【作者】 吴秀祯；

【导师】 娄红祥；

【作者基本信息】 山东大学 ， 药物化学， 2010， 博士

【摘要】 近年来,随着广谱抗生素的大量应用、艾滋的蔓延、癌症化疗和器官移植所引起的免疫抑制的增加、以及器官插管术的广泛开展,临床真菌感染的发病率和死亡率呈逐年上升趋势。特别是持续性免疫缺陷患者,其真菌感染率更高。真菌感染,特别是深部真菌感染难以治疗。临床常用抗真菌药物主要是唑类药物和两性霉素B,这些抗真菌药物均对人体有不同程度的毒副作用；且随着抗真菌药物的广泛应用,临床上出现越来越严重的真菌耐药现象。因此,临床上迫切需要寻找新的抗真菌药物来治疗真菌感染。苔藓植物是植物界中仅次于被子植物的高等植物,全世界约有23000种。其中藓(mosses) 14000余种,苔(liverworts) 6000余种,角苔(hornworts)300余种。其在系统发育学上位于藻类和蕨类植物之间。在中国和印度,苔藓植物作为一种传统药物,被用来治疗心血管疾病、扁桃体炎、支气管炎、中耳炎、膀胱炎、皮肤病以及烧伤等。近年来植物学家从苔藓植物中分离获得了大量结构新颖且活性显著的萜类化合物和芳香族化合物,其中许多可作为新药研究的优良先导化合物。对苔类植物活性成分的研究表明苔类植物是生物活性天然产物的重要来源。地钱属植物为叶状体苔类。从地钱中分离得到的大环双联苄具有广泛的生物学活性,如抗氧化、抗真菌、抗病毒、抗细菌和细胞毒活性等,因此它们受到越来越多的关注。羽苔素E是从地钱(Marchantia polymorpha L. (Marchantiaceae))中分离得到的一种双联苄类化合物。药理学活性实验表明2～12μg/ml羽苔素E可以逆转阿霉素诱导的K562/A02细胞的多药耐药性,而对正常细胞仅表现微弱毒性。进一步研究发现,羽苔素E有抗白色念珠菌的作用,并通过提高耐药真菌细胞内氟康唑药物浓度而逆转真菌耐药。提示羽苔素E具有非常好的药物开发价值。但是羽苔素E的抗真菌作用机制尚未阐明。因此,本课题的目的就是通过一系列实验阐明羽苔素E的抗真菌机制。基因表达谱分析技术为羽苔素E的抗真菌机制研究提供了全新和有效的研究手段。利用白色念珠菌基因组芯片绘制了羽苔素E作用后白色念珠菌的基因表达谱,分析比较相互间的不同,在全基因范围内寻找受羽苔素E影响的相关基因。基因芯片结果显示多个被羽苔素E上调或下调的基因与线粒体功能有关。而线粒体是细胞内的重要细胞器,在氧代谢、ATP产生和钙平衡中发挥重要作用。提示线粒体是羽苔素E的潜在抗真菌靶点。因此,实验进一步测定了羽苔素E对白色念珠菌线粒体功能的影响。在实验中,采用罗丹明染色法测定羽苔素E作用后白色念珠菌的线粒体膜电势,高效液相色谱法测定白色念珠菌线粒体内ATP含量,分光光度法测定线粒体F0F1-ATPase和脱氢酶活性,荧光法测定线粒体内活性氧含量。实验结果显示羽苔素E导致白色念珠菌线粒体膜电势超极化和线粒体内ATP耗竭。线粒体F0F1-ATPase水解ATP的活性被显著提高,使得线粒体内ATP被过度水解,导致线粒体内ATP耗竭。在水解ATP的同时,F0F1-ATPase将质子由线粒体胞浆泵入线粒体膜间隙,进而导致线粒体膜电势超极化。线粒体脱氢酶的活性被显著抑制,引起真菌细胞内能量代谢异常,导致ATP合成减少。羽苔素E所致线粒体功能的异常导致线粒体内活性氧累积。抗氧化剂半胱氨酸不但可以抑制羽苔素E诱导的线粒体内活性氧累积,而且可以抑制羽苔素E的抗真菌活性。提示活性氧是羽苔素E发挥抗真菌作用的关键介质。活性氧是真菌凋亡的关键调节因子。因此,实验进一步研究羽苔素E诱导的白色念珠菌线粒体活性氧累积是否导致真菌凋亡。实验采用流式细胞术检测羽苔素E作用后细胞周期的变化,超薄透射电镜观察真菌超微结构的改变,DAPI染色法观察细胞核形态变化,FITC-annexin V染色法观察白色念珠菌磷脂酰丝氨酸的外翻, RT-PCR法测定羽苔素E对白色念珠菌CDC28、CLB2和CLB4表达水平的影响,FITC-VAD-FMK荧光染色法测定羽苔素E作用后白色念珠菌metacaspase活性,分光光度法测定线粒体内细胞色素C含量。实验结果显示羽苔素E导致白色念珠菌出现典型的凋亡特征,包括细胞周期阻滞在G2／M期、染色体凝集、细胞核裂解和磷脂酰丝氨酸的外翻。提示羽苔素E导致了白色念珠菌细胞凋亡。CDC28、CLB2和CLB4的表达被羽苔素E下调,导致白色念珠菌细胞周期阻滞。羽苔素E促进白色念珠菌线粒体内细胞色素C释放,并激活了metacaspase o抗氧化剂半胱氨酸抑制羽苔素E导致的细胞核裂解、磷脂酰丝氨酸的外翻以及metacaspase的活化,提示活性氧是羽苔素E所致细胞凋亡的重要介质。实验结果表明羽苔素E通过引起白色念珠菌线粒体活性氧累积,进而激活metacaspase诱导真菌凋亡,从而发挥其抗真菌作用。细胞壁是真菌细胞的重要结构,完整的细胞壁为刚性结构,作为物理化学屏障保护细胞及其内的酶类,并控制细胞内膨胀压力以维持菌体的完整性。细胞壁还参与调节营养物质的吸收和代谢产物的分泌,在维持真菌的生长和正常的生理功能中起到重要作用。几丁质是真菌细胞壁的主要成分之一。几丁质合成酶催化N-乙酰氨基葡萄糖在细胞膜上聚合成几丁质,在真菌细胞的分裂和成熟中发挥重要作用。在前期实验中,透射电镜结果显示羽苔素E处理后的白色念珠菌细胞壁出现明显损伤,并且细胞分裂受阻。提示羽苔素E可能对细胞壁几丁质合成有抑制作用。采用荧光法测定羽苔素E对几丁质合成酶活性以及几丁质原位合成的影响,并用荧光定量PCR法测定羽苔素E对三种几丁质合成酶基因表达的影响。实验结果显示羽苔素E抑制了几丁质合成酶活性和几丁质的原位表达。细胞壁合成被抑制导致细胞壁损伤,使得真菌细胞对渗透压敏感,导致真菌细胞死亡,从而发挥羽苔素E的抗真菌作用。此外,真菌细胞壁的损伤有益于氟康唑进入耐药真菌细胞内,提高了真菌细胞内氟康唑的药物浓度,从而发挥逆转真菌耐药的作用。本课题表明羽苔素E通过诱导线粒体内活性氧累积导致真菌凋亡以及抑制白色念珠菌细胞壁几丁质的合成而发挥抗真菌作用,为羽苔素E作用机制的阐明提供了理论依据。更多还原

【Abstract】 Over the last decade, the incidence of fungal infections has increased dramatically, causing high morbidity and mortality. Patients with defective immune systems, due to AIDS (Acquired Immune Deficiency Syndrome), cancer chemotherapy, or immunosuppressive drugs, are significantly affected by fungi. Additionally, with the large scale application of broad-spectrum antifungal agents and the introduction of protocols for antifungal prophylaxis in patients at risk, there has been a notable increase in drug resistance. The development of more effective antifungal therapies is therefore of paramount importance.Natural products are still major sources of innovative therapeutic agents in various conditions, including infectious diseases. The bryophytes [Musci (mosses), Marchantiophyta (liverworts) and Anthocerotae (hornworts)], are morphologically placed between the algae and the pteridophytes (fern). Liverworts have been used in various remedies of folk medicine