【作者】 凌敏；

【导师】 梁健；

【作者基本信息】 广西大学 ， 生物学， 2022， 硕士

【摘要】 念珠菌性阴道炎(Vulvovaginal candidiasis,VC)是育龄期女性最常见的外生殖系统感染类型之一。据统计,四分之三的女性在一生中至少会感染一次VC,接近半数VC患者出现病情反复发作的现象。该病严重地降低了患者的生活质量。目前,临床上主要使用唑类抗真菌药物治疗VC,但由于抗菌药物长期、过度地使用,使得真菌耐药、药物副作用等问题日益严峻。因此,迫切需要安全、有效且副作用低的治疗方案来应对VC。近年来许多研究发现,使用传统中药外洗方治疗VC,临床效果显著且副作用少,但其相关的现代药理作用及机制不明确。因此,本课题通过检索诸多数据库中关于中药外洗方治疗VC的文献,收集相关药方,选择高频出现的核心组分,包括苦参、蛇床子、地肤子、穿心莲、岗松、黄柏、枯矾、冰片、栀子、龙胆草、花椒、五倍子十二种中药材,并将该十二种中药组成的复方命名为祛湿止痒方(QSZYF)。通过体内外实验、超高效液相色谱-质谱联用技术(UHPLC-MS/MS)结合网络药理学的方法,系统阐明QSZYF治疗VC的药理作用及机制。研究方法:首先,采用微量稀释法,考察通过水提醇沉法和醇提水沉法得到的QSZYF的不同提取物的体外抗白色念珠菌作用。通过对十二种中药进行减方配伍,研究不同配伍方式的抗白色念珠菌作用,并测定其最低杀菌浓度(MFC)值。其次,采用UHPLC-MS/MS分析QSZYF中的化学成分,并结合网络药理学方法,对QSZYF抗VC作用的关键靶点和信号通路等进行初步预测。然后,研究QSZYF体内抗白色念珠菌效果。通过建立小鼠VC疾病模型,采用阴道真菌计数、革兰氏染色法和HE染色法,检测给药后小鼠阴道内的真菌负荷、菌丝形成情况及阴道组织的炎症水平,从而评价QSZYF体内治疗效果。最后,通过酶联免疫吸附测定(ELISA)实验,验证QSZYF对网络药理学预测出的关键作用靶点表达水平的影响。体外药敏实验结果:QSZYF使用醇提水沉法提取所获得的醇提液,抗白色念珠菌效果优于使用水提醇沉法。三种减方配伍方中,QSZYF抗白色念珠菌效果最佳,其MFC值为5 mg/mL。采用UHPLC-MS/MS方法分析QSZYF中的化学成分,识别并筛选出98种活性成分,这些成分主要类别为萜类、黄酮类、生物碱类等化合物,涉及的药理活性主要包括抗炎、抗菌、抗病毒和免疫调节等。通过数据库查找并筛选得到98个活性成分相关的作用靶点1059个。从DrugBank、GeneCards以及OMIM数据库获得与VC相关的靶点420个。通过构建QSZYF成分-VC靶点网络,得到共同靶点1 10个。通过蛋白互作网络(PPI)分析,获得得分排名前10的关键靶点,包括ALB、IL-6、TNF、IL1B、CXCL8、IL-10、INS、STAT3、CCL2、TLR4。KEGG 富集分析显示,QSZYF抗VC的靶点富集在19信号通路上,包括细胞因子-细胞因子受体互作信号通路、IL-17信号通路、NF-κB信号通路等。GO分析显示,QSZYF抗VC的靶点涉及的生物过程包括对细菌来源分子的反应、正向调节细胞因子的产生、白细胞分化、炎症反应的调节、急性炎症反应等。体内抗白色念珠菌结果:采用腹腔注射苯甲酸雌二醇(1.25 mg/kg)+阴道接种白色念珠菌(1× 106 CFU/ml)方法造模。造模成功后将小鼠分成七组,采用阴道灌洗的方式用药物治疗7天。给药方案:对照组(100μL无菌水/只/d)、模型组(100μL无菌水/只/d)、QSZYF高剂量治疗组(40 mg/mL,100 μL/只/d)、QSZYF 中剂量治疗组(20 mg/mL,100 μL/只/d)、QSZYF 低剂量组(10 mg/mL,100 μL/只/d))、克霉唑组(15 mg/mL,100 μL/只/d)、肤阴洁洗液治疗组(100 μL/只/d)。阴道真菌计数结果显示:QSZYF高剂量组抗菌效果最佳。抗菌效果排序:QSZYF高剂量组>QSZYF低剂量>肤阴洁组>克霉唑组>QSZYF中剂量组(P<0.01);治疗结束7天后再次测定阴道中真菌负荷。抗菌效果排序:QSZYF低剂量组>QSZYF高剂量>QSZYF中剂量组>克霉唑组>肤阴洁组(P<0.01)。结果提示QSZYF可有效且持续地抑制白色念珠菌在阴道内增殖,作用效果优于克霉唑和肤阴洁;革兰氏染色结果显示:模型组阴道灌洗液在整个实验过程中均可见到假菌丝。QSZYF高、中、低剂量组和克霉唑、肤阴洁组阴道灌洗液中未见到假菌丝,提示QSZYF可有效抑制白色念珠菌在阴道内入侵和粘附。HE染色结果显示:和对照组相比,模型组阴道组织中可见大量中性粒细胞浸润,其粘膜层形成肉眼可见的微脓肿。QSZYF高、中、低剂量组和克霉唑组、肤阴洁组中性粒细胞数量显著减少,组织结构恢复平整,提示QSZYF可改善阴道组织炎症水平;ELISA结果显示:QSZYF可以显著降低小鼠血清中促炎细胞因子IL-6、IL-1β和TNF-α的表达水平,体内实验结果与网络药理学PPI分析预测结果相一致。该结果提示QSZYF有效治疗VC的作用机制可能与下调促炎症细胞因子IL-6、IL-1β和TNF-α的表达有关。更多还原

