目的 随着社会发展，过敏性疾病的发病率也在逐年上升。花粉症是由植物花粉诱发特异性免疫球蛋白E（specific Immunoglobulin E，sIgE）介导的过敏性疾病，季节性及地区性特点明显。多数花粉症患者都会经历由过敏性鼻炎向哮喘，由呼吸道过敏向食物过敏及严重过敏反应逐步进展的过敏性疾病自然进程。蒿属花粉是我国多数地区空气中含量最多的夏秋季花粉，致敏率高且诱发哮喘比例高。本研究旨在解析蒿属花粉的组分致敏模式，寻求可靠的生物标志物用于过敏性疾病自然进程的风险预测和评估，探寻蒿属花粉损伤气道上皮屏障、促进过敏性哮喘的机制，以期早识别、早预防、早干预，实现防治关口前移。

方法 首先通过大肠杆菌表达系统、镍柱亲和层析纯化和凝胶过滤层析技术，制备7种蒿属花粉重组过敏原组分蛋白Art v 1-6和Art an 7。接下来，招募符合蒿属花粉过敏诊断的患者，收集血清并采集相关临床信息。利用间接ELISA法，以7种重组蒿属花粉组分蛋白作为抗原，建立蒿属花粉过敏的组分解析诊断（component-resolved diagnostic, CRD）方案，探究7种组分蛋白sIgE阳性率。使用卡方检验、Mann-Whitney U检验或Kruskal-Wallis H检验以及二元logistic回归分析等方式判断合并常见过敏性疾病与蒿属花粉致敏组分间的相关性。最后，使用蒿属花粉处理气道上皮细胞A549，通过转录组、细胞凋亡试验、细胞蛋白提取、免疫印迹试验等方法探究蒿属花粉对于气道上皮屏障破坏的作用机制。

结果 成功获得蒿属花粉7种重组致敏组分蛋白，且与蒿属花粉过敏患者血清sIgE存在特异性结合。收集蒿属花粉过敏患者血清共411例，用建立的最佳试验方法对其进行检测，结果显示Art v 1、Art v 2、Art v 3、Art v 4、Art v 5、Art v 6和Art an 7的sIgE结合率分别为66.2（272/411）、43.6%（179/411）、38.2%（157/411）、26.8%（110/411）、4.9%（20/411）、25.1%（103/411）、22.6%（93/411）。病史大于5年的320例患者中，Art v 3（OR = 1.78，95%CI: 1.07–2.97，P = 0.027）和Art an 7（OR = 1.82，95%CI: 1.05–3.18，P = 0.034）sIgE阳性是合并哮喘发生的独立危险因素，且过敏性鼻炎伴哮喘组患者Art v 3（51.6% vs. 36.9%, X² = 5.948, P = 0.015）和Art an 7（33.7% vs. 19.6%, X² = 7.363, P = 0.007）两种组分的率均显著高于单纯过敏性鼻炎组。411例患者中，Art v 1（OR = 2.02，95%CI: 1.07–3.84，P = 0.031）、Art v 3（OR = 2.24，95%CI: 1.39–3.63，P = 0.001）和Art v 6（OR = 1.97，95%CI: 1.08–3.59，P = 0.027）sIgE阳性是合并食物过敏的独立危险因素，且伴发食物过敏组患者Art v 3（50.9% vs. 33.4%, X² = 10.231, P = 0.001）、Art v 4（34.0% vs. 23.9%, X² = 4.059, P = 0.044）和Art v 6（35.8% vs. 21.0%, X² = 9.316, P = 0.002）三种组分的阳性率均显著高于不伴食物过敏组；Art v 3（OR = 2.30，95%CI: 1.09–4.85，P = 0.029）和Art v 6（OR = 2.29，95%CI: 1.08–4.86，P = 0.030）sIgE阳性是合并严重过敏反应的独立危险因素，且伴发严重过敏反应患者的Art v 3（57.9% vs. 36.2%, X² = 5.865, P = 0.015）和Art v 6（42.4% vs. 23.3%, X² = 5.961, P = 0.015）组分阳性率均显著高于不伴严重过敏反应组；Art v 4（OR = 2.58，95%CI: 1.27–5.25，P = 0.009）sIgE阳性是合并湿疹的独立危险因素，且伴发湿疹组患者Art v 4组分的阳性率显著高于不伴湿疹组（45.7% vs. 24.7%, X² = 7.232, P = 0.007）；Art an 7（OR = 2.40，95%CI: 1.21-4.74，P = 0.012）sIgE阳性是合并慢性荨麻疹的独立危险因素，且伴发荨麻疹组患者Art an 7组分的阳性率显著高于不伴发荨麻疹组（39.0% vs. 20.8%, X² = 6.994, P = 0.008）。此外，患者蒿属花粉致敏组分数每增加1个，合并哮喘的风险增加21%（OR = 1.21，95%CI: 1.03-1.41，P = 0.021），合并食物过敏的风险增加29%（OR = 1.29，95%CI: 1.07-1.55，P = 0.007），合并慢性荨麻疹的风险增加42%（OR = 1.42，95%CI: 1.12-1.79，P = 0.004）。蒿属花粉刺激使得A549细胞在形态上出现了胞体皱缩、胞间连接减少等变化，但是并未出现明显的凋亡情况。在转录层面，蒿属花粉刺激使得A549细胞的HMOX1基因表达上调，但在蛋白水平的验证中，蒿属花粉的刺激不会导致A549细胞HO-1蛋白表达升高以及ZO-1、E-cadherin、Occludin三种连接蛋白表达的降低。

结论 CRD可用于评估蒿属花粉的组分致敏模式，且多种蒿属花粉致敏组分sIgE阳性与患者合并哮喘、食物过敏、严重过敏反应、湿疹、慢性荨麻疹等常见过敏性疾病存在显著相关性。此外伴/不伴常见过敏性疾病的患者多种蒿属花粉致敏组分sIgE阳性率和水平也存在显著差异。本研究未能证实蒿属花粉可以通过破坏气道上皮连接蛋白或诱导气道上皮细胞铁死亡的方式诱发患者过敏性哮喘的发生。

Objective With the ongoing development of society, the prevalence of allergic diseases has been increasing year by year. Pollinosis, a seasonal and region-specific allergic disorder, is mediated by specific immunoglobulin E (sIgE) in response to plant pollen exposure. Most patients with pollinosis experience a progressive allergic march, transitioning from allergic rhinitis to asthma, and from respiratory allergies to food allergies and even severe systemic reactions. Artemisia pollen, the most abundant pollen in the air during late summer and autumn across many regions of China, exhibits a high sensitization rate and strong asthma-inducing potential. This study aims to elucidate the component-resolved sensitization patterns of Artemisia pollen, identify reliable biomarkers for predicting and assessing the risk of allergic disease progression, and investigate the mechanisms by which Artemisia pollen disrupts airway epithelial barrier function and promotes allergic asthma. The ultimate goal is to enable early identification, prevention, and intervention, thereby advancing the window of allergic disease management.

Methods Seven recombinant allergen components of Artemisia pollen (Art v 1–6 and Art an 7) were prepared using an Escherichia coli expression system, followed by nickel-affinity and size-exclusion chromatography. Serum samples and clinical data were collected from patients diagnosed with Artemisia pollen allergy. Indirect enzyme-linked immunosorbent assay (ELISA) was used to establish a component-resolved diagnostic (CRD) system, with the seven recombinant allergens serving as antigens, to determine sIgE sensitization rates. Chi-square test, Mann–Whitney U test, Kruskal–Wallis H test, and binary logistic regression were employed to assess the correlations between specific component sensitization and comorbid allergic diseases. Finally, A549 airway epithelial cells were stimulated with Artemisia pollen to explore the underlying mechanisms of epithelial barrier disruption, using transcriptomic analysis, apoptosis assays, protein extraction, and immunoblotting.

Results All seven recombinant Artemisia allergenic components were successfully obtained and showed specific IgE-binding activity with sera from patients allergic to Artemisia pollen. A total of 411 serum samples were collected and tested using the optimized ELISA system, and the sIgE positivity rates for Art v 1, Art v 2, Art v 3, Art v 4, Art v 5, Art v 6 and Art an 7 were 66.2% (272/411), 43.6% (179/411), 38.2% (157/411), 26.8% (110/411), 4.9% (20/411), 25.1% (103/411), and 22.6% (93/411), respectively. Among 320 patients with a disease duration of more than five years, positive IgE responses to Art v 3 (OR = 1.78, 95%CI: 1.07–2.97, P = 0.027) and Art an 7 (OR = 1.82, 95%CI: 1.05–3.18, P = 0.034) were identified as independent risk factors for asthma comorbidity. The sensitization rates to Art v 3  (51.6% vs. 36.9%, X² = 5.948, P = 0.015) and Art an 7 (33.7% vs. 19.6%, X² = 7.363, P = 0.007) were significantly higher in patients with allergic rhinitis and asthma compared with those with allergic rhinitis alone. In the full cohort of 411 patients, positive IgE responses to Art v 1 (OR = 2.02, 95% CI: 1.07–3.84, P = 0.031), Art v 3 (OR = 2.24, 95% CI: 1.39–3.63, P = 0.001), and Art v 6 (OR = 1.97, 95% CI: 1.08–3.59, P = 0.027) were identified as independent risk factors for comorbid food allergy. The patients with food allergy showed significantly higher sensitization rates to Art v 3 (50.9% vs. 33.4%, X² = 10.231, P = 0.001), Art v 4 (34.0% vs. 23.9%, X² = 4.059, P = 0.044), and Art v 6 (35.8% vs. 21.0%, X² = 9.316, P = 0.002) compared to the patients without food allergy. Art v 3 (OR = 2.30, 95% CI: 1.09–4.85, P= 0.029) and Art v 6 (OR = 2.29, 95% CI: 1.08–4.86, P = 0.030) were identified as independent risk factors for comorbid anaphylaxis. Patients with anaphylaxis exhibited significantly higher sensitization rates to Art v 3 (57.9% vs. 36.2%, X² = 5.865, P = 0.015) and Art v 6 (42.4% vs. 23.3%, X² = 5.961, P = 0.015) compared with the patients without anaphylaxis. