【作者】 黄杉；

【导师】 姜远英；

【作者基本信息】 第二军医大学 ， 药理学， 2009， 博士

【摘要】 近年来,随着艾滋病的广泛传播,癌症的放疗、化疗,器官移植后免疫抑制剂的使用,广谱抗生素的大量使用,以及导管等越来越多的生物材料应用于人体,真菌感染率的不断增加,尤其以白念珠菌的感染最为普遍。全球很多中心检测统计资料表明,1996至2002年期间,在系统性真菌感染病例中,白念珠菌感染占50%-60%。由于长期广泛地预防性和治疗性使用唑类抗真菌药物(如氟康唑),白念珠菌的耐药现象越来越普遍,耐药程度也越来越高,日益严重的耐药现象和有限的治疗药物使白念珠菌感染成为临床上迫切需要解决的问题。黄芩素(黄芩苷元,Baicalein,BE)是从唇形科植物黄芩的干燥根中提取的有效成份之一。黄芩是常用中药,其味苦、性寒,归肺、心、肝、胆、大肠经,具有清热燥湿、泻火解毒、止血安胎等功效。黄芩素具有抗氧化、抗炎、抗菌、抗肿瘤、抗病毒等药理活性,且对血液细胞及肝脏细胞等正常细胞无毒性。虽然有研究报道黄芩素具有抗真菌活性,但由于其活性较弱,一直未应用于临床。鉴于目前白念珠菌普遍的多药耐药现象,本课题从联合用药角度出发,探索治疗白念珠菌感染的新策略。本课题首先采用微量液基稀释法考察黄芩素对白念珠菌的抑制作用,并以棋盘式微量稀释法测定黄芩素与氟康唑合用对白念珠菌的抑制作用。其结果显示:黄芩素单独使用时对敏感型白念珠菌具有抑制作用,其有效抗菌浓度为1μg/ml-2μg/ml,但对氟康唑耐药的白念珠菌无效。黄芩素与氟康唑合用于敏感型白念珠菌时,氟康唑的MIC80值在合用后均有降低,黄芩素的MIC80值在合用后有50%菌株呈现降低,但所有受试菌株的FIC指数均>0.5,两药相互作用表现为无关。在对氟康唑耐药的白念珠菌中,氟康唑与BE合用显示出显著的协同作用,原本对氟康唑耐药(MIC80≥64μg/ml)的菌株变得敏感,合用BE后氟康唑的MIC80值降低了32至1028倍,MIC80值分别在≤0.125至2μg/ml之间;BE的MIC80值也明显下降,受试菌株的FIC指数位于0.037-0.098之间,两药相互作用表现为协同。应用Time-kill curves实验对临床耐药白念珠菌0603433进一步验证氟康唑与黄芩素合用的协同作用。菌液起始浓度选用体外药敏实验的1~5×103CFU/ml和时间-杀菌曲线常用的1~5×105CFU/ml。当菌液起始浓度为1~5×103CFU/ml时,药物作用24h后,16μg/ml黄芩素单用对生长曲线没有影响,未见抗菌作用,其曲线与对照组生长曲线几乎重合。10μg/ml氟康唑显示较弱的抑菌作用,合用16μg/ml黄芩素24h后则大大增强了抑菌效果,表明两药合用具有协同抑菌作用。当菌液起始浓度为1~5×105CFU/ml时,单用16μg/ml黄芩素或单用10μg/ml氟康唑对生长曲线都没有明显的抑制作用,两药合用24h后,菌浓度比单用氟康唑明显下降。在对氟康唑耐药的白念珠菌中,以棋盘式微量稀释实验考察了BE与咪康唑、酮康唑的抗真菌相互作用。结果表明,咪康唑和酮康唑与BE合用均具有显著协同作用,合用BE后两者的MIC80值全部降到0.5μg/ml以下,FIC指数均<0.5,两药相互作用表现为协同。另外,BE与两性霉素B(Amphotericin B,AmB)合用具有协同抗真菌的作用。合用BE后两性霉素B的MIC值由1-2μg/ml降到0.062以下,明显提高了两性霉素B的杀菌作用。但对于近平滑念珠菌和光滑念珠菌中,黄芩素与氟康唑合用FIC值均在1.5以上,表现为相互作用无关。为了比较正常的与经黄芩素处理的白念珠菌生物被膜形态差异,以FUN-1和ConA染色后,激光共聚焦显微镜观察显示:正常生物被膜中含有大量菌丝,经8μg/ml黄芩素处理的生物被膜明显受到抑制,结构疏松,菌丝成分明显减少,极易从附着物上脱落。将不同浓度的黄芩素处理白念珠菌,48小时后XTT法测定生物被膜的生长动力学,结果表明,4μg/ml至32μg/ml的黄芩素可对白念珠菌生物被膜的抑制率达70%,2μg/ml的黄芩素对白念珠菌生物被膜的抑制率为15%,1μg/ml的黄芩素对白念珠菌生物被膜生长几乎无抑制作用。刮取经不同浓度的黄芩素处理的白念珠菌,测定CSH值,结果可见,在黄芩素浓度为32μg/ml时,白念珠菌的CSH值最小,为0.11,随着BE浓度的降低,CSH值逐渐增大,呈现出剂量依赖性,经1μg/ml BE处理的白念珠菌CSH值为0.85。采用实时定量RT-PCR法测定白念珠菌CSH相关基因CSH1的mRNA表达水平,结果表明,与对照组相比,经黄芩素处理的白念珠菌CSH1 mRNA表达水平明显下降。将不同浓度的黄芩素处理白念珠菌SC5314,12h后以流式细胞仪测定细胞凋亡率,同时采用已知真菌细胞凋亡诱导药物两性霉素B(AmB)作为阳性对照。结果显示,与空白对照组相比,黄芩素可明显诱导白念珠菌细胞凋亡,4μg/ml、8μg/ml黄芩素分别可诱导10%及20%的细胞凋亡。DAPI染色进一步确证了白念珠菌细胞凋亡的发生。应用透射电镜技术观察8μg/ml黄芩素作用于白念珠菌后细胞超微结构的变化。在白念珠菌的正常细胞超微结构中,细胞形状规则,细胞膜和细胞壁完整、光滑、边界清晰可见,胞核形完整,染色质均匀分布。经8μg/ml黄芩素处理的白念珠菌细胞核异常,染色质固缩,向核四周凝集,细胞质中有空泡产生,另外分裂细胞多处于两细胞粘连状态。另外,黄芩素对白念珠菌细胞内ROS产生的促进作用具有剂量依赖性,并随着时间的延长对ROS产生的促进作用也随之增强。32μg/ml黄芩素处理48h后ROS产生量是对照组的5倍。将16μg/ml的黄芩素处理白念珠菌SC5314,于不同时间点测定线粒体膜电位,同时以两性霉素B作为阳性对照。结果显示,与空白对照组相比,黄芩素可明显降低白念珠菌线粒体膜电位,经黄芩素处理12h的白念珠菌线粒体膜电位下降至未经处理组一半值。经32μg/ml黄芩素作用3h后,CAP1、SOD2及TRR1的mRNA表达水平明显上调,尤其是CAP1基因上调了近24倍,SOD2和TRR1基因的表达也分别上调了3倍和9倍。由于黄芩素可诱导白念珠菌氧化应激相关转录因子CAP1的mRNA高表达,本实验考察黄芩素对野生型菌(CAF2-1)、CAP1基因敲除菌(CJDADH)及高表达菌(CJDCAP1)细胞存活率的影响,结果显示,经32μg/ml黄芩素处理3h后,CJDADH、CAF2-1和CJDCAP1的存活率分别为35%、41.7%和54%。基因芯片杂交实验结果表明,黄芩素作用于白念珠菌以后,可引起多种相关基因的上调或下调,差异基因功能主要涉及细胞周期、跨膜物质转运、毒力因子及热休克蛋白等。更多还原