【Abstract】 Vulvovaginal candidiasis(VC)is one of the most prevalent and easy recur-rent gynecological diseases in women.In clinical practice,the antibiotics are usually used to treat VC.However,the prolonged and excessive use of antibiot-ics has led to increase the fungal resistance.At present,so many clinical studies have shown that treating vaginitis by Traditional Chinese medicine has good therapeutic effects.However,the relevant pharmacological studies and mecha-nisms are inadequate.Therefore,we collected twelve common herbs,including Ku Shen,She Chuang Zi,Chuan Xin Lian,Gang Song,Huang Bo,Bing Pian,Long Dan Cao,Hua Jiao,Wu Bei Zi,Di Fu Zi and Zhi Zi,which were used to treat in clinical practice and named as QSZYF.We investigated the anti-candida albicans effect of QSZYF in vitro and in vivo.The pharmacological effects and mechanisms of QSZYF in treating VC were investigated by combining Ultra-performance liquid chromatography-mass spectrometry(UHPLC-MS/MS)and network pharmacology.Methods:we examined the drug sensitivity of QSZYF in vitro and assessed the effectiveness of QSZYF for VC in vivo.Using UHPLC-MS/MS and network pharmacology identified the key compounds,core targets and pathways in QSZYF for VC.In animal experiments,Kunming mice were randomly divided into seven groups,including the control group,model group,three QSZYF-treatment groups(including low-dose group,medium-dose group,and high-dose group),and positive control group((including clotrimazole group and FYJ group).We adopted“estradiol+Candida albicans”to induce VC mice models and examined the therapeutic effectiveness by Colony counting,Gram staining and Hematoxylin eosin(H&E)staining.ELISA was applied to determine the se-rum inflammatory cytokines levels.Results of antibacterial test in vitro:The antifungal effect of QSZYF for VC,by ethanol extracting and water deposition,is better.The MFC of QSZYF for VC is 5 mg/ml.Furthermore,we identified 98 chemical components and1059 relevant potential gene targets of QSZYF for VC by UHPLC-MS/MS and network pharmacology.420 targets of VC were obtained from Drug Bank,Gene Cards and OMIM databases.110 common targets were obtained by con-structing the QSZYF-VC targets network.The top 10 scoring targets were ob-tained by the PPI analysis,including ALB,IL-6,TNF,IL1B,CXCL8,IL-10,INS,STAT3,CCL2,TLR4.The KEGG enrichment analysis showed that the targets of QSZYF-VC were enriched in 19 signaling pathways,including Cyto-kine-cytokine receptor interaction,IL-17 signaling pathway,NF-k B signaling pathway etc.The biological process of GO analysis showed that the targets of QSZYF for VC were cytokine receptor binding response to molecule of bacteri-al origin,positive regulation of cytokine production,leukocyte differentiation,regulation of inflammatory response,acute inflammatory response etc.We inte-grated the datasets and performed network analysis to distinguish the hub genes and key pathways.Results of experiments in vivo:We adopted“estradiol+Candida albicans”to induce VC mice models.After building the models,the mice were divided in-to seven groups and treated with drugs for 7 days by vaginal irrigation.The dos-ing regimens:control group(100μL sterile water/d),model group(100μL ster-ile water/d),QSZYF high-dose group(40 mg/m L,100μL/d),QSZYF medium-dose group(20 mg/m L,100μL/d),QSZYF low-dose group(10 mg/m L,100μL/d),Clotrimazole group(15 mg/m L,100μL/d),and FYJ group(100μL/d).The results of vaginal fungal counting showed that QSZYF high-dose group had the best antibacterial effect,and the effect ranking:QSZYF high-dose group>QSZYF low-dose group>FYJ group>Clotrimazole group>QSZYF medium-dose group(P<0.01).It suggesting that QSZYF was significantly decreased the vaginal fungal loading.The Gram staining results showed that hyphae could be seen in the vaginal irrigation solution of the model group throughout the exper-iment.In the other treatment groups,no hyphae were observed in the vaginal ir-rigation solution,which suggested that QSZYF disturbed the formation of hy-phae.The H&E staining results showed that the administration of QSZYF sig-nificantly reduced the neutrophil infiltration in the vaginal epithelium and the inflammatory response of the tissue.The ELISA results showed that the levels of IL-6,IL-1b and TNF-a in the model group were observably higher than the control group(P<0.01).The levels of IL-1b,IL-6 and TNF-a were decreased in QSZYF high-dose group and QSZYF medium-dose group compared with the model group.The results confirmed that QSZYF inhibited inflammatory re-sponse-induced high expression of IL-6,IL-1b and TNF-a,which suggested a strong correlation between the actual experiments in pharmacology and analysis of network pharmacology.更多还原