to treat illnesses of the cardiovascular system, tonsillitis, bronchitis, tympanitis, cystitis, as well as skin diseases and burns in China and Japan, In the past decades, various types of lipophilic terpenoids and aromatic compounds, which show significant biological activities, are isolated from the bryophytes. Now the bryophytes have been well know as a source of biologically active, naturally occurring compounds.Macrocyclic bis(bibenzyls) isolated from liverworts were found to have a wide range of biological activities such as antioxidation, antifungi, antivirus, antibacteria and cytotoxicity. As a result, they are attracting more and more attention. Plagiochin E (PLE), a macrocyclic bis(bibenzyl) isolated from liverwort Marchantia polymorpha L. (Marchantiaceae), has been found to have the reversal effect on multidrug resistance in adriamycin-induced resistant K562/A02 cells at concentration of 2 to 12μg/ml, with little cytotoxicity to normal cells. Furthermore, it exhibited in vitro antifungal activity against Candida albicans, and also found to reverse fungal resistance to fluconazole by increasing accumulation of fluconazole in the C. albicans. However, the underlying mechanism of action is unknown. The present study was designed to investigate the antifungal mechanism of PLE in C. albicans.The genome-scale cDNA microarray platform provides an effective research tool to investigate the antifungal target of PLE. To get a panoramic view of the responses of yeast cells to the PLE at the molecular level, cDNA microarray analysis was conducted. The global gene expression changes in C. albicans responding to PLE treatment were measured. A lot of genes significantly up-regulated or down-regulated by PLE were related to mitochondria, which are important organelles in fungi. Mitochondria play very important roles in the life cycle of cells, which are involved in a range of processes, such as the oxygen consumption, ATP production, and calcium mobilization. The results suggested that the mitochondria are a potential target of PLE. As a consequence, the effects of PLE on mitochondria function in C. albicans were determined. We assayed the mitochondrial membrane potential (mt△Ψ) using rhodamine 123, measured ATP level in mitochondria by HPLC, and detected the activities of mitochondrial F0F1-ATPase and dehydrogenases. Besides, the mitochondrial dysfunction-induced reactive oxygen species (ROS) production was determined by a fluorometric assay, and the effects of antioxidant L-cysteine on PLE-induced ROS production and the antifungal effect of PLE on C. albicans were also investigated. Results showed that exposure to PLE resulted in an elevation of mtA△Ψ, and a decrease of ATP level in mitochondria. The ATP depletion owed to PLE-induced enhancement of mitochondrial F0F1-ATPase and inhibition of the mitochondrial dehydrogenases. These dysfunctions of mitochondria caused ROS accumulation in C. albicans, and this increase in the level of ROS production and PLE-induced decrease in cell viability were prevented by addition of L-cysteine, indicating that ROS was an important mediator of the antifungal action of PLE. In summary, PLE exerts its antifungal activity through mitochondrial dysfunction-induced ROS accumulation in C. albicans.ROS are a key regulator to yeast apoptosis. As a consequence, the further study was designed to investigate whether PLE induced apoptosis in C. albicans. We assayed the cell cycle by flow cytometry using PI staining, observed the ultrastructure by transmission