【作者】 李志鸿；

【导师】 杨素清；

【作者基本信息】 黑龙江中医药大学 ， 中医外科学（专业学位）， 2023， 博士

【摘要】 目的:1.通过中医传承辅助平台及数据挖掘技术,对杨素清教授治疗痤疮的临床医案进行回顾性研究,探究其治疗痤疮的用药规律、组方特色、辨证思路,总结临证经验。2.通过网络药理学及分子对接方法,初步预测导师治疗痤疮最具代表性的中医证型的核心药物组合,及其发挥治疗作用的潜在分子机制。3.结合中医传承辅助平台及网络药理学的研究结果,以网络药理学理论通路为基础,建立病证结合的痤疮动物模型,将痤疮痰瘀互结型核心方进行实验验证,系统分析核心方的微观作用靶点。方法:1.依据诊断、纳入、排除标准,收集2013年9月-2021年12月间黑龙江中医药大学附属第一医院杨素清教授门诊的痤疮有效医案751例。将收录的痤疮医案数据经初步处理后,采取双人录入方式上传至中医传承辅助平台(V2.5),归纳总结导师诊疗痤疮的常见中医证型、高频中药、性味归经、核心药物组合、用药规律及临证经验等。2.选取痰瘀互结型痤疮的核心药物组合进行网络药理学及分子对接分析。利用TCMSP数据库检索并获取该处方中药物的化学成分及潜在作用靶点,通过Cytoscape 3.7.1将其作用关系进行网络展示。在Genecards、OMIM、TTD及Drugbank数据库中获得痤疮的疾病靶点。以STRING数据库进行蛋白质相互作用网络分析并构建PPI网络,运用Cytoscape 3.7.1软件进行网络拓扑分析筛选该方治疗痤疮的核心靶点。借助DAVID数据库对交集靶点进行GO及KEGG通路富集分析;通过Autodock软件将主要活性成分与核心靶点进行分子对接,以预测该核心方治疗痤疮的潜在作用机制。3.采用油酸涂抹+痤疮杆菌注射+冰水游泳+高脂饮食法构建痰瘀互结型痤疮大鼠模型。将大鼠随机分为6组,每组5只大鼠,分别为空白对照组、模型对照组、西药对照组(异维A酸软胶囊)、中药低剂量组(核心方低剂量)、中药中剂量组(核心方中剂量)、中药高剂量组(核心方高剂量)。药物灌胃后,观察各组大鼠的皮损表观形态,记录灌胃过程中皮损的变化情况,并分别采用HE染色法对比各组大鼠皮损的微观病理形态改变;采用ELISA法对各组大鼠血清中mTOR、HIF-1α、VEGF的含量水平进行检测;采用 RT-PCR法对皮损中 mTORmRNA、HIF-1αmRNA、VEGFmRNA的表达情况进行检测;采用Western Blot法对皮损中mTOR、HIF-1α、VEGF蛋白的含量进行检测;采用免疫组化染色法对皮损中mTOR、HIF-1α、VEGF蛋白的阳性表达进行对比分析;同时,采用流式细胞法对各组大鼠血清中Th17细胞的表达情况进行检测,从而具体分析中药核心方对mTOR/HIF-1 α/VEGF通路及Th 17细胞的靶点作用情况。结果:1.中医传承辅助平台相关结果751例医案中,女性患者617例,男性患者134例。年龄分布情况:20-29岁,352例;30-39岁,203例;10-19岁,132例;其他年龄段共64例。有家族史的患者约占总人数的56%。602例患者自觉存在诱发因素,其中,饮食、情绪、晚睡熬夜、月经、化妆品等因素皆占较高比例。中医证型与痤疮程度分级情况:痰瘀互结型中重度Ⅳ级最多;肝郁化火型皆为中度Ⅲ级;冲任不调型以中度Ⅲ级为最多;血热风盛型中度Ⅱ级最多;寒凝气滞型中皆为重度Ⅳ级;热毒蕴结型中度Ⅲ级最多;脾虚湿盛型及风湿热蕴型皆为中度Ⅱ级。频次最多的10个症状由高到低依次为结节、脓疱、面部油腻、纳差、脉滑、月经后期、急躁易怒、腹胀、囊肿、苔白腻。中医证型分布情况:痰瘀互结型354例(47.14%)、肝郁化火型144例(19.17%)、冲任不调型108例(14.38%)、血热风盛型48例(6.39%)、寒凝气滞型42例(5.59%)、热毒蕴结型23例(3.06%)、风湿热蕴型17例(2.26%)脾虚湿盛型15例(2.00%)。药物四气频次从高到低依次为温、寒、平、热、凉。药物五味频次从高到低依次为辛、苦、甘、咸、酸。药物归经频次从高到低依次为肝经、脾经、肺经、胃经、肾经、心经、胆经、膀胱经、大肠经、小肠经、心包经、三焦经。用药频次排在前10位的药物依次为甘草、半夏、陈皮、浙贝母、柴胡、黄芩、乌药、青皮、炒白术、川芎。结合用药频次、关联规则、复杂熵聚类等网络分析的研究,确定导师诊疗痤疮所用核心药物组合为:黄芩、乌药、半夏、柴胡、青皮、陈皮、党参、甘草、炒白术、浙贝母、川芎。2.网络药理学及分子对接相关结果通过网络药理学分析,筛选出痰瘀互结型痤疮核心方药物化学成分228个,获取潜在靶点280个,检索得到痤疮的疾病靶点1466个,药物-疾病的交集靶点113个,核心靶点有STAT3、RELA、JUN、TNF、IL-6、AKT1、MAPK1、TP53、IL10、ESR1、MYC、IL4、IL1B、MAPK8、IL2、IL1A、CXCL8等。GO基因注释条目涉及492个BP、93个MF、44个CC(P<0.0 1);KEGG信号通路114条,涉及TNF信号通路、HIF-1信号通路、FoxO信号通路、Toll受体信号通路、T细胞受体信号通路、PI3K-AKT信号通路、NF-κB信号通路、MAPK信号通路、ErbB信号通路、VEGF信号通路、mTOR信号通路、类固醇激素生物合成、神经营养素信号通路、雌激素信号通路、Jak-STAT信号通路、p53信号通路、AMPK信号通路等。分子对接结果可见度值最高的2个活性成分与核心靶点AKT1、JUN、MAPK1、TNF、IL-6均具有较佳结合性。3.用核心方干预痰瘀互结型痤疮大鼠模型mTOR/HIF-1 α/VEGF通路及Th17细胞机制相关研究结果:使用制备好的痰瘀互结型痤疮大鼠模型,进行免疫通路与免疫细胞的机制研究。经核心方干预后,在宏观肉眼观察下,痰瘀互结型痤疮大鼠皮损表现出显著的修复过程,西药对照组≈中药高剂量组>中药中剂量组>中药低剂量组;在微观病理观察下,痰瘀互结型痤疮大鼠模型对照组中皮损仍存在明显的表皮角化异常、棘层增厚、炎性细胞浸润、毛细血管扩张等病理改变,而中药中剂量组、高剂量组及西药对照组的大鼠皮损组织基本恢复正常,结构完整,表皮细胞排列规则、整齐,与空白对照组相比,mTOR/HIF-1α/VEGF通路、Th17细胞在上述不同血清及皮损组织中均呈现出高表达状态。中药中剂量组、中药高剂量组、西药对照组可以显著降低血清中 mTOR、HIF-1α、VEGF、CD4+IL-17+的含量水平,抑制皮损中mTORmRNA、HIF-1αmRNA、VEGFmRNA的表达。同时,中药高剂量组和西药对照组可以显著抑制皮损中mTOR蛋白、HIF-1α蛋白、VEGF蛋白的含量水平。对于定位于细胞核和细胞质中的上述阳性蛋白的表达,中药中剂量组、中药高剂量组、西药对照组均能够显著抑制其蛋白含量的阳性表达率。中药低剂量组亦有一定的抑制mTOR/HIF-1α/VEGF通路相关指标及CD4+IL-17+表达的作用。由此说明,中药核心方可以显著影响mTOR/HIF-1α/VEGF通路及CD4+IL-17+由血清分布、到皮损基因、再到皮损蛋白合成的动态连续性过程。结论:1.导师治疗痤疮多从阳虚痰阻立论、重视温阳化痰。临证常将痤疮分为8个中医证型,其中“痰瘀互结型”为导师辨治最多的证型,常治以“温阳健脾、行气祛痰、活血化瘀”。2.通过对“痰瘀互结型”痤疮核心方的网络药理学机制的分析,初步推测其通过参与调节机体炎症反应、免疫机制、激素水平、皮脂代谢、氧化应激等多个生物过程,来达到治疗痤疮的作用。3.本研究建立了“痰瘀互结型”痤疮大鼠模型,从皮损和中医证候学角度对该模型进行了系统评价,为构建“痰瘀互结型”痤疮大鼠模型提供了科学、合理的依据。4.本研究在网络药理学基础上,发现了“痰瘀互结型”痤疮核心方对“痰瘀互结型”痤疮大鼠模型的干预机制靶点。其可能是通过抑制血清及皮损组织中mTOR/HIF-1α/VEGF信号通路传导与Th17细胞表达,来改善皮脂腺环境,抑制炎症反应,影响血管新生等,最终达到治疗痤疮的目的。更多还原