【Abstract】 With the wide spread of human immunodeficiency virus (HIV), misusing of tremedous antibiotic and immunosuppressive therapy to treat cancer or organ transplant, and also increasing number of biological material applying in human beings, the incidence of systemic fungal infections has been risen dramatically in recent years. Candida albicans infection is the most common isolates among them. It shows that C. albicans accounts about 50-60% in the major systemic fungal pathogen during 1996 to 2002. The triazole fluconazole is the most widely used antifungal drug to treat Candida infections. Unfortunately, wide spread uses of fluconazole have led to the rapid development of drug resistance which has severely hindered antifungal therapy.Baicalein (BE) is a major component originally isolated from the roots of Scutellaria baicalensis Georgi. Several different functions of BE have been reported. In addition to its inhibition effect on lipoxygenase, BE was found to have antioxidant, neuroprotective, antibacterial, antiviral activities. It was reported that BE also has antifungal activities, but could not be applied in clinical therapy because of its few effect. Attempts have been made to cope with treatment failures by using combination therapy. To seek for a novel combination therapy, we investigated the in vitro interaction of fluconazole and BE against fluconazole-resistant clinical isolates of C. albicans.In this study, we first investigated the effect of BE on C. albicans by microdilution assay and the synergistic activities between FLC and BE against FLC-resistant C. albicans by checkerboard microdilution assay. We found that the drug-sensititive C. albicans but not the fluconazole-resistant C. albicans was inhibited when baicalein was used alone,the effective concentration was 1μg/ml-2μg/ml. When used in drug-sensitive C. albicans with the combination of fluconazole and BE, each drug showed the little change comparing with drugs used alone. The fractional inhibitory concentration (FIC) index was all above 0.5,meaning no synergism in fluconazole-BE combination. However, the fluconazole-BE combination markedly reduced MIC80s, especially the MIC80 of either individual agent while used in fluconazole-resistant C. albicans. The corresponding median FIC index was 0.069 (range, 0.037 to 0.098). Synergism was observed in all isolates .Further time-kill studies were conducted with fluconazole and BE against one chosen clinical fluconazole-resistant C. albicans 0603433. BE did not affect the growth curve at 16mg/ml after 24 h, regardless of the initial inoculum is 103 or 105 CFU/ml,the fluconazole showed a little antifungal activity for initial inoculum is 103 CFU/ml . The fungistatic activity of fluconazole was dramatically enhanced by addition of BE when used in combination for 24h.Then we further investigated whether BE could play a same role with other antifungal agent or in other fungus. Respectively,the combination of baicalein and KCZ or MCZ reduced the MIC below to 0.5μg/ml both and showed significant synergistic activities against fluconazole-resistant C. albicans (FIC index <0.5).And the combination of BE and AmB reduced the MIC from 1-2μg/ml to 0.062μg/ml and also showed the synergistic activities .When used in Candida parapsilosis and Candida glabrata with the combination of fluconazole and BE, no synergism was observed (FIC>1.5).In order to examine the effect of baicalein (BE) on C. albicans biofilm formation,we dyed the BE-treated biofilm and untreated biofilm with the fluorescent stains FUN-1 and concanavalin A-Alexa Fluor 488 conjugate (ConA).Confocal scanning laser microscopy (CSLM) showed that normal C. albicans biofilm exhibited a typical three-dimensional nature, composed mainly of true hyphae. When cells were treated with 8μg/mL BE, biofilm development was inhibited and growth was predominantly composed of yeast cells and pseudohyphae.True hyphae were rarely observed, a factor that contributed to the poor biofilm architectures. To provide a more quantitative assessment of the extent of cell growth of biofilms in the presence or absence of BE, the ability of cells to reduce XTT was measured after the treatment for biofilms with different concentration of BE for 48h .The inhibitory effect on biofilms appeared to be dose-related. Over 70% inhibition was observed at BE concentrations between 4μg/ml and 32μg/ml A lower concentration (2μg/ml) produced 15% inhibition and 1μg/ml BE had almost no effect on biofilm formation.To examine the mechanism by which BE inhibits biofilm formation, the CSH of biofilm was measured by the biphasic separation method. Negative correlations were observed between BE concentration and the CSH of biofilm. High CSH was observed in normal mature biofilm. When the cells were treated with BE, CSH decreased. The relative CSH was 0.85 and 0.11 at BE concentrations of 1μg/ml and 32μg/ml, respectively.The mRNA expression levels of CSH1, which codes for the CSH-associated protein in C. albicans, were determined by real-time RT-PCR. The results showed that BE-treated cells expressed lower levels of CSH1 mRNA than the cells grown in the absence of BE, which correlated with the CSH exhibited by the cells.Here, we describe cellular changes that accompany death in C. albicans after exposure 12h to a range of different concentrations of BE , including treatment with the antifungal agent amphotericin B (AMB) as a positive control .Flow cytometry (FACS) assay showed that 10% or 20% apoptosis of C. albicans were induced respectively at baicalein concentrations of 4μg/ml or 8μg/ml.DAPI-staining reassured the occurrence of the apoptosis. Cells dying under 8μg/ml BE treated displayed several markers characteristic of apoptosis. These include the rapid exposure of phosphatidylserine (PS) at the outer cell membrane, cellular dyskaryosis,the generation of vacuole in cytoplasm, the margination of chromatin in nuclei, nuclear fragmentation, and the degradation of DNA.Compared to the normal cells, the apoptotic cells induced by BE showed increased endogenous reactive oxygen species (ROS) generation, and it shows the dose-dependent and time-dependent activity.The generation of ROS induced by 32μg/ml BE for 48h was 5 times higher than control . 16μg/ml BE induced cells shows less mitochondrial membrane potential and for 12h decreased 50% comparing with control. After treated with 32μg/ml BE for 3h , the mRNA expression of CAP1, SOD2 and TRR1 were apparently up-regulated.Considered the expression of CAP1 was up-regulated more than 24times than control, further experiment we did to investigate survival of wild type strains ,CAP1 knock-out strains and overexpression CAP1strains treated by BE. Reaults showed after induced by 32μg/ml BE for 3h,the survival of three strains were 35%,41.7%and54%.That means CAP1 played a role in baicalein-induced apoptosis. CAP1-deleted cells was very sensitive to baicalein.Gene chip analysis showed that the genes affected by baicalein were related to cell cycle, drug efflux, virulence, substrate transport and heat shock.更多还原