electron microscopy, studied the nuclear fragmentation by DAPI staining, and investigated the exposure of phosphatidylserine at the outer layer of the cytoplasmic membrane by the FITC-annexin V staining. The effect of PLE on expression of CDC28, CLB2, and CLB4 was determined by RT-PCR. Besides, the activity of metacaspase was detected by FITC-VAD-FMK staining, and the release of cytochrome c from mitochondria was also determined. Furthermore, the effect of antioxidant L-cysteine on PLE-induced apoptosis in C. albicans was also investigated. The results showed that cells treated with PLE showed typical markers of apoptosis: G2/M cell cycle arrest, chromatin condensation, nuclear fragmentation, and phosphatidylserine exposure. The expression of CDC28, CLB2, and CLB4 was down-regulated by PLE, which may contribute to PLE-induced G2/M cell cycle arrest. Besides, PLE promoted the cytochrome c release and activated the metacaspase, which resulted in the yeast apoptosis. The addition of L-cysteine prevented PLE-induced nuclear fragmentation, phosphatidylserine exposure, and metacaspase activation, indicating the ROS was an important mediator of PLE-induced apoptosis. In summary, PLE induced apoptosis in C. albicans through a metacaspase-dependent apoptotic pathway.The fungal cell wall plays an important role in the growth and viability of fungi. Chitin is indispensable for the construction of cell wall, and therefore, for fungal survival. Inhibition of chitin polymerization may affect cell wall maturation, septum formation and bud ring formation, damaging cell division and cell growth. Observation under the TEM showed the structure of cell wall and cell division in C. albicans were seriously damaged by PLE, which suggested the antifungal activity of PLE was associated with its effect on chitin synthesis. As a result, the effect of PLE on chitin synthesis in C. albicans was investigated at cellular and molecular levels. The effect of PLE on chitin synthetases (Chs) activities in vitro were assayed using 6-O-dansyl-N-acetylglucosamine (DNAG) as a fluorescent substrate, and its effect on chitin synthesis in situ was assayed by spheroplast regeneration. Reverse transcription-PCR (RT-PCR) was performed to assay its effect on expression of chitin synthetase genes (CHS). Enzymatic assays and spheroplast regeneration showed PLE inhibited chitin synthesis in vitro and in situ. Results of RT-PCR showed PLE significantly down-regulated the expression of CHS1, and up-regulated the expression of CHS2 and CHS3. Because different Chs is regulated at different stages of transcription and posttranslation, the down-regulation of CHS1 would decrease the level of Chsl and inhibit its activity, and the changes on CHS2 and CHS3 would not affect the activities of Chs2 and Chs3. These results indicate that the antifungal activity of PLE would be attributed to its inhibitory effect on chitin synthesis of cell wall in C. albicans. The inhibition of PLE on cell wall biosynthesis affects the continuity of fungal cell wall, makes fungi sensitive about osmotic pressure, causing fungi death. This PLE-induced cell wall damage would contribute to the antifungal action of PLE, and also can promote the inflow of fluconazole into fungal cell, resulting in reversal of fungal resistance.Above results indicated that PLE inhibited cell wall chitin synthesis and induced apoptosis in C. albicans through activating the metacaspase by ROS accumulation. These results would conduce to elucidate its underlying antifungal mechanism.更多还原