【作者】 郭佳；

【导师】 粟娟； 刘洪；

【作者基本信息】 中南大学 ， 临床医学（专业学位）， 2024， 博士

【摘要】 研究背景和目的:银屑病是角质形成细胞过度增殖和免疫细胞异常活化的慢性炎性皮肤疾病。尽管目前生物制剂的出现给银屑病患者带来希望,但是依然存在易复发、部分患者响应差或由于禁忌症不能使用生物制剂的情况。因此深入探寻银屑病发病机制,寻找有效治疗手段仍然迫在眉睫。近些年来,越来越多的研究发现在银屑病皮损病灶处由于细胞异常增殖出现糖酵解增强、能量代谢紊乱等特征,提示了能量代谢在银屑病中的重要作用。腺苷作为能量代谢分子ATP的重要代谢产物,腺苷及其下游受体在银屑病中的作用及机制尚不明确。研究方法:1.通过LC/MS-MS法检测银屑病患者、健康志愿者血浆中腺苷的含量并与临床严重程度进行相关性分析。2.利用多组学数据及人组织免疫荧光明确腺苷下游作细胞及细胞表面受体ADORA2B在银屑病中的表达情况。3.构建Adora2b全敲小鼠,在咪喹莫特诱导的银屑病样小鼠皮炎模型上验证Adora2b对表型及炎症的影响。4.qRT-PCR检测腺苷通过ADORA2B对人原代角质形成细胞炎症因子表达情况。5.构建角质形成细胞Adora2b条件性敲除小鼠,在银屑病样小鼠皮炎模型验证角质形成细胞Adora2b对表型及炎症的影响。6.利用ADORA2B的特异性小分子抑制剂PSB1115,评估其对银屑病样皮炎模型表型及炎症的作用。7.RNA-seq探究ADORA2B下游可能通路。8.线粒体压力测定、透射电镜、流式细胞术等检测腺苷及ADORA2B对角质形成细胞线粒体功能、形态、数目的影响。9.流式细胞术等检测ADORA2B对人原代角质形成细胞线粒体ROS的影响。10.qRT-PCR以及体内实验检测Mito-tempo清除线粒体ROS对ADORA2B促进角质形成细胞炎症的影响。11.qRT-PCR和Western Blot实验检测ADORA2B对HO-1的RNA和蛋白水平的影响。12.荧光素酶报告基因、染色质免疫共沉实验明确ADORA2B通过p-CREB对HO-1的转录调控作用。13.通过回复实验验证ADORA2B通过调控HO-1影响线粒体功能从而影响角质形成细胞炎症。结果:1.银屑病患者血浆中腺苷水平升高,与PASI评分呈正相关。2.ADORA2B在银屑病中升高且与PASI评分呈正相关。3.小鼠Adora2b敲除可以减轻咪喹莫特诱导的银屑病模型的红斑、鳞屑、厚度,减少Th17细胞的浸润及Il-17,Il-23,Tnf-α,S100a8,Cxcl1等银屑病相关炎症因子的表达。4.腺苷通过ADORA2B促进角质形成细胞IL-23,S100A8,S100A9等炎症因子表达。5.角质形成细胞条件性敲除ADORA2B能减轻减轻银屑病模型的红斑、鳞屑、厚度,减少Th17细胞的浸润及Il-17,Il-23,Cxcl2,S100a8,S100a9等炎症因子的表达。6.PSB1115能减轻银屑病模型的红斑、鳞屑、厚度,减少Th17细胞的浸润及Il-17,Il-23,Tnf-α,S100a8,Cxcl1等炎症因子的表达。7.ADORA2B下游氧化磷酸化、氧化应激等通路明显改变。腺苷通过ADORA2B介导角质形成细胞线粒体基础耗氧量和储备耗氧量降低。流式细胞术和透射电镜结果提示ADORA2B激动剂Bay60-6583导致了线粒体结构改变但不影响线粒体数目。Bay60-6583可以促进线粒体ROS生成。8.qRT-PCR结果显示Mito-tempo能抑制Bay60-6583导致的角质形成细胞炎症因子表达升高,体内动物实验也显示Mito-tempo能逆转Bay60-6583导致的银屑病样皮炎小鼠表型加重。9.Bay60-6583促进HO-1的m RNA和蛋白水平表达,Bay60-6583促进p-CREB表达。敲低CREB可以抑制Bay60-6583导致的HO-1的m RNA和蛋白水平表达升高。p-CREB与HO-1启动子区域直接结合。10.敲低HO-1可以减轻Bay60-6583导致的角质形成细胞线粒体功能障碍和银屑病相关炎症因子表达的升高。结论:1.银屑病中腺苷水平升高且与疾病严重程度呈正相关。2.腺苷通过作用于角质形成细胞上ADORA2B促进炎症。3.ADORA2B通过激活p-CREB-HO-1通路介导角质形成细胞线粒体功能障碍促进炎症。4.靶向ADORA2B有望成为银屑病治疗的有效途径。图27幅,表15个,参考文献91篇更多还原