【Abstract】 Objective:1.Through the Traditional Chinese Medicine Inheritance Support System(TCMISS)and data mining technology,this paper makes a retrospective study on Professor Yang Suqing clinical medical records in the treatment of acne,analyzes the medication law,prescription characteristics and syndrome differentiation ideas in the treatment of acne,and refines her clinical experience.2.To preliminarily predict the core drug combination of the most representative TCM syndrome type of acne and the potential molecular mechanisms by which they exert their therapeutic effects.3.by network pharmacology and molecular docking methods,Combined with the results of traditional Chinese medicine inheritance support system and network pharmacology,the core prescriptions of acne key syndromes were used in the leading theoretical pathway of network pharmacology for experimental verification.To establish an animal model of acne with combination of disease and syndrome,and to analyze the micro targets of core prescriptions.Methods:1.According to the diagnosis,inclusion and exclusion criteria,751 valid acne medical records of Professor Yang Suqing outpatient clinic in the the First Affiliated Hospital of Heilongjiang University of traditional Chinese medicine from September 2013 to December 2021 were collected.After preliminary processing,the collected acne medical records were uploaded to the traditional Chinese medicine inheritance support system(V2.5)by double input.The common TCM syndrome types,high-frequency Chinese medicine,nature and taste tropism,core drug combination,medication rules and clinical experience of the tutor’s diagnosis and treatment of acne were summarized.2.Network pharmacology and molecular docking analysis were performed to select the core drug combination of acne with phlegm blocking collaterals.TCMSP database was used to retrieve and obtain the chemical composition and potential targets of the drugs in the prescription,and Cytoscape 3.7.1 was used to network display their interaction.The acne targets were obtained from Genecards,OMIM,TTD and Drugbank databases.Protein-protein interaction network analysis was performed using the STRING database to construct the PPI network.Cytoscape 3.7.1 software was used to analyze the network topology to screen the core targets of the formula in the treatment of acne.The DAVID database was used to perform GO and KEGG pathway enrichment analysis of intersection targets.Autodock software was used to perform molecular docking between the main active ingredients and the core targets to predict the potential mechanism of action of the core recipe in the treatment of acne.3.A rat model of phlegm and blood stasis syndrome acne was established by oleic acid smearing,Bacillus acnes injection,swimming in ice water and highfat diet.The rats were randomly divided into 6 groups,with 5 rats in each group.The rats were divided into blank control group,model control group,western medicine control group(isotretinoin soft capsule),Chinese medicine low-dose group(core prescription low dose),Chinese medicine medium-dose group(core prescription medium dose),Chinese medicine high-dose group(core prescription high dose).After drug gavage,the apparent morphology of skin lesions in each group was observed,and the changes of skin lesions during gavage were recorded.HE staining was used to compare the micropathological changes of skin lesions in each group;ELISA was used to detect the levels of mTOR,HIF-1α,and VEGF in serum of each group;RT-PCR was used to detect the expression of mTORmRNA,HIF-1αmRNA,VEGFmRNA,and Western Blot was used to detect the expression of mTOR,HIF-1α,and VEGF protein in the lesions.The expressions of mTOR,HIF-1α and VEGF proteins in the skin lesions were detected by immunohistochemical staining,and the expression of Th17 cells in the serum of rats in each group was detected by flow cytometry.The target effects of the core prescription of traditional Chinese medicine on mTOR/HIF-1α/VEGF pathway and Th17 cells were analyzed.Results:1.Related results of traditional Chinese medicine inheritance support systemAmong the 751 cases hace 617 female patients and 134 male patients.Age distribution:20-29 years old has 352 cases;30-39 years old has 203 cases;1019 years old has 132 cases;other age groups have 64 cases in total.Patients with family history accounted for about 56%of the total.602 patients felt that there were predisposing factors,among which diet,mood,staying up late,menstruation,cosmetics and other factors accounted for a high proportion.IIn terms of the relationship between the degree classification and TCM syndrome types,the phlegm turbidity Blocking Collaterals type has the most moderate and severe grade Ⅳ;the liver depression transforming fire type has the most moderate grade Ⅲ;the Chong Ren disharmony type has the most moderate grade Ⅲ;the blood hot wind type has the most moderate grade Ⅱ;the cold coagulation and qi stagnation type has the most severe grade Ⅳ;and the heat toxin accumulation type has the most moderate grade Ⅲ;spleen deficiency dampness excess type and rheumatism heat accumulation type are moderate grade Ⅱ;The 10 most frequent symptoms from high to low were nodules,pustules,greasy face,poor appetite,slippery pulse,late menstruation,irritability,abdominal distension,cysts,white and greasy fur.Statistical results of TCM syndrome types:Among 751 cases of acne,354 cases(47.14%)were phlegm blocking collaterals;144 cases(19.17%)were liver stagnation and fire;108 cases(14.38%)were Chong Ren disharmony;48 cases(6.39%)were blood hot wind;42 cases(5.59%)were cold coagulation and qi stagnation;23 cases(3.06%)were heat toxin accumulation;17 cases(2.26%)were rheumatic heat accumulation;15 cases(2.00%)were spleen deficiency and dampness accumulation.The four qi frequencies of drugs from high to low were warm,cold,flat,hot and cool.The frequency of the five flavors of the drugs used was from high to low,followed by spicy,bitter,sweet,salty and sour.The frequency of the drugs used was liver meridian,spleen meridian,lung meridian,stomach meridian,kidney meridian,heart meridian,gallbladder meridian,bladder meridian,large intestine meridian,small intestine meridian,pericardium meridian and Sanjiao meridian from high to low.The top 10 drugs with medication frequency are licorice,Pinellia ternata,tangerine peel,Fritillaria thunbergii,bupleurum,Scutellaria baicalensis,black medicine,green bark,fried Atractylodes macrocephala with bran and Ligusticum chuanxiong.Based on the study of frequency,clustering,association rules,and complex network analysis,the core drug combinations for the diagnosis and treatment of acne were determined as Scutellaria baicalensis,Radix aconiti,Pinellia pinellia,Radix bupleurum,Radix phellodendri,Radix codonopsis,Radix glycyrrhizae,Rhizoma atractylodes rhizoma,Fritillaria fritillaris,Phellodendri officinalis,Ligusticum chuanxiong.2.Research Results on Network Pharmacology and Molecular Docking:Through network pharmacology analysis,228 medicinal chemical components in the core recipe of phlegm-turbidium-blocking collateralblocking pattern were screened,280 potential targets were obtained,1466 disease targets of acne and 113 drug-disease intersection targets were retrieved.The core targets included STAT3,RELA,JUN,TNF,IL-6,AKT1,MAPK1,TP53,IL10,ESR1,MYC,IL4,IL1B,MAPK8,IL2,IL1A,CXCL8,etc.GO gene annotation entries involved 492 BP,93 MF and 44 CC(P<0.01).114 KEGG signaling pathways,Involved in TNF signaling pathway,HIF-1 signaling pathway,FoxO signaling pathway,Toll receptor signaling pathway,T cell receptor signaling pathway,PI3K-AKT signaling pathway,NF-kB signaling pathway,MAPK signaling pathway,ErbB signaling pathway,VEGF signaling pathway,mTOR signaling pathway,steroid hormone biosynthesis,Through nutrient signaling pathway,estrogen signaling pathway,Jak-STAT signaling pathway,p53 signaling pathway,AMPK signaling pathway,etc.The two active ingredients with the highest visibility value of molecular docking results had good binding with the core targets AKT1,JUN,MAPK1,TNF,and IL-6.3.Related research results of core prescription intervention on mTOR/HIFlα/VEGF pathway and Th17 cell mechanism:Under macroscopic observation,after the core prescription intervention,the acne lesions of rats showed a significant repair process,and western medicine control group≈Chinese medicine high dose group≈Chinese medicine medium dose group>Chinese medicine low dose group.After gavage,the pathological changes of microscopic skin lesions in each group showed that there were still obvious abnormal keratinization of epidermis,spinous layer thickening,inflammatory cell infiltration,telangiectasia and other pathological changes in the model control group,while the skin lesions of rats in the middle dose and high dose of Chinese medicine groups and the western medicine control group basically returned to normal,with complete structure and regular and neat arrangement of epidermal cells.Compared with the blank control group,mTOR/HIF-1α/VEGF pathway and Th17 cells were highly expressed in the above different serum and skin lesions.The levels of mTOR,HIF-1α,VEGF and CD4+IL-17+in serum were significantly decreased,and the expressions of mTORmRNA,HIF-1αmRNA and VEGFmRNA in skin lesions were inhibited in the TCM middle-dose group,the TCM high-dose group and the western medicine control group.At the same time,the high dose of traditional Chinese medicine group and the western medicine control group could significantly inhibit the expression levels of mTOR protein,HIF-1α protein and VEGF protein in skin lesions.For the expression of the above positive proteins located in the nucleus and cytoplasm,the TCM middle-dose group,the TCM high-dose group and the western medicine control group could significantly inhibit the positive expression rate of their protein content.The low dose of traditional Chinese medicine also inhibited the related indicators of mTOR/HIF-1α/VEGF pathway and CD4+IL17+expression.Therefore,the core prescription of traditional Chinese medicine can significantly affect the dynamic and continuous process of mTOR/HIF-1α/VEGF pathway and CD4+IL-17+from serum distribution,to skin lesion genes,and then to skin lesion protein synthesis.Conclusion:1.The tutor’s treatment of acne is mainly based on Yang deficiency and phlegm obstruction,emphasizing warming Yang and eliminating phlegm.The mentor’s clinical syndrome usually divides acne into 8 TCM syndrome types."Phlegm-stasis mutual knot type" is the most common syndrome type for the teacher to distinguish and treat,and is often treated with "warming Yang strengthening spleen,Qi removing phlegm,promoting blood circulation and removing blood stasis".2.Through the analysis of the network pharmacological mechanism of"phlegm-stasis internode type" acne core prescription,it is preliminatively speculated that it can achieve the effect of acne treatment by participating in the regulation of inflammatory response,immune mechanism,hormone level,sebum metabolism,oxidative stress and other biological processes.3.In this study,the acne rat model of "phlegm-stasis interjunction type"was established,and the model was systematically evaluated from the perspective of skin lesions and TCM syndromes,which provided a scientific and reasonable basis for the construction of the acne rat model of "phlegmstasis interjunction type".4.Based on network pharmacology,this study found the intervention mechanism target of "Phlegm-stasis Hu-jietype" acne core prescription on the acne rat model of "phlegm-stasis Hu-jietype".It may inhibit mTOR/HIFlα/VEGF pathway conduction and Th17 cell expression in serum and skin tissue,thus improving the environment of sebaceous glands,inhibiting inflammatory response,affecting angiogenesis and so on,and finally achieving the purpose of acne treatment.更多还原