呋喃喹啉类生物碱白鲜碱体外抗真菌活性及作用机制研究
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摘要
真菌性感染是临床上重要的感染性疾病。然而随着抗真菌剂的广泛应用,临床耐药菌株不断增多,使得真菌耐药性问题日趋严重,因此急需开发新型抗真菌药物和寻找新型治疗方案。目前,从中草药中寻找有效抗真菌成分已成为研究热点。本研究旨在明确植物白鲜皮的活性成分呋喃喹啉类生物碱白鲜碱的体外抗真菌活性,考察其与常用抗真菌制剂氟康唑对白色念珠菌和红色毛癣菌等致病真菌协同抗菌效果,并以模式真菌酿酒酵母为研究对象力求从分子生物学角度阐明其作用机制。首先通过微量稀释法药物敏感性实验、棋盘式微量稀释实验、体外琼脂扩散实验和时间-杀菌曲线研究了白鲜碱对临床分离耐药真菌的体外敏感性。在此基础上,针对模式真菌酿酒酵母,运用基因芯片技术、实时荧光定量PCR、Western blotting技术、药物荧光吸收检测、激光扫描共聚焦、流式细胞术等研究了白鲜碱对模式真菌的作用机制。本研究在国内外首次将白鲜碱与氟康唑联合应用抗临床分离致病性真菌,结果表明,两药联合对26株耐药白色念珠菌中的20株菌具有显著协同效果,对红色毛癣菌和酿酒酵母菌株也具有显著的协同作用,FICI(Fractional inhibitory concentration index)值在0.25-1.5之间;两药合用药片的抑菌圈直径明显扩大且抗真菌活性的增强大于2 log10 CFU/mL。本研究在国际上首次利用DNA芯片技术从全基因组水平评价白鲜碱对模式真菌酿酒酵母基因表达谱的影响,揭示白鲜碱的抗菌作用分子机制,结果表明在白鲜碱诱导下,酵母细胞中的889个基因差异表达(表达倍数≥2),511个上调,378个下调,其中多药耐药、脂质生物合成、DNA复制和重组等pathway受到白鲜碱的影响。实时荧光定量RT-PCR和western blotting从基因水平和蛋白水平较好的检测了部分芯片结果。本研究还表明,白鲜碱能促使酿酒酵母细胞中若丹明6G外排增加,这与PDR5基因表达增高相关;64μg/mL的白鲜碱能明显提高酵母DNA含量(P<0.05),与DNA复制和重相关基因高表达相一致;32和64μg/mL的白鲜碱能显著破坏酿酒酵母细胞膜(P<0.05),这与细胞膜麦角固醇基因ERG高表达的芯片结果相一致。本研究为临床治疗真菌感染提供了新型治疗方案,为白鲜碱临床应用打下良好的基础。
Clinical infectious diseases are usually caused by three main types of micro-organisms including viruses, bacteria and fungi, so the treatment of fungal infections is an important part of clinical anti-infective treatment. At present, the application of drugs against fungi are restricted, because the drugs have high toxicity and are easy to produce drug resistance resulting in the risng of the recurrence rate and re-infection rates. There was an urgent need to develop new antifungal drugs with high efficiency and low toxicity. Currently, it is an important research direction and hot spot in the search for antifungal active ingredient from Chinese herbal medicine. In the past century, people have found more than 300 kinds of Chinese herbal medicines with anti-fungal activity. Furoquinoline alkaloids dictamnine, isolated from the root bark of Dictamnus dasycarpus Turcz (Rutaceae plants dry root bark), which has the activities of inhibiting platelet aggregation, anti-insects, vascular relaxation and so on. Recent studies have shown that dictamnine can induce the cytotoxicity of cervical cancer, colon cancer and oral cancer cell in human. It is worth noting that dictamnine has obvious antifungi activity.
At present, the combined application of antifungal agents has become one of the clinical important development of the clinical anti-fungal treatment. However, combination therapy at home and abroad reported are limited in a small number of combination between the antifungal agents and some combinations can produce the antagonism. So, screening the antifungal composition from Chinese herbal medicine and combining with current antifungal agents is an effective treatment program. Through the combination of antifungal agents can improve the therapeutic effect and reduce of the drug toxicity.
This study investigated the in vitro drug sensitivity of dictamnine alone and in combination with fluconazole (FLC) against clinical isolates fungi and by using the tests of minimal inhibitory concentration recommended by CLSI and checkerboard microdilution method, agar diffusion test, time-kill curves. The results showed that dictamnine have the antifungal activity. The interaction between FLC and dictamnine was synergistic in 20 out of 26 Candida albicans strains, Trichophyton rubrum and Saccharomyces cerevisiae (S. cerevisiae). At the same time, the two drugs found no antagonism. FICIs (Fractional inhibitory concentration index) ranging from 0.25 to 1.5. The MIC value of two drugs used in combination was lower than that of each drug used alone, the result showed that fluconazole and dictamnine have significant synergy antifungal activity in vitro. The results of agar diffusion test show synergy more intuitively. When fluconazole at 16μg combinated with different dose of dictamnine, the inhibition zone expanded significantly compared to the control. The boundary was clear and there is no colony growth in inhibition circle. To our knowledge, this is first report on evaluation of in vitro antifungi activity against clinical pathgenic fungi in combination with fluconazole and dictamnine. MTT cytotoxicity experiment showed that high-dose of dictamnine (≥1024μg/mL)had the the toxic effects on spleen cells of health mice, and this dose of dictamnine was hihger than MIC value of dictamnine against clinical drug-resistant fungal. So, viewing from the cytotoxic, dictamnine can be used as lead compounds for drug design. The experimental results can provide a reference of clinical medicine with dictamnine.
DNA microarray have a great value in drug screening, target gene identification, drug testing and administration of personalized delivery, gene discovery, genomic library mapping, species identification of traditional Chinese medicine, DNA research, computer applications, and so on. Fortune forecasted that bio-chip will has more impact on human than the micro-electronic chip in the 21st century. Gene chip provides a powerful tool in the study of gene function for the“post-genome project”and in modern medical science and medical diagnostics.
The yeast S. cerevisiae is an ideal organism for the study of antifungal action, because S. cerevisiae has strong ability of adapting to environmental changes, which has similar characteristics to higher organisms, most notably because the genome sequence of S. cerevisiae has already been completed and the function of almost 70% of the genes is known. Additional, S. cerevisiae DNA chips commercialized can be used to indicate putative gene function, understand biochemical pathways, and investigate relationships between regulators and their target genes. The advantages mentioned above make the possibility of studying the mechanism of antifungal agents on whole-genome by using S. cerevisiae as a model. In this study, the chips will provide a powerful tool for studying the global gene expression profile of S. cerevisiae induced by dictamnine. Microarray data analysis revealed that a large number of genes (889) were differentially expressed in response to dictamnine treatment. 511 genes increased in expression and 378 genes were inhibited. Transcriptome data was interpreted using the hierarchical cluster tool, T-profiler. The genes included in multidrug resistance response, lipid biosynthesis pathway, sulfate assimilation, DNA replication and DNA recombination were significantly regulated by dictamnine. The transcription factors rRPE, PAC, Novel Filamenta, PDR3, MSN2/4, RPN4, TBP and UPC2 were also affected by dictamnine. The results of microarray can lay the foundation for the study on the mechanism of dictamnine against other pathogenic fungi, especailly yeast-like fungi.
In addition, quantitative real-time RT-PCR and western-blotting were performed to verify the microarray results of selected genes. There was positive correlation between microarray data and real time RT-PCR data for all 11 genes, six genes were induced and five genes were reduced in response to dictamnine. The western-blotting results showed that the quantity of protein multidrug efflux protein Pdr5p was increased, which was consistent with the results of microarray. The results lays the foundation for further study on drug-resistance mechanisms of pathogenic fungi treated by dictamnine.
In order to furtherly verify the microarray results and clarify the action mechanism of dictamnine against fungi, we studied the effect of dictamnine on model fungus S. cerevisiae using the methods of glucose-dependent efflux of rhodamine 6G, laser scanning confocal microscopy and flow cytometry. The results showed that dictamnine can inhibit the growth of S. cerevisiae, the higher drug concentration, the stronger inhibition. Large dosage of dictamnine (2×MIC) can effect the intra-cellular hereditary substance, the contents of DNA was increased and inhibit the budding and sporing of S. cerevisiae; dictamnine can activate the efflux pump of S. cerevisiae, this is consistent with the result of microarray and western-blotting; the results of flow cytometry showed that dictamnine can fracture the cell membrane. Tests mentioned above are to lay the foundation in further explore the mechanism of the active ingredient from anti-fungal medicine.
This paper provides the new treatment protocols to clinical treatment of fungal infections; provides a useful theoretical basis for studying the action mechanisms of dictamnine against pathogenic fungi; and lays the foundation for the development and utilization of dictamnine.
Clinical infectious diseases are usually caused by three main types of micro-organisms including viruses, bacteria and fungi, so the treatment of fungal infections is an important part of clinical anti-infective treatment. At present, the application of drugs against fungi are restricted, because the drugs have high toxicity and are easy to produce drug resistance resulting in the risng of the recurrence rate and re-infection rates. There was an urgent need to develop new antifungal drugs with high efficiency and low toxicity. Currently, it is an important research direction and hot spot in the search for antifungal active ingredient from Chinese herbal medicine. In the past century, people have found more than 300 kinds of Chinese herbal medicines with anti-fungal activity. Furoquinoline alkaloids dictamnine, isolated from the root bark of Dictamnus dasycarpus Turcz (Rutaceae plants dry root bark), which has the activities of inhibiting platelet aggregation, anti-insects, vascular relaxation and so on. Recent studies have shown that dictamnine can induce the cytotoxicity of cervical cancer, colon cancer and oral cancer cell in human. It is worth noting that dictamnine has obvious antifungi activity.
At present, the combined application of antifungal agents has become one of the clinical important development of the clinical anti-fungal treatment. However, combination therapy at home and abroad reported are limited in a small number of combination between the antifungal agents and some combinations can produce the antagonism. So, screening the antifungal composition from Chinese herbal medicine and combining with current antifungal agents is an effective treatment program. Through the combination of antifungal agents can improve the therapeutic effect and reduce of the drug toxicity.
This study investigated the in vitro drug sensitivity of dictamnine alone and in combination with fluconazole (FLC) against clinical isolates fungi and by using the tests of minimal inhibitory concentration recommended by CLSI and checkerboard microdilution method, agar diffusion test, time-kill curves. The results showed that dictamnine have the antifungal activity. The interaction between FLC and dictamnine was synergistic in 20 out of 26 Candida albicans strains, Trichophyton rubrum and Saccharomyces cerevisiae (S. cerevisiae). At the same time, the two drugs found no antagonism. FICIs (Fractional inhibitory concentration index) ranging from 0.25 to 1.5. The MIC value of two drugs used in combination was lower than that of each drug used alone, the result showed that fluconazole and dictamnine have significant synergy antifungal activity in vitro. The results of agar diffusion test show synergy more intuitively. When fluconazole at 16μg combinated with different dose of dictamnine, the inhibition zone expanded significantly compared to the control. The boundary was clear and there is no colony growth in inhibition circle. To our knowledge, this is first report on evaluation of in vitro antifungi activity against clinical pathgenic fungi in combination with fluconazole and dictamnine. MTT cytotoxicity experiment showed that high-dose of dictamnine (≥1024μg/mL)had the the toxic effects on spleen cells of health mice, and this dose of dictamnine was hihger than MIC value of dictamnine against clinical drug-resistant fungal. So, viewing from the cytotoxic, dictamnine can be used as lead compounds for drug design. The experimental results can provide a reference of clinical medicine with dictamnine.
DNA microarray have a great value in drug screening, target gene identification, drug testing and administration of personalized delivery, gene discovery, genomic library mapping, species identification of traditional Chinese medicine, DNA research, computer applications, and so on. Fortune forecasted that bio-chip will has more impact on human than the micro-electronic chip in the 21st century. Gene chip provides a powerful tool in the study of gene function for the“post-genome project”and in modern medical science and medical diagnostics.
The yeast S. cerevisiae is an ideal organism for the study of antifungal action, because S. cerevisiae has strong ability of adapting to environmental changes, which has similar characteristics to higher organisms, most notably because the genome sequence of S. cerevisiae has already been completed and the function of almost 70% of the genes is known. Additional, S. cerevisiae DNA chips commercialized can be used to indicate putative gene function, understand biochemical pathways, and investigate relationships between regulators and their target genes. The advantages mentioned above make the possibility of studying the mechanism of antifungal agents on whole-genome by using S. cerevisiae as a model. In this study, the chips will provide a powerful tool for studying the global gene expression profile of S. cerevisiae induced by dictamnine. Microarray data analysis revealed that a large number of genes (889) were differentially expressed in response to dictamnine treatment. 511 genes increased in expression and 378 genes were inhibited. Transcriptome data was interpreted using the hierarchical cluster tool, T-profiler. The genes included in multidrug resistance response, lipid biosynthesis pathway, sulfate assimilation, DNA replication and DNA recombination were significantly regulated by dictamnine. The transcription factors rRPE, PAC, Novel Filamenta, PDR3, MSN2/4, RPN4, TBP and UPC2 were also affected by dictamnine. The results of microarray can lay the foundation for the study on the mechanism of dictamnine against other pathogenic fungi, especailly yeast-like fungi.
In addition, quantitative real-time RT-PCR and western-blotting were performed to verify the microarray results of selected genes. There was positive correlation between microarray data and real time RT-PCR data for all 11 genes, six genes were induced and five genes were reduced in response to dictamnine. The western-blotting results showed that the quantity of protein multidrug efflux protein Pdr5p was increased, which was consistent with the results of microarray. The results lays the foundation for further study on drug-resistance mechanisms of pathogenic fungi treated by dictamnine.
In order to furtherly verify the microarray results and clarify the action mechanism of dictamnine against fungi, we studied the effect of dictamnine on model fungus S. cerevisiae using the methods of glucose-dependent efflux of rhodamine 6G, laser scanning confocal microscopy and flow cytometry. The results showed that dictamnine can inhibit the growth of S. cerevisiae, the higher drug concentration, the stronger inhibition. Large dosage of dictamnine (2×MIC) can effect the intra-cellular hereditary substance, the contents of DNA was increased and inhibit the budding and sporing of S. cerevisiae; dictamnine can activate the efflux pump of S. cerevisiae, this is consistent with the result of microarray and western-blotting; the results of flow cytometry showed that dictamnine can fracture the cell membrane. Tests mentioned above are to lay the foundation in further explore the mechanism of the active ingredient from anti-fungal medicine.
This paper provides the new treatment protocols to clinical treatment of fungal infections; provides a useful theoretical basis for studying the action mechanisms of dictamnine against pathogenic fungi; and lays the foundation for the development and utilization of dictamnine.
引文
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