基于“营卫”理论探讨银屑病与代谢免疫的关系

【作者】 刘勇；

【导师】 韩东燃；

【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2023， 博士

【摘要】 研究目的:银屑病是一种T细胞介导的慢性炎症性皮肤病,发病机制复杂,尚未完全阐明。目前生物制剂已经成为银屑病的主要治疗方法之一,直接从免疫角度,通过阻断免疫系统产生的特定炎症因子来治疗银屑病,其见效快,疗效好,但其也存在无法根治,仍会复发和有一定副作用等问题,按照中医的整体观念和治未病思想,我们既要关注治疗后皮损改善程度,也得关注患者生存时间、生存质量和复发问题。随着对银屑病研究的深入,逐渐认为银屑病也是系统性疾病,常伴发多种代谢相关疾病,其是影响银屑病患者生存预后的重要因素。因此为了更好的防治银屑病,我们对于银屑病的研究,既要关注免疫问题,也要考虑代谢因素。随着现代医学中代谢免疫学的发展,诸多学者认为“营卫”理论与代谢免疫生理病理有诸多类似之处,“营卫”早在《内经》中有诸多论述,不仅是人体基本生命活动物质之一,也是重要的中医基础理论之一。因此本研究的目的是依照中医“营卫”理论,探索银屑病患者代谢和免疫相关生物指标对于银屑病患者发病、死亡风险来说具有怎样的主次关系,为了更好的从代谢免疫角度治疗银屑病提供理论支持,也为了阐释从“营卫”论治银屑病的科学内涵。方法:第一部分为理论探讨,通过查阅有关银屑病和营卫的研究文献进行总结归纳,探讨了营卫的理论基础,营卫倾移动与银屑病的关系,从银屑病病机特点解读营卫与五脏、营卫与玄府、营卫与六经和营卫与湿热的关系,同时探讨了营卫的实质以及如何从“营卫”论治银屑病。第二部分为横断面研究,这项研究使用了 2003-2006年和2009-2014年美国国家健康和营养调查(NHANES)的银屑病数据,使用COX比例风险回归分析、随机森林生存分析和限制性三次样条模型来评估银屑病患者外周血实验室检查免疫代谢相关指标和银屑病死亡率之间的相关性。第三部分为生物信息学研究,从基因表达综合数据库(GEO)下载银屑病皮肤组织基因表达谱数据集和DNA甲基化数据集,通过免疫浸润分析、功能富集分析、蛋白-蛋白相互作用(PPI)网络构建、机器学习算法分析(LASSO、随机森林、SVM-RFE)等多种分析方法分析调控网络,挖掘与银屑病相关的靶基因。第四部分为实验研究,基于第三部分研究结果,通过Western blotting、Real-time PCR和免疫荧光等方法研究松香酸对极化态人外周血中性粒细胞的相关基因以及中性粒细胞胞外诱捕网(neutrophil extracellular traps,NETs)生成的影响。结果:第一部分理论探讨发现内外邪侵扰人体导致五脏、六经、玄府功能异常,进而“卫阳”奋起抗邪于脉外,同时牵动脉内“营阴”,最终形成营卫偏倾、分布异常,形成“营卫倾移态”,导致出现银屑病样皮损,久之则生湿生热伏于营卫,是银屑病难治易复发的原因。第二部分研究纳入了 528例银屑病患者,基于银屑病人群和性别亚组的研究结果,发现代谢指标尿酸(UA)、红细胞分布宽度(RDW)和糖化血红蛋白(HBA1C)是潜在的与银屑病死亡相关的生物标志物。在总体人群中,调整年龄、性别、种族、教育程度、吸烟状况、饮酒状况和收入后,UA(HR1.59,95%CI:1.24-2.04,P<0.001);RDW(HR 1.55,95%CI:1.16-2.07,P<0.001)和 HBAIC(HR1.47,95%CI:1.21—1.80,P<0.001)与死亡风险增加显著相关。血糖(RG,非线性P=9E-04)和糖化血红蛋白(HBAIC,非线性P=0.0014)也与死亡风险有很强的U形关联,分别在89mg/dl和5.1%时死亡风险最低。第三部分研究分析发现97个高甲基化和表达下调(Hyper-down)基因占比最高(47%)为银屑病主要甲基化基因,Hyper-down基因主要富集在葡萄糖稳态、AMP活化蛋白激酶(AMPK)信号通路、脂质储存疾病、部分脂肪营养不良和胰岛素抵抗等方面。胰岛素受体底物1(IRS1)、Rho鸟嘌呤核苷酸交换因子10(ARHGEF10)和维甲酸诱导蛋白14(RAI14)被确定为银屑病潜在靶点。第四部分研究发现松香酸治疗银屑病可能与通过上调ARHGEF10的表达抑制中性粒细胞迁移进而拮抗NETs的生成有关。讨论:通过理论探讨发现皮肤健康离不开营卫和谐,营卫失衡导致的“营卫倾移”是银屑病基本病机,“营卫倾移”而致的“营卫湿热伏邪”是银屑病加重或复发的重要因素。红细胞分布宽度(RDW)尿酸(UA)和糖化血红蛋白(HBA1C)与银屑病患者死亡风险高度相关,且均为代谢相关指标,这些指标廉价易得,早期监测,并根据其变化采取相应措施可有效降低银屑病患者轻症变重症最终死亡的风险。生物信息学分析发现与银屑病相关的基因主要有IRS1、RAI14和ARHGEF10,其中IRS1直接与代谢相关,RAI14和ARHGEF10与细胞运动相关。通过细胞实验研究初步发现治疗银屑病的中药松香,其有效成分松香酸可上调人外周血极化状态下的中性粒细胞ARHGEF10的表达,进而可抑制NETs的生成。从营卫理论角度说,红细胞分布宽度(RDW)可反应营阴的运行情况,尿酸(UA)、糖化血红蛋白(HBA1C)和胰岛素受体底物1(IRS1)可反应营阴的功能,因此银屑病病机可能具有“营本卫标”的特点。RAI14和ARHGEF10可能与“营卫倾移”病理过程中的“倾移”运动有关,而松香作为清湿热、调节代谢和免疫的中药,其主要成分松香酸可能通过上调极化态中性粒细胞ARHGEF10表达进而影响NETs生成来逆转“营卫倾移态”为“营卫调和态”。基于上讨论,我们认为对于银屑病的治疗,在兼顾代谢和免疫因素的同时,更应该关注代谢因素对银屑病患者生存时间、生存质量以及复发的影响。中医药防治上,应在“营卫”理论的指导下,认识到湿邪和热邪的辩证统一关系,根据银屑病发病不同阶段标本缓急情况,确立防治时清热和除湿的主次关系,将除湿贯穿银屑病防治始终。本研究的创新点主要在于:1)将营卫理论应用于银屑病病因病机研究,依照此理论我们探索了银屑病代谢和免疫的主次关系,阐述了传统中医营卫理论的科学性。2)通过对银屑病患者生存数据分析发现,代谢免疫(营卫)指标中,与银屑病患者死亡风险显著相关的指标主要是代谢指标(营),为银屑病“营本卫标”的病机特点和银屑病代谢免疫的主次关系提供了理论依据。3)通过对银屑病患者甲基化芯片数据集和表达谱数据集进行联合分析发现,主要是以IRS1为代表的代谢相关基因(营)与银屑病发病显著相关,再次论证了银屑病代谢免疫的主次关系,同时筛选出的与细胞运动相关的银屑病潜在靶点基因ARHGEF10和RAI14可能是银屑病“营卫倾移”病理改变中“倾移”的物质基础。4)通过实验研究发现了松香酸治疗银屑病可能与松香酸通过激活ARHGEF10基因的表达,从而抑制中性粒细胞的迁移能力进而拮抗了 NETs的形成有关,证明了ARHGEF10可能是银屑病“营卫倾移”病理改变中“倾移”的物质基础,为从“营卫”论治银屑病提供了理论依据。未来,我们一方面会在第四部分研究的基础上继续探索代谢和免疫之间的交互关系,进而阐释基于“营卫”理论指导银屑病辨证论治的科学内涵,另一方面会在银屑病代谢免疫主次关系和“营本卫标”的理论基础上,探索银屑病患者除湿法贯穿防治始终,对银屑病患者生存质量以及“瘥后防复”的影响。本研究的不足之处主要在于:1)受限于与银屑病相关的生存数据难以获得且质量不高,第二部研究中纳入的病例数据来源于美国,可能种族差异会使结果出现偏倚,研究最终纳入了 528例样本,虽然符合样本量要求,但更多纳入样本所分析的结果将会有更强的可信度,未来我们将构建国内银屑病生存预后队列数据并纳入更多相关变量以研究中国银屑病人群生存预后影响因素。2)第三部分研究中不同数据集可能存在批次效应,虽然可以通过一些方法去除批次效应,但无法完全消除,因此可能存在一定偏倚,未来我们会构建同批次更多样本的芯片数据以探索银屑病潜在靶点基因。3)第四部分研究仅从细胞水平做了初步研究,需要更多的回复实验验证结果的可靠性,同时若从动物实验层面进行结果验证会有更强的说服力。更多还原

【Abstract】 Objective:Psoriasis is a chronic inflammatory skin disease mediated by T cells,and its pathogenesis is complex and has not been fully clarified.At present,biological agents have become one of the main treatment methods for psoriasis.From the point of view of immunity,treating psoriasis by blocking specific inflammatory factors produced by the immune system is quick and effective,but it also has some problems,such as incurable disease,recurrence and some side effects.According to the overall concept of traditional Chinese medicine and the idea of preventing diseases,we should not only pay attention to the improvement of skin lesions after treatment,but also pay attention to the survival time,quality of life and recurrence of patients.With the in-depth study of psoriasis,it is gradually considered that psoriasis is also a systemic disease,often accompanied by a variety of metabolic related diseases,which is an important factor affecting the survival and prognosis of psoriasis patients.Therefore,in order to better prevent and treat psoriasis,we should not only pay attention to immune problems,but also consider metabolic factors in the study of psoriasis.With the development of metabolic immunology in modern medicine,many scholars think that there are many similarities between the theory of "Ying-wei”and the physiological pathology of metabolic immunity."Ying-wei"has been discussed in "Neijing" as early as,which is not only one of the basic life activities of human body,but also one of the important basic theories of traditional Chinese medicine.Therefore,the purpose of this study is to explore the primary and secondary relationship between metabolic and immune-related biological indicators of psoriasis patients and the risk of morbidity and mortality in accordance with the theory of "Ying-wei" in traditional Chinese medicine,in order to provide theoretical support for better treatment of psoriasis from the perspective of metabolic immunity,and also to explain the scientific connotation of treating psoriasis from "Ying-wei".Methods:The first part was a theoretical study,which summarized the research literature about psoriasis and Ying-wei,discussed the theoretical basis of Ying-wei,the relationship between Ying-wei’s tilting movement and psoriasis,interpreted the relationship between Ying-wei and five internal organs,Ying-wei and Xuanfu,Ying-wei and six meridians,and Ying-wei and damp heat from the pathogenesis characteristics of psoriasis,and discussed the essence of Ying-wei and how to treat psoriasis from Ying-wei.The second part was a cross-sectional study using psoriasis data from the 2003-2006 and 2009-2014 National Health and Nutrition Examination Survey(NHANES).COX proportional hazards regression analysis,random forest survival analysis,and restricted cubic spline model were used to evaluate the association between immune-metabolic indicators in peripheral blood and psoriasis mortality.Section three was a bioinformatics study.The gene expression profile dataset and DNA methylation dataset of psoriatic skin tissues were downloaded from the Gene Expression Omnibus(GEO)database.Immune infiltration analysis,functional enrichment analysis,protein-protein interaction(PPI)network construction,machine learning algorithm analysis(LASSO,random forest,SVM-RFE)and other analysis methods were used to analyze the regulatory network and explore the target genes related to psoriasis.Section four was an experimental study based on section three,Western blotting,Real-time PCR and immunofluorescence were used to study the effects of rosin acid on the related genes of polarized human peripheral blood neutrophils and the formation of neutrophil extracellular traps.Results:In the first part,it was found that internal and external pathogens invaded the human body,which led to abnormal functions of the five internal organs,six meridians and Xuanfu,and then Wei-Yang rose up to resist pathogens outside the pulse,and at the same time pulled the Yin in the artery,eventually forming a tilt and abnormal distribution of Ying-wei.which led to psoriasis-like lesions.For a long time,it was the reason why psoriasis was difficult to treat and easy to recur.In the second part,528 patients with psoriasis were included.Based on the research results of psoriasis population and sex subgroups,it was found that uric acid(UA),red blood cell distribution width(RDW)and glycosylated hemoglobin(HBA1C)were potential biomarkers related to psoriasis death.In the general population,UA(HR 1.59,95%CI:1.242.04,P<0.001)after adjusting age,sex,race,education level,smoking status,drinking status and income;RDW(HR 1.55,95%CI:1.16-2.07,P<0.001)and HBA1C(HR 1.47,95%CI:1.21-1.80,P<0.001)were significantly correlated with the increased risk of death.Blood glucose(RG,nonlinear P=9E-04)and glycosylated hemoglobin(HBA1C,nonlinear P=0.0014)also have a strong U-shaped correlation with the risk of death,and the risk of death is the lowest at 89 mg/dl and 5.1%respectively.In the third part,it was found that 97 hypermethylated and Hyper-down genes accounted for the highest proportion(47%),and the hyper-down genes were mainly enriched in glucose homeostasis,AMPK signaling pathway,lipid storage diseases,partial lipodystrophy and insulin resistance.Insulin receptor substrate(IRS1),Rho guanine nucleotide exchange factor 10(ARHGEF10)and retinoic acid-induced 14(RAI14)were identified as potential targets of psoriasis.In the fourth part,it was found that rosin acid could affect the expression of ARHGEF10 in polarized neutrophils in human peripheral blood and inhibit the production of NETs.Discussion:Through theoretical research,it is found that skin health can’t be separated from the harmony between camp and health,and the shift of camp and health caused by the imbalance of camp and health is the basic pathogenesis of psoriasis,and the damp heat and evil of camp and health caused by the shift of camp and health is an important factor for the aggravation or recurrence of psoriasis.Red blood cell distribution width(RDW),uric acid(UA)and glycosylated hemoglobin(HBA1C)are highly correlated with the risk of death in patients with psoriasis,and they are both metabolic indicators.These indicators are cheap and easy to obtain.Early monitoring and taking corresponding measures according to their changes can effectively reduce the risk of death in patients with psoriasis.Bioinformatics analysis showed that the genes related to psoriasis mainly included IRS1,RAI14 and ARHGEF10,in which IRS1 was directly related to metabolism,RAI14 and ARHGEF10 were related to cell motility.Through cell experiment,it was found that rosin acid,the effective component of rosin,which is a traditional Chinese medicine for treating psoriasis,can affect the expression of ARHGEF10 in neutrophils in polarized human peripheral blood and inhibit the production of NETs.According to the theory of Ying-wei,red blood cell distribution width(RDW)can reflect the operation of Yingyin,while uric acid(UA),glycosylated hemoglobin(HBA1C)and insulin receptor substrate(IRS1)can reflect the function of Ying-Yin,so the pathogenesis of psoriasis may have the characteristics of "Ying-Ben-Wei-Biao".RAI14 and ARHGEF10 may be related to the "tilting"movement in the pathological process of "Ying-wei tilting".Rosin,as a traditional Chinese medicine to remove dampness and heat,regulate metabolism and immunity,the main component of rosin,may reverse the "tilting state of Ying-wei" to "Ying-wei balance state" by up-regulating the expression of ARHGEF10 in polarized neutrophils and then affecting the formation of NETs.Based on the above discussion,we think that for the treatment of psoriasis,we should pay attention to the influence of metabolic factors on the survival time,quality of life and recurrence of psoriasis patients while taking into account both metabolic and immune factors.In the prevention and treatment of traditional Chinese medicine,under the guidance of the theory of "Ying-wei",we should recognize the dialectical unity relationship between dampness and heat,and establish the primary and secondary relationship between heat clearing and dehumidification according to the priority of specimens in different stages of psoriasis,so that the method of dehumidification can be used throughout the prevention and treatment of psoriasis.Innovations of this study mainly include:1)Applying the theory of Ying-wei to the etiology and pathogenesis research of psoriasis,exploring the primary and secondary relationship between metabolism and immunity in psoriasis,and demonstrating the scientific nature of traditional Chinese medicine theory of Ying-wei.2)Through the analysis of survival data from psoriasis patients,it was found that among the indicators of metabolic immunity(Ying-wei),the ones significantly associated with the risk of death in psoriasis patients were mainly metabolic indicators(Ying).This provides theoretical evidence for the pathogenesis characteristics of psoriasis as the "Ying-ben-wei-biao" and the primary and secondary relationship between metabolism and immunity in psoriasis.3)Through the joint analysis of methylation chip data set and expression profile data set of psoriasis patients,it was found that metabolic-related genes(represented by IRS1)were significantly associated with the occurrence of psoriasis,further confirming the primary and secondary relationship between metabolism and immunity in psoriasis.Additionally,potential target genes related to cell movement.ARHGEF10 and RAI14,which were screened out,may serve as the material basis for the pathological changes of "Ying-wei imbalance" in psoriasis.4)The experimental results have revealed that the treatment of psoriasis with abietic acid may be related to the inhibition of neutrophil migration by abietic acid through activating the expression of ARHGEF10 gene,thus antagonizing the formation of NETs.These results have proved that ARHGEF10 may be the material basis of "leaning" in the pathological change of "tilting state of Ying-wei" of psoriasis.and provided a theoretical basis for the treatment of psoriasis from Ying-wei.In the future,on the one hand,we will continue to explore the interaction between metabolism and immunity based on the study four,and then explain the scientific connotation of guiding the syndrome differentiation and treatment of psoriasis based on the theory of "Ying-wei".On the other hand,we will explore the effect of dehumidification method on the quality of life and"prevention of the recrudescence of disease" of psoriasis patients based on the theory of "Yingben-wei-biao" of psoriasis.The main shortcomings of this study are:1)Limited by the difficulty of obtaining survival data related to psoriasis and the low quality,the case data included in study 2 came from the United States,and racial differences may bias the results.The study finally included 528 samples,although it met the sample size requirements,but the results analyzed by more included samples will have stronger credibility.In the future,we will construct the Chinese psoriasis survival and prognosis cohort data and include more relevant variables to study the survival and prognosis factors of Chinese psoriasis population.2)There may be batch effect in different data sets in study 3.Although the batch effect can be removed by some methods,it cannot be eliminated,so there may be a certain bias.In the future,we will construct chip data of more samples in the same batch to explore potential target genes of psoriasis.3)Study 4 was only a preliminary study at the cellular level,so more experiments are needed to verify the reliability of the results.At the same time,it would be more convincing to verify the results at the animal level.更多还原