【Abstract】 Backgrouds and objective:Psoriasis is a chronic inflammatory skin disease characterized by excessiveproliferation of keratinocytes and abnormal activation of immune cells.Although the emergence of biologics has brought new hope to patients with psoriasis,there are lots of problems,such as the cases of relapse,poor response an the limitation use of biologics due to contraindications,still exist.Therefore,it is still urgent to explore the pathogenesis of psoriasis and find effective treatment.In recent years,more and more studies have found that hypoxia,enhanced glycolysis,and energy metabolism disorders occur in psoriasis lesions due to abnormal cell proliferation,revealing the important role of energy metabolism in psoriasis.Among this process,adenosine is an important metabolite of energy metabolism molecule ATP.But the role and mechanism of adenosine metabolism and its downstream receptors in psoriasis are still unclear.Methods:1.The correlation between plasma adenosine concerntration and PASI score of psoriasis patients was analyzed.2.multi-omics dataset and Immunofluorescence were used to identify adenosine’s target cell keratinocytes and cell related receptor ADORA2B,and to evaluate the correlation between ADORA2B and PASI score.3.To explore the effect of ADORA2B on psoriasis pathologenesis,Adora2b KO mice were used to investigate the change of phenotype and inflammatory in the imiquine-induced psoriasis-like mouse model.4.qRT-PCR was used to detect the expression of inflammatory factors in human primary keratinocytes after treated with adenosine with/or inhibition ADORA2B.5.Keratinocyte-specific Adora2b knockout mice were constructed to test the Adora2b on keratinocytes effect of phenotype and inflammatory on IMQ-induced psoriasis-like mouse model.6.A specific small molecule inhibitor of ADORA2B were used to investigated the therapy effect in a IMQ-induced psoriasis like model.7.RNA-sequencing(RNA-Seq)was performed to analyse differentially expressed genes and pathways of human primary keratinocytes after inhibition or activation of ADORA2B.8.Effects of adenosine and ADORA2B on mitochondrial function,morphology and number of mitochondrial in primary keratinocytes were detected by Seahorse mitochondrial stress test assay,electron microscopy and Mito-Tracker green stain.9.The effect of ADORA2B on mitochondrial ROS of human primary keratinocytes was detected by flow cytometry.10.qRT-PCR and in vivo experiments were used to verify the proinflammation effect of ADORA2B on keratinocytes via scavenging mitochondrial ROS by Mito-tempo.11.qRT-PCR and Western Blot experiments were used to detect the effect of ADORA2B on the RNA and protein levels of HO-1.12.Luciferase reporter gene assay and Chl P assay were employed to reveal the transcriptional regulation of p-CREB on HO-1.13.Through the rescue experiment,ADORA2B was verified to affect mitochondrial function by regulating HO-1,leading to facilating keratinocyte inflammation.Results:1.The level of adenosine in the plasma of psoriasis patients were significantly elevated compared to those in the healthy control group and showed a significant positive correlation with the Psoriasis Area and Severity Index(PASI)score.2.ADORA2B significantly upregulated in psoriasis.Its expression levels correlated with the PASI score in patients with psoriasis.3.Adora2b knockout can significantly ameliorated IMQ-induced skin scaling,erythema,and thickeness,as well as the Th17 cells infiltration and the expression of inflammatory cytokines such as Il-17,Il-23,Tnf-α,S100a8,Cxcl1.4.qRT-PCR results showed adenosine promotes the expression of inflammatory molecular associated with psoriasis like IL-23,S100A8 and S100A9 in keratinocytes via targeting on ADORA2B.5.Keratinocyte-specific deficiency of Adora2b can significantly ameliorated IMQ-induced skin scaling,erythema,and thickeness,as well as the Th17 cells infiltration and the expression of inflammatory cytokines such as Il-17,Il-23,Cxcl2,S100a8,S100a9.6.PSB1115 could can significantly ameliorated IMQ-induced skin scaling,erythema,and thickeness,as well as the Th17 cells infiltration and the expression of inflammatory cytokines such as Il-17,Il-23,Tnf-α,S100a8,Cxcl1.7.ADORA2B leaded to significant changes in downstream pathways such as oxidative phosphorylation and oxidative stress.Mitochondrial stress test via Seahorse experiments revealed that adenosine mediates mitochondrial dysfunction through ADORA2B in keratinocytes,characterized by decreased basal and spare oxygen consumption rates.Flow cytometry and transmission electron microscopy results suggested that ADORA2B agonist Bay60-6583 leaded to alterations in mitochondrial structure without affecting mitochondrial quantity.Bay60-6583 could promote mitochondrial ROS generation.9.The qRT-PCR results indicated that Mito-tempo could suppress the elevated expression of psoriasis-related inflammatory factors induced by ADORA2B agonists in keratinocytes.In vivo animal experiments further confirmed that Mito-tempo could reverse the phenotype aggravation of psoriasis-like dermatitis mice caused by Bay60-6583.10.Bay60-6583 promoted the expression of m RNA and protein levels of HO-1,and Bay60-6583 could enhance the expression of p-CREB.Knockdown of CREB could inhibit the elevated expression of HO-1 m RNA and protein levels induced by ADORA2B agonists.Luciferase reporter gene assay and Chl P assay demonstrated direct binding of p-CREB to the HO-1promoter region.11.Inhibition of HO-1 restored mitochondrial dysfunction and alleviated inflammation in Bay60-6583-treated keratinocytes.Conclusions:1.Adenosine levels were significantly elevated in patients with psoriasis and were positively related with the disease severity.2.Adenosine promoted keratinocytes inflammation via targeting on ADORA2B.3.ADORA2B induced mitochondrial dysfunction through activation of p-CREB-HO-1 axis,leading to keratinocytes inflammation.4.Targeting ADORA2B might be a promising therapy for psoriasis.更多还原