因的表达,阻断白念珠菌从酵母态向菌丝态的转变,最终破坏了白念珠菌生物被膜的形成；线粒体有氧呼吸链的转换与内源性ROS产生的增加可能是10b降低成熟白念珠菌生物被膜细胞代谢活性的重要作用机制。体外细胞毒试验结果表明：虽然10b剂量依赖性地抑制小鼠胚胎成纤维细胞(BALB/C 3T3)以及人正常肝细胞(L-02)的代谢活性,但其对哺乳动物细胞的IC50值远远高于对真菌细胞的MIC50值,这为该类化合物能够继续深入研究提供了基本的安全保证。我们的实验数据为开发针对新的作用靶点,结构新颖的抗真菌药物,克服目前临床日益严重的真菌感染,提供了有意义的信息。更多还原

【Abstract】 Candida infections have become a serious medical problem due to high incidence and mortality in AIDS patients, transplant recipients and other immunosuppressed individuals. Despite continuous expansion in the arsenal of antifungal drugs, antifungal drugs available cannot meet the increasing requirements for managing infections in medically complex patients.2-aminotetralin derivates were synthesized as novel chemical structural antifungal agents. Of them,10b was found to have the strongest antifungal activity in vitro. The aim of the present work was to further investigate the antifungal activity of 10b as well as its mechanism of action in vitro. Broth microdilution analytic results showed that 10b possessed potent activities against almost all C. albicans isolates, which was similar to or more powerful than that of ketoconazole (KCZ) or fluconazole (FLC) and MIC80 value ranged from 0.063 to 8μg/ml. Despite high resistance to FLC and ICZ (both MIC80 value 256μg/ml) for isolate SC5314R,10b displayed powerful antifungal activity against this strain, MICgo value being 0.125μg/ml. Agar disk diffusion test further confirmed that 10b possesses more powerful antifungal activity against FLC-resistant isolates than that of FLC and KCZ and exhibits a fungicidal activity to some extent. Growth curve test, Half Inhibitory Concentration (IC50) and Minimal Fungicidal Concentration (MFC) determination also confirmed the potent antifungal activity of 10b. In addition, crystal violet staining method and XTT reduction assay were performed to investigate the anti-biofilm activity of 10b. The analytic results showed 10b dramatically inhibit biofilm formation and siginificantly reduced mature (48 h) biofilm metabolic activity. Moreover, the inhibitory effect of 10b was more powerful than that of farnesole. Confocal laser scanning microscopy (CLSM) showed that preincubated with 0.1μM 10b, the typical architecture of biofilms (intertwining mycelial structures and a basal layer of blastospores) was destructed although pseudohyphae and true hyphae were observed. When the concentration was increased by 10-fold, the adherent yeast cells were successfully prevented from germination and resulted in scant or nonexistent biofilms. To clarify the underlying action mechanism, Thin-Layer Chromatography (TLC) assay and Gas Chromatograph-Mass Spectrometer (GC/MS) assay were performed to investigate the targets of 10b in the ergosterol biosynthetic pathway amd transmission electron micrograph was used to observe the ultrastructure of C. albicans cells. Then, cDNA microarray study and realtime RT-PCR assay were conducted to study and research the genes expression profiles of yeast C. albicans or C. albicans biofilms treated or untreated with 10b. Finally, cytotoxicity of 10b in vitro was assessed by MTS/PMS reduction assay. TLC analytic results showed that sterols in C. albicans strain treated by 10b were evidently different from that of treated by KCZ. Their major differences in sterol classes were most pronounced in lanosterol and its analogues. The spottednesses of lanosterol and its analogues in KCZ-grown cells were significantly bigger and deeper than that of control-grown cells while disappeared in 10b-grown cells. It is presumed that 10b possesses a mode of action different from KCZ in ergosterol biosynthetic pathway.10b might inhibit the activities of ergosteol metabolic enzymes except for lanosterol 14a-demethylase and resulted in lanosterol changing into other intermediate sterols. GC/MS analytic results showed that 10b produced a profile similar to that of homozygous erg24 strain, in which ignosterol and ergosta-8,14,22-trienol were major sterols and no trace of 24-methylene-lanost-8-en-3-ol was found. However, KCZ-grown cells had a profile with 24-methylene-lanost-8-en-3-ol like the predominant sterol. We therefore postulate that sterol C-14-reductase, encoded by ERG24, instead of lanosterol 14α-demethylase, encoded by ERG11, is an important target for 10b in this pathway.10b also showed an action mechanism different from that of morpholines in the ergosterol biosynthetic pathway, although both of them are sterol C-14-reductase