【作者】 李涛；

【导师】 李吉； 张亦雅；

【作者基本信息】 中南大学 ， 临床医学（八年制）（专业学位）， 2024， 博士

【摘要】 背景:玫瑰痤疮是一种好发于面中部的慢性反复发作性炎症性皮肤病。由于该病机制不明,临床上缺乏有效的根治药物,故造成病情易反复发作,极大地影响了患者的身心健康和生活质量。感觉神经致密地分布在表皮和真皮,不仅能感受外界刺激,还能通过与免疫细胞进行通讯在多种皮肤病中发挥重要作用。过去数年间,神经免疫成为探讨皮肤稳态维持和疾病发生的研究热点。玫瑰痤疮患者极容易受到外界温度变化、日晒、辣椒素等多种因素刺激而出现症状反复发作。目前研究认为神经免疫功能紊乱是导致玫瑰痤疮反复发作的关键因素,但其具体机制尚不明确。目的:探究感觉神经在玫瑰痤疮发病中的作用和机制,为玫瑰痤疮的临床治疗及药物研发提供新的靶点及方向。第一部分感觉神经激活加重玫瑰痤疮炎症方法:（1）小鼠背部涂抹辣椒素凝胶,并用抗菌肽LL37构建玫瑰痤疮样模型。观察皮肤红斑并测量皮肤厚度;收取皮损,苏木精-伊红（HE）染色观察组织病理学改变;实时定量聚合酶链式反应（RT-qPCR）检测玫瑰痤疮疾病相关因子的表达情况;免疫荧光检测皮肤组织CD4⁺T细胞和CD31⁺血管的表达情况。同时收取小鼠背根神经节（DRG）,利用RT-qPCR和免疫荧光检测DRG中转录激活因子3（ATF3）的表达情况。（2）构建树脂毒素（RTX）诱导的感觉神经消融小鼠、利多卡因N-乙基溴（QX314）诱导的感觉神经抑制小鼠、感觉神经特异性敲除的Nav1.8-DTR小鼠,以上小鼠背部涂抹辣椒素凝胶并进行玫瑰痤疮造模,观察玫瑰痤疮表型、炎症水平及免疫浸润情况。（3）通过RT-qPCR检测辣椒素刺激的玫瑰痤疮样小鼠皮肤和DRG中神经肽的表达情况。（4）感觉神经特异性标记小鼠背部涂抹辣椒素凝胶并进行玫瑰痤疮造模,取皮损进行Whole-mount和免疫荧光染色,观察降钙素基因相关肽（CGRP）的表达及其与感觉神经的共定位情况。结果:（1）辣椒素可加重玫瑰痤疮样小鼠的皮肤炎症,表现为红斑评分、红斑面积和皮肤厚度的增加,真皮炎症细胞的浸润增多,皮损中玫瑰痤疮相关的炎症因子表达升高,同时CD4⁺T细胞和CD31⁺血管数量的增加。此外,在辣椒素刺激的玫瑰痤疮样小鼠DRG中,ATF3的表达水平显著升高,ATF3⁺细胞明显增多。（2）感觉神经消融、抑制和敲除均缓解了辣椒素刺激引起的玫瑰痤疮样小鼠皮炎的加重,表现为红斑评分、红斑面积和皮肤厚度的减少,真皮炎症细胞的浸润减轻,皮损中玫瑰痤疮相关的炎症因子表达降低,同时CD4⁺T细胞和CD31⁺血管数量的减少。（3）辣椒素刺激的玫瑰痤疮样小鼠的DRG中,神经肽CGRP的表达显著增加。尽管CGRP的表达可以在玫瑰痤疮样小鼠皮肤组织中检测到,但其表达水平远低于DRG中的表达水平。（4）Nav1.8⁺神经元与CGRP存在明显的共定位,辣椒素刺激明显增加了皮肤感觉神经中CGRP的表达。结论:辣椒素激活皮肤感觉神经,可能通过分泌CGRP来加重玫瑰痤疮炎症。第二部分感觉神经通过分泌CGRP激活γδT细胞参与玫瑰痤疮发病方法:（1）小鼠皮内注射CGRP并进行玫瑰痤疮造模;使用CGRP受体拮抗剂,CGRP_8-37处理小鼠,同时背部涂抹辣椒素激活神经并进行玫瑰痤疮造模;观察玫瑰痤疮表型、炎症水平及免疫浸润情况。（2）利用单细胞转录组分析小鼠皮肤中表达CGRP受体的细胞,确认为γδT细胞;再次对感觉神经特异性标记小鼠背部涂抹辣椒素凝胶并进行玫瑰痤疮造模,取皮损进行Whole-mount和免疫荧光染色,观察γδT细胞的浸润及其与感觉神经的共定位情况。（3）γδT细胞功能缺失(Tcrd^-/-)小鼠背部涂抹辣椒素激活神经并进行玫瑰痤疮造模;观察玫瑰痤疮表型、炎症水平及免疫浸润情况。（4）分选出小鼠的γδT细胞并用CGRP刺激,利用流式细胞术检测γδT细胞分泌的细胞因子。利用蛋白质组学探究CGRP激活γδT细胞调节炎症反应的相关途径。（5）使用CGRP受体拮抗剂瑞美吉泮处理小鼠,同时涂抹辣椒素激活神经并进行玫瑰痤疮造模,观察玫瑰痤疮表型、炎症水平及免疫浸润情况,评估瑞美吉泮的治疗效果。同时,利用免疫荧光染色观察小鼠DRG中ATF3的表达以及皮损中γδT细胞的浸润情况。结果:（1）皮内注射CGRP导致玫瑰痤疮样小鼠皮炎的加重,包括红斑加重、皮肤增厚,炎症细胞浸润增多,炎症因子表达升高,CD4⁺T细胞和CD31⁺血管也有所增多。而CGRP_8-37处理缓解了辣椒素诱导的皮炎加重,包括红斑和皮肤增厚的减轻,炎症浸润减轻,炎症因子表达降低,CD4⁺T细胞和CD31⁺血管也有所减少。由此可见,CGRP对于感觉神经激活引起的皮炎加重是必需的。（2）γδT细胞表达CGRP受体,并且在玫瑰痤疮皮损中γδT细胞数量增加。皮肤中γδT细胞与感觉神经存在空间接触,感觉神经激活后玫瑰痤疮皮损中γδT细胞数量进一步增加。（3）在Tcrd^-/-小鼠中,玫瑰痤疮样皮炎得到了缓解,辣椒素诱导的皮炎加重也同样被减轻。具体表现为红斑评分、红斑面积以及皮肤厚度的减低。此外,炎症细胞的浸润也减少,皮损中炎症因子的表达也明显降低。另外,CD4⁺T细胞和CD31⁺血管的数量也减少。（4）体外实验表明,CGRP可能通过炎症和代谢相关途径激活并诱导γδT细胞分泌白细胞介素17A（IL-17A）,γδT细胞是受感觉神经调控的靶细胞。（5）瑞美吉泮可以有效缓解感觉神经介导的玫瑰痤疮皮炎加重。包括红斑评分、红斑面积和皮肤厚度的减少,炎症细胞的浸润减轻,炎症因子的表达降低,CD4⁺T细胞和CD31⁺血管数量的减少。此外,瑞美吉泮不影响玫瑰痤疮样小鼠DRG中ATF3的表达。然而,它却显著挽救了辣椒素处理的玫瑰痤疮样小鼠中γδT细胞的增加。结论:受到辣椒素刺激后,感觉神经被激活并分泌CGRP,随后直接激活真皮γδT细胞,从而加重玫瑰痤疮的免疫紊乱。更多还原

【Abstract】 Background:Rosacea is a chronic,recurrent inflammatory skin disease that is common in the central face region.Due to the unclear pathogenesis of the disease and the lack of effective curative treatments in clinical practice,the condition tends to recur,greatly impacting the physical and mental health as well as the quality of life of patients.Sensory nerves are densely distributed in the epidermis and dermis and play an important role not only in sensing external stimuli but also in communicating with immune cells in various skin diseases.In recent years,neuroimmunology has become a hot topic in research on maintaining skin homeostasis and the onset of diseases.Patients with rosacea are hypersensitive to symptom recurrence triggered by external factors such as changes in temperature,sunlight,capsaicin,among others.Current research suggests that the dysregulation of neuroimmune function is a key factor leading to the recurrent episodes of rosacea,although the specific mechanism is not yet clear.Objective:Explore the role and mechanism of sensory nerves in the pathogenesis of rosacea,providing new targets and directions for the clinical treatment and drug development of rosacea.Part Ⅰ Sensory nerve activation exacerbates inflammation in rosaceaMethods:（1）We topically applied capsaicin gel to the back of mice and established a rosacea-like model using LL37.Then we observed skin erythema and measured skin thickness;collected skin lesions,performed HE staining to observe histopathological changes in the tissue;used RT-qPCR to detect the expression of rosacea-related factors;conducted immunofluorescence to examine the expression of CD4⁺T cells and CD31⁺blood vessels in skin tissue.Simultaneously,we collected dorsal root ganglia（DRG）from mice and utilized RT-qPCR and immunofluorescence to analyze the expression of transcription factor 3（ATF3）in the DRG.（2）We conducted sensory nerve ablation mice induced by RTX,sensory nerve inhibition mice induced by QX314,and Nav1.8-DTR mice with sensory nerve specific knockout.Then we topically applied capsaicin gel to the back of these mice and induced a rosacea-like model,observing the phenotypes of rosacea,levels of inflammation,and immune infiltration in these mice.（3）The expression of neuropeptides in the skin and dorsal root ganglia（DRG）of capsaicin-stimulated rosacea-like mice was detected using RT-qPCR.（4）We topically applied capsaicin gel to the back of sensory neuron-specific marker mice and induced a rosacea-like model.Skin lesions were collected for whole-mount and immunofluorescence staining to observe the expression of CGRP and its colocalization with sensory neurons.Results:（1）In rosacea-like mice,capsaicin administration further increased the redness score,area of redness,and skin thickness,as well as increased infiltration of inflammatory cells in the dermis.The expression of rosacea-related factors was elevated in the skin lesions,along with an increase in the number of CD4⁺T cells and CD31⁺blood vessels.Furthermore,in the dorsal root ganglia（DRG）of capsaicin-stimulated rosacea-like mice,the expression level of ATF3 and ATF3⁺cells were further induced by capsaicin treatment in LL37-induced rosacea-like DRG.（2）The sensory nerve ablation,inhibition,and knockout all alleviated the exacerbation of rosacea-like mouse dermatitis induced by capsaicin stimulation,manifested by a decrease in erythema score,erythema area,and skin thickness;a reduction in infiltration of dermal inflammatory cells;attenuated expression of inflammatory factors in skin lesions;as well as reduced numbers of CD4⁺T cells and CD31⁺blood vessels.（3）In the DRG of capsaicin-stimulated rosacea-like mice,the expression of the neuropeptide CGRP significantly increased.While CGRP expression was detectable in the skin tissue of these mice,the levels were considerably lower compared to those observed in the DRG.（4）There is significant co-localization between Nav1.8+neurons and CGRP,with capsaicin stimulation markedly increasing the expression of CGRP in skin sensory nerves.Conclusion:Capsaicin activates skin sensory nerves and may exacerbate rosacea inflammation by secreting CGRP.Part Ⅱ Activation ofγδT cells by CGRP secreted from sensory nerves is involved in the pathogenesis of rosaceaMethods:（1）We injected CGRP intravenously and conducted rosacea-like mouse model;we treated the mice with a CGRP receptor antagonist,CGRP_8-37,and simultaneously applied capsaicin on the back to activate the nerves and induced rosacea-like model,observing the rosacea phenotype,levels of inflammation,and immune infiltration.（2）We performed single-cell transcriptomic analysis to identify the cells expressing CGRP receptors in the mouse skin asγδT cells.Subsequently,we topically applied capsaicin gel to the back of sensory neuron-specific marker mice and induced a rosacea-like model.Skin lesions were collected for whole-mount and immunofluorescence staining to observe the infiltration ofγδT cells and their colocalization with sensory neurons.（3）γδT cell-deficient(Tcrd^-/-)mice were topically treated with capsaicin on the back to activate nerves and established a rosacea-like model,observing the phenotype of rosacea,inflammation levels,and immune infiltration.（4）γδT cells were isolated and stimulated with CGRP,and the cytokines secreted byγδT cells were detected using flow cytometry.Proteomics was utilized to investigate the pathways through which CGRP activatesγδT cells to regulate inflammatory responses.（5）Mice were treated with the CGRP receptor antagonist,rimegepant,while concurrently receiving topical application of capsaicin to activate nerves and induced a rosacea-like model.The rosacea phenotype,inflammation levels,and immune infiltration were observed to evaluate the therapeutic effect of rimegepant.Additionally,immunofluorescence staining was employed to assess the expression of ATF3 in the DRG of mice and the infiltration ofγδT cells in the skin lesions.Results:（1）Subcutaneous injection of CGRP exacerbates rosacea-like skin inflammation in mice,characterized by increased redness,skin thickening,augmented inflammatory cell infiltration,elevated expression of inflammatory mediators,and increased CD4⁺T cells and CD31⁺blood vessels.Conversely,treatment with CGRP8-37 alleviates capsaicin-induced dermatitis by reducing redness and skin thickening,mitigating inflammation infiltration,decreasing inflammatory mediator expression,and lowering CD4⁺T cells and CD31⁺blood vessels.Thus,it is evident that CGRP is essential for exacerbating skin inflammation induced by sensory nerve activation.（2）γδT cells express the CGRP receptor,and the quantity ofγδT cells increases in rosacea skin lesions.Within the skin,γδT cells spatially interact with sensory nerves,and following sensory nerve activation,the number ofγδT cells in capsacin-stimulated rosacea lesions further escalates.（3）In Tcrd^-/-mice,rosacea-like dermatitis was ameliorated,and capsaicin-induced dermatitis exacerbation was also reduced.Specifically,this was evidenced by decreased redness score,reduced redness area,and skin thickness.Furthermore,there was a reduction in inflammatory cell infiltration,a notable decrease in the expression of inflammatory mediators in skin lesions.Additionally,the numbers of CD4⁺T cells and CD31⁺blood vessels were also decreased.（4）In vitro experiments indicated that CGRP may activate and induce IL-17A secretion inγδT cells through inflammation and metabolism-related pathways.γδT cells are target cells regulated by sensory nerves.（5）Rimegepant effectively alleviates sensory nerve-mediated exacerbation of rosacea-like dermatitis.This includes reduction in redness score,redness area,and skin thickness,as well as amelioration of inflammatory cell infiltration,decreased expression of inflammatory mediators,and reduction in the numbers of CD4⁺T cells and CD31⁺blood vessels.Furthermore,Rimegepant does not affect ATF3 expression in DRG of rosacea-like mice;however,it significantly rescues the increase inγδT cells in capsaicin-treated rosacea-like mice.Conclusion:After being stimulated by capsaicin,sensory nerves are activated and secrete CGRP,which then directly activates dermalγδT cells,thus exacerbating the immune dysregulation of rosacea.更多还原