【作者】 安毛毛；

【导师】 姜远英；

【作者基本信息】 第二军医大学 ， 药理学， 2011， 博士

【摘要】 近年来临床上深部真菌感染率大幅上升,已成为免疫功能低下患者死亡的主要原因之一。目前可供临床选择的治疗深部真菌感染药物较少且毒副作用大,再加上日益严重的真菌耐药现象使其防治成为临床上的棘手难题。目前深部真菌感染的主要病原菌仍然是白念珠菌,耐药现象也最为突出,因此白念珠菌深部感染的防治也是抗真菌感染研究领域的重点。我国中药资源丰富,来源广泛,而且中药用于真菌感染(主要是浅部真菌感染)具有悠久的历史,从中药中筛选抗真菌药物并研究其作用机制已经成为目前抗真菌药物研究领域的重要方向。大蒜是百合科葱属植物的鳞茎,含复杂的化学成分,主要包括大蒜素、蒜氨酸、微量元素等多种活性成分。大蒜素是从大蒜的球形鳞茎中提取的挥发性油状物,是大蒜中主要生物活性成分的总称。Allicin又名大蒜新素,化学名二烯丙基二硫化物,是大蒜素的主要活性成分之一,具有抗病原微生物、抑制肿瘤细胞生长、降血脂、抗血小板、降血压、预防心血管疾病、抗氧化等多种药理学作用。早在上世纪70年代就有研究表明allicin具有抗真菌活性,由于其活性较弱一直未应用于临床,但有文献研究表明allicin具有协同Cu2+抗白念珠菌和协同两性霉素B抗酿酒酵母菌的活性,提示allicin可能也具有协同抗真菌药物对常见致病真菌的抗菌活性。本课题从联合用药的角度出发,考察allicin与临床常用抗真菌药物的相互作用。本研究采用美国临床实验室标准化协会(Clinical and Laboratory Standards Institute,CLSI)CLSI-M27A3和M38A2文件所推荐的微量液基稀释法(Broth Microdilution)以棋盘法设计检测allicin与临床常用抗真菌药物联合应用对致病真菌的体外最低抑菌浓度(Minimal Inhibitory Concentration,MIC),以部分抑制浓度指数(fractional inhibitory concentration index,FICI)为主要指标考察药物之间的相互作用,实验结果表明allicin与两性霉素B、氟康唑合用对白念珠菌存在较强的协同作用(FICI <0.5),allicin与两性霉素B合用或者与氟康唑合用后,两性霉素B与氟康唑的MIC值均显著降低,allicin的MIC值也降低到1-8μg/ml之间,并且allicin与两性霉素B的协同作用强于氟康唑;allicin与两性霉素B、氟康唑合用对丝状真菌不存在协同作用,表现为无关作用(0.5<FICI<4);allicin与5-氟尿嘧啶、特比萘芬合用对常见致病真菌均不存在协同作用,表现为无关作用(0.5<FICI<4)。本课题深入研究了allicin协同两性霉素B抗白念珠菌的作用及可能的作用机制。两性霉素B (amphotericin B,AmB)属于多烯类抗真菌药物,其耐药率最低,抗菌谱广,被称为治疗深部真菌感染的“金标准”,但是严重的毒副作用尤其是肾毒性大大限制了两性霉素B在临床上的应用。本课题研究了allicin协同两性霉素B抗白念珠菌作用,结果发现allicin与两性霉素B合用对40株临床分离白念珠菌FICI均小于0.5,全部表现为协同作用;allicin能使两性霉素B对白念珠菌的时间-杀菌曲线明显下移;allicin能够协同两性霉素B对白念珠菌芽管及菌丝形成的抑制作用;动物体内实验结果研究发现,与单独应用两性霉素B相比,allicin与两性霉素B合用能够显著延长系统性白念珠菌感染的免疫功能低下小鼠的生存时间(P<0.05),并且显著降低肝脏、脾脏、肾脏主要脏器的真菌负荷量(P<0.05)。目前为止尚未有文献提示allicin协同两性霉素B抗白念珠菌可能的作用机制,本课题考察了两性霉素B单用和allicin与两性霉素B合用对白念珠菌基因表达谱和总代谢谱的差异,旨在整合比较转录组学和代谢组学研究结果初步发现allicin协同两性霉素B抗白念珠菌可能的作用机制。研究结果提示allicin是通过多途径、多靶点发挥协同作用的,可能的作用机制包括氧化损伤、抑制麦角甾醇合成通路、抑制细胞电子传递链影响能量代谢。根据转录组学和代谢组学研究结果,本课题主要从以上三方面验证了allicin协同两性霉素B抗白念珠菌作用机制,进一步的研究结果发现:①allicin通过氧化损伤作用发挥协同两性霉素B抗白念珠菌作用:allicin单独作用于白念珠菌对其细胞内活性氧水平及活性氧所导致的细胞膜的脂质过氧化损伤程度并无显著影响,allicin与两性霉素B联合作用于白念珠菌时细胞内活性氧水平及其所导致的氧化损伤(如细胞膜脂质过氧化损伤)程度均较单用两性霉素B显著升高,同时白念珠菌细胞内抗氧化损伤活性物质还原型谷胱甘肽水平显著降低,也提示allicin能够协同两性霉素B对白念珠菌的氧化损伤;②allicin通过抑制麦角甾醇生物合成通路发挥协同作用:allicin与两性霉素B联合应用和单独应用两性霉素B相比白念珠菌细胞内角鲨烯含量显著升高而麦角甾醇含量显著降低,提示allicin可能通过抑制白念珠菌角鲨烯环氧化酶阻断角鲨烯向羊毛甾醇的合成,从而抑制白念珠菌细胞膜重要成分麦角甾醇的合成,发挥与两性霉素B的协同作用;③allicin与两性霉素B联合应用和单独应用两性霉素B相比细胞内ATP含量降低,能量代谢障碍;④allicin在真菌体外培养基中可代谢生成活性化合物Diallyl disulfide(DADS),发挥协同抗白念珠菌作用。综上所述,本课题研究发现allicin具有体外和动物体内协同两性霉素B抗白念珠菌作用;allicin是通过多途径、多靶点发挥协同抗真菌作用的,其主要作用机制包括氧化损伤、抑制麦角甾醇生物合成、抑制细胞内ATP产生及产生活性代谢产物等,但是allicin协同抗白念珠菌作用的具体靶点及动物体内的药代动力学尚有待于进一步研究。更多还原

【Abstract】 With the increasing immunocompromised patients in recent years, the incidence of systemic fungal infections has been raised dramatically with a heavy mortality. Owing to the adverce effect of antifungal drugs and the severely drug resistance of fungal pathogen, the therapy systemic fungal infections is becomes more and more difficult. Candida albicans (C.albicans) is the major opportunistic fungal pathogen of humans and investigation on the treatment of candidiasis is an important issue.Screening active component from traditional Chinese drug is an important strategy for antifungal drug investigation. Allicin (diallyl thiosulfinate) is the main biologically active component of freshly crushed garlic extract. Allicin exerts various biological activities such as antimicrobial and anticancer activities in addition to the capacity to lower serum lipid levels, particularly cholesterol levels, and ocular pressure. Previous published data indicated that allicin had certain in vitro antifungal activity, but the minimal inhibitory concentration (MIC) was relatively high, limiting its clinical utility. Researchers demonstrated that Cu2+ exerted fungicidal activity by promoting endogenous ROS production, and recent studies found that allicin could enhance the fungicidal activity of Cu2+. It is suggested that allicin may also be able to enhance the fungicidal activity of antifungal drugs.In the present study, we investgated the interaction effect beteen allicin and antifungal drugs including fluconazole, amphotericin B, terbinafine and 5-fluorouracil. The in vitro MICs of the compounds against isolates of fungal pathogen were determined by the microbroth dilution method according to the Clinical and Laboratory Standards Institute (formerly the National Committee for Clinical Laboratory Standards). The fractional inhibitory concentration index (FICI) is defined as the sum of the MIC of each drug when used in combination divided by the MIC of the drug used alone. Synergy and antagonism were defined by FICIs of≤0.5 and >4, respectively. A FICI result of >0.5 but≤4 was considered indifferent. Our results indicated that AmB+ allicin combination and FLC+allicin combination markedly reduced MIC values of either individual agent for C.albicans and synergism was observed in all isolates of C.albicans (FICI <0.5).The polyene macrolide antibiotic AmB is the gold standard of antifungal treatment for the most severe invasivemycoses. However, owing to its poor permeability across membranes, an increased amount of AmB must be administered to patients in clinical situations, often resulting in severe side effects such as renal damage. To lessen the severity of the side effects, AmB is often combined with other antifungal drugs. So in the study, we further confirmed the synergic effect beteen allicin and AmB. Our results indicated that Synergism was observed in all 40 isolates (all the FICI<0.5) .The synergic effect of allicin with AmB was also confirmed in time-kill curves. The fungistatic activity of AmB was dramatically enhanced by addition of allicin. To assess the antifungal activity of AmB combined with allicin, initial experiments were performed by challenging mice. The adddtion of allicin (1 mg/kg) to AmB (0.5 mg/kg) treatment for mice infected by C.albicans significantly improved the survival time (P<0.05) and significantly reduced the fungal burden in kidney tissues, liver tissues and spleen tissues.There is no study suggested the possible mechanism of the the synergic effect beteen allicin and AmB. So in the present study, we first investigated the change of gene expression profiling with a complementary DNA microarray and metabolite profile with GC/MS analysis in C.albicans after addtition of alllicin to AmB treatment. Our results suggested that oxidative damage, ergosterol biosynthesis inhibition and electron transport chain blocking may be involved in the mechanism of the the synergic effect beteen allicin and AmB.Our further resaeach work indicated: (1) ROS production significantly elevated in C.albicans after addtition of alllicin to AmB treatment. And MDA level, representing the phospholipid peroxidation reaction, was significantly elevated in C. albicans treated by AmB in the presence of allicin, indicating that allicin could accelerate the endogenous ROS production and thus induced oxidative damage such as phospholipid peroxidation; (2) addition of allicin to AmB treatment could inhibit ergosterol production and lanosterol, while elevated squalene level. The above results suggested that maybe target squalene epoxidase to block ergosterol biosynthesis signaling; (3) addition of allicin to AmB treatment induced ATP production inhibited, exerting synergic effectwith AmB; (4) the breakdown products of allicin, diallyl disulfide(DADS), also posees the synergic anticandidal effect with AmB.To conclusion,the present study shows that allicin has a synergistic effect with AmB against C. albicans in vitro and in vivo. Oxidative damage, ergosterol biosynthesis inhibition and inhibition of mitochondrial ATP production may be involved in the mechanism of the the synergic effect. Our study suggested that allicin was a promising and safe agent to combine with AmB for efficacy and to reduce the AmB dose to lessen side effects, although the molecular mechanisms need further study.更多还原