inhibitors. Morpholines inhibit sterol C-8 isomerase, encoded by ERG2 gene, resulting in ergosta-8-en-3-ol accumulation. As sterol C-8 isomerase is situated in the downstream of sterol C-14-reductase, Fm-grown erg24 strain produced a profile completely identical to that of erg24 strain. Although the sterol composition of 10b-grown cells was completely identical to that of erg24 strain, the ratio of ergosta-8,14, 22-trienol to ignosterol was different. The content of ergosta-8,14,22-trienol in 10b-treated erg24 strain was much more than that of in erg24 strain without 10b treatment, indicating that there must be another important target upstream sterol C-14 reductase. We deduced that another target was an enzyme related to sterol C-5 desaturase, which catalyzes the conversion of ergosta-8,14,22-trienol to ergosta-5,8,14,22-tetraenol. In addition, we found that the content of ignosterol built up in the wake of increase in the concentration of 10b, indicating that sterol C-5 desaturase related enzyme was the dominant target of 10b at a low concentration, while the affinity of 10b with sterol C-14 reducase augmented along with the increase in the concentration of 10b and finally changed into the fundamental target at a higher concentration. Although the ERG24 gene, encoding the C-14 sterol reductase, has been reported to be essential to the aerobic growth of Saccharomyces cerevisiae, the erg24 mutant of C. albicans is capable of growth under normal aerobic conditions on standard defined and enriched media. Transmission electron micrographs show that 10b treatment cannot changes the integration and permeability of the fungal membrane. This indicates that the major mechanism of action of 10b against C. albicans is uncorrelated to inhibiting the activity of sterol C-14 reducase in ergosterol biosynthetic pathway. Then a cDNA microarray study and real-time RT-PCR assay were performed to further clarify the mechanism of action of 10b. Yeast C. albicans cDNA microarray and realtime RT-PCR analytic results showed that 10b treatment resulted in marked down-regulation in metabolism-related genes, including glycolysis-related genes (e.g., PFK1, CDC19 and HXK2), fermentation-related genes (e.g., PDC11, ALD5 and ADH1) and respiratory electron transport chain-related genes (e.g., CBP3, COR1 and QCR8). Real-time fluorimetric assay revealed that 10b treatment increased generation of endogenous reactive oxygen species (ROS), which was similar to or more powerful than that of miconazole (MCZ). Function analytic results shawed that 10b treatment decreased mitochondrial membrane potential (ΔΨm), ubiquinone-cytochrome C reductase (complex III) activity and intracellular ATP level in C. albicans SC5314 strain. Besides, addition of antioxidant ascorbic acid (AA) reduced the antifungal activity of 10b significantly. These results suggest that mitochondrial aerobic respiration shift and endogenous ROS augmentation might contribute to the antifungal activity of 10b against yeast C. albicans. C. albicans biofilm cDNA microarray and realtime RT-PCR results showed that 10b treatment resulted in a striking down-regulation of hypha-specific gene ECE1 and a marked up-regulation of transcriptional repressors NRG1 in C. albicans biofilms, which is directly linked to inhibit biofilm formation. Moreover, the expressions of glycolysis related genes (e.g., HXK2 and PFK1), fermentation-related genes (e.g., ADH1) and antioxidant defense (e.g., SOD5) were decreased markedly. Functional analysis indicated that addition of anti-oxidant ascorbic acid reduced inhibitory efficiency of 10b on mature biofilm. These results indicated that inhibition of 10b on biofilm formation possibly depends on impairing the ability of C. albicans to change its morphology via altering the expression of biofilm formation genes. Mitochondrial aerobic respiration shift and endogenous ROS augmentation might be a major contribution to reduce mature biofilm metabolic activity. Although 10b seemed to reduce MTS/PMS reduction in a dose dependent manner, IC50 value for mammalian cells was much higher than MIC50 value for C. albicdns. This indicates that the formulation is preliminarily safe and warrants further study for possible human applications. Our data provide useful information for the development of new antifungal agents with novel chemical structure and distinct targets in ergosterol biosynthetic pathway and of new strategies to reduce candida infections.更多还原