【作者】 杨帆；

【导师】 茆灿泉；

【作者基本信息】 西南交通大学 ， 生物学， 2023， 硕士

【摘要】 马拉色菌(Malassezia.furfur,M.furfur)属于担子菌类酵母菌,当前由马拉色菌感染诱发的疾病患者数量众多,临床治疗却存在药物种类少、易复发以及存在副作用等问题,兼具抑菌活性与抗耐药特性的新型抗菌药物的研究与发现成为当务之急。基于当前马拉色菌感染的严重性和药物研发的现状,以及植物药具有的多种优势,本研究从植物药角度,开展了抗马拉色菌天然植物药材的筛选、植物药组方MYF-20的设计与药效确定并进行了抗马拉色菌作用机制的初步研究,其目的为抗马拉色菌植物药的研发提供重要的前期工作基础与参考。在本研究中,我们首先以糠秕马拉色菌(Malassezia furfur,M.furfur)为研究主体,基于课题组前期抗菌药物的研究结合我们对294种植物药醇提物的抗马拉色菌作用筛选结果,最终优选出侧柏叶、当归、红花等多种植物药合理配比的组方MYF-20。通过对组方MYF-20的体外抑菌性能与体内药效验证,我们发现MYF-20具有很强的抑菌作用,其对马拉色菌的最低抑菌浓度为7.0%,药物浓度7.83 mg/m L,并且抑菌效果具有明显的浓度与时间依赖性。马拉色菌的活性检测以及Ph Loxin B荧光染色结果表明,MYF-20可以在低至10 h内对马拉色菌展现出强的杀伤作用。在昆虫实验中,体内注射2.0×10~6 CFU马拉色菌的大蜡螟死亡率高达80%,但经10.0%MYF-20体内给药后成功将大蜡螟生存率提高了60.0%,最终大蜡螟的存活率稳定在80.0%以上。大蜡螟健康系统的评分结果显示,给药组健康水平变化趋势由对照组的急剧恶化趋于平稳,并在培养期间始终稳定。同时血淋巴真菌计数结果显示,MYF-20使大蜡螟体内的真菌数量降低了100倍,并且抑制了马拉色菌在大蜡螟体腔内的扩散。以上结果中,组方处理组结果均显著优于阳性药伊曲康唑处理组,证明MYF-20的体内抑菌活性高于伊曲康唑。而进一步的研究表明,健康大蜡螟注射10.0%MYF-20,对大蜡螟的健康与生存状态并无显著影响,显示组方具有低毒的特性。随后,我们又通过体外实验与转录组学分析,初步研究了MYF-20组方对马拉色菌的抑制作用机制。电导率测定与扫描电镜结果表明,组方可以作用于马拉色菌细胞膜,通过破坏细胞膜的完整性改变其通透性,引起胞内物质外流致使马拉色菌死亡;同时MYF-20的作用导致了马拉色菌芽孢断裂、细胞坍缩,细胞发生形态上的崩解。对马拉色菌生物膜形成的结晶紫染色显示,5.0%、10.0%、15.0%浓度的MYF-20均可破坏生物膜完整性,10%浓度的组方处理后生物膜几乎完全消失,说明组方在10.0%的浓度下就可以达到对生物膜的最大抑制作用。我们也分析了组方处理对马拉色菌脂肪酶活性的影响,与对照组相比,组方处理组脂肪酶在24 h、48 h活性分别降低了67.43%、86.75%,这进一步解释了组方对马拉色菌的强抑制作用及机制。在探究了MYF-20的作用途径后,我们又在转录组水平分析了组方可能的抑菌机制。差异基因GO、KEGG、GSEA富集结果显示,组方可能通过对MAPK信号通路的抑制,干扰马拉色菌的细胞周期,阻碍马拉色菌减数分裂形成孢子,从而减弱马拉色菌对药物的耐受并发挥其抑菌作用。最后,我们采用网络药理学与分子对接的计算机辅助研究方法,进一步探索MYF-20是否能作用于马拉色菌相关疾病靶点对疾病起到治疗作用。通过数据库检索共收集到组方有效活性成分17种及作用靶点214个,疾病靶点350个;通过网络药理学分析后筛选到核心靶点IL6、TP53、TNF、CXCL8、PTGS2、IL1B、CCL2、IL10、CTNNB1共9个,核心活性成分Quercetin、Kaempferol、Luteolin、beta-sitosterol、Pyrethrin II、lignan、qt＿carthamone、baicalein、DNOP、Hinokinin、Stigmasterol共11个。组方作用靶点GO富集分析结果显示,组方可能通过作用于细胞膜以及胞内膜细胞器,参与调控细胞信号识别、传导、炎症响应等途径,影响免疫系统活化、蛋白活性与蛋白结合活性等生物功能;KEGG富集分析结果显示,组方作用靶点主要富集结果包括对微生物感染的识别与免疫响应(疟疾、新冠病毒、结核、甲流感等)、炎性疾病(类风湿关节炎、炎症性肠病)与免疫炎症相关通路(Toll样受体信号通路、T细胞受体信号通路、JAK-STAT信号通路、NF-κB信号通路、TNF信号通路以及IL-17信号通路等)以及细胞因子相互作用与C型凝集素受体通路等生物学过程。分子对接结果证明组方活性成分Kaempferol、Qurecetin、Luteolin都可以与模式受体Mincle蛋白结合,结合能分别为-59.371 kcal/mol、-59.481 kcal/mol、-25.392 kcal/mol。综上所述,本研究以马拉色菌为研究主体,基于实验室研究基础,结合大量抗马拉色菌植物药筛选结果,挑选具有良好抑菌效果的单味植物药进行组方和科学配比得到植物药组方MYF-20,并通过体内外抑菌实验证明了组方具有良好的抑菌作用及安全性。随后结合实验与数据库数据分析初步探索了MYF-20抗马拉色菌的作用机制,分析显示MYF-20组方可以破坏马拉色菌细胞膜完整性,抑制生物膜形成与脂肪酶活性,并可能通过对MAPK信号通路的调控干扰马拉色菌的细胞周期与减数分裂进程,阻止马拉色菌以孢子形式对不利环境的耐受进而发挥抑菌作用。网络药理学与分子对接结果显示MYF-20可能通过槲皮素、山奈酚、木犀草素等活性成分作用于Toll样受体、C型凝集素受体等信号通路抑制免疫激活,同时通过对NF-κB信号通路、TNF信号通路以及IL-17信号通路等炎症通路的调控达到抑制炎症的效果。更多还原

【Abstract】 Malassezia furfur(M.furfur)belongs to basidiomycete yeast.There are many patients with diseases induced by Malassezia infection,but the clinical treatment suffers from few drugs,easy recurrence and side effects so far.Based on the current slow development of anti-Malassezia drugs,this study is expected to screen anti-Malassezia natural botanicals through antibacterial activity assay,select some of them for scientific formulation as MYF-20,carry out in vitro/in vivo antibacterial activity verification,and explore the potential antibacterial mechanism of MYF-20 with the help of transcriptomic analysis,network pharmacology analysis and molecular docking.Based on the previous antifungal drug research of our lab,combined with the screening results of 294 botanicals for anti-Malassezia effect,we finally selected a reasonable ratio of various botanicals such as Phellodendron leaf,Angelica sinensis and Safflower,and the formula was MYF-20.We found that MYF-20 has a strong antifungal effect,and the minimum inhibitory concentration for Malassezia was 7.0%,drug concentration was 7.83 mg/m L,and the inhibitory effect has concentration dependence and time dependence.The results of Malassezia activity assay and Ph Loxin B fluorescence staining showed that MYF-20 could show strong inhibition against Malassezia in as low as 10 hours.In the in vivo test,after the in vivo injection of 1.0×10~8 CFU/m L high concentration Malassezia solution to construct the model of Galleria mellonella,the mortality rate of the model group was as high as 80%,but after 10.0%MYF-20 in vivo administration successfully increased the survival rate of Galleria mellonella by 60.0%,and finally the survival rate of Galleria mellonella was stabilized at more than 80.0%.The scoring results of the health system of Galleria mellonella showed that the trend of health level change in the MYF-20 group tended to be stable from the sharp deterioration in the control group and remained stable during the incubation period.The results of the hemolymph fungal count also showed that the group reduced the fungal population in Galleria mellonella by 100-fold and inhibited the spread of Malassezia in the body cavity of Galleria mellonella.In the above results,all the results of the MYF-20treatment group were significantly better than those of the positive drug itraconazole treatment group,which proved that the in vivo antibacterial activity of MYF-20 was higher than that of itraconazole.On the other hand,the healthy Galleria mellonella was injected with PBS and10.0%MYF,and the results showed that the in vivo injection of MYF-20 had no significant effect on the health and survival status of Galleria mellonella,which also reflected the low toxicity of MYF-20.Secondly,we further investigated the mechanism of action of MYF-20 by in vitro experiments and transcriptome sequencing.The results of conductivity measurements and scanning electron microscopy indicated that MYF-20 could act on the cell membrane of Malassezia and change its permeability by disrupting the integrity of the cell membrane,causing the outflow of intracellular material and resulting in the death of Malassezia;meanwhile,the action of MYF-20 led to the breakage of Malassezia budding spores,cell collapse and morphological disintegration of cells.The crystalline violet staining of the biofilm formation of Malassezia showed that 5.0%,10.0%and 15.0%MYF-20 could destroy the biofilm integrity,and the biofilm almost completely disappeared after the 10%concentration of the formula,indicating that the maximum inhibitory effect of the formula on biofilm could be achieved at 10%concentration.We measured the activity of lipase of Histoplasma marcescens treated group,and compared with the control group,the lipase activity of Histoplasma marcescens treated group decreased by 67.43%and 86.75%at 24 h and 48 h,respectively,which further explained the strong inhibitory property and mechanism of action of Histoplasma marcescens.After exploring the pathway of action of MYF-20,we analyzed the possible inhibition mechanism of Histoplasma at the gene level by transcriptomics.The enrichment results of differential genes GO,KEGG,and GSEA showed that the histone may interfere with the cell cycle of Malassezia through the inhibition of MAPK signaling pathway and impede the meiosis of Malassezia to form spores,thus weakening the tolerance of Malassezia to the drug and exerting its inhibitory effect.Finally,we used methods of network pharmacology and molecular docking to continue to explore whether MYF-20 can act on Malassezia-related disease targets to play a therapeutic role in the disease.A total of 17 active ingredients and 214 active targets of the formula were collected through database search,and 350 disease targets;9 core targets of IL6,TP53,TNF,CXCL8,PTGS2,IL1B,CCL2,IL10,CTNNB1 were screened through network pharmacology analysis,and the core active ingredients of Quercetin,Kaempferol,Luteolin,beta-sitosterol,Pyrethrin II,lignan,qt＿carthamone,baicalein,DNOP,Hinokinin,Stigmasterol,11 in total.The results of GO enrichment analysis showed that the histone may act on cell membrane and intracellular membrane organelles to regulate cellular signal recognition,transduction,inflammatory response and other pathways,affecting biological functions such as immune system activation,protein activity and protein binding activity;the results of KEGG enrichment analysis showed that the main enrichment results of the histone targets include recognition and immune response to microbial infections(malaria The results of KEGG enrichment analysis showed that the main enrichment results of the histone targets include recognition and immune response to microbial infections(malaria,neocoronavirus,tuberculosis,influenza A,etc.),inflammatory diseases(rheumatoid arthritis,inflammatory bowel disease)and immune inflammation-related pathways(Toll-like receptor signaling pathway,T-cell receptor signaling pathway,JAK-STAT signaling pathway,NF-κB signaling pathway,TNF signaling pathway and IL-17 signaling pathway,etc.)and cytokine interactions with C-type lectin receptor pathway.Biological processes.The molecular docking results demonstrated that the active ingredients of the formula,Kaempferol,Qurecetin and Luteolin,could bind to Mincle protein with binding energies of-59.371 kcal/mol,-59.481 kcal/mol and-25.392 kcal/mol,respectively.In summary,our research focusd on Malassezia and based on the results of laboratory research and high-throughput screening of anti-Malassezia botanicals,botanicals with good antibacterial effect was selected and formulated to obtain the botanical formula MYF-20,which was proved to have good antifungal activity by in vitro and in vivo inhibition experiments,and then combined with experimental and database data analysis to explore the preliminary mechanism of action of MYF-20 against Malassezia.The analysis showed that MYF-20 could disrupt the integrity of Malassezia cell membrane,inhibit biofilm formation and lipase activity,and possibly interfere with the cell cycle and meiosis of Malassezia through the regulation of MAPK signaling pathway,preventing Malassezia from tolerating the unfavorable environment in the form of spores and thus exerting antibacterial effects.The results of network pharmacology and molecular docking showed that MYF-20 may act on Toll-like receptors,C-type lectin receptors and other signaling pathways through quercetin,kaempferol and lignocaine to inhibit immune activation,and through the regulation of NF-κB signaling pathway,TNF signaling pathway and IL-17 signaling pathway and other inflammatory pathways to achieve the effect of inhibiting inflammation.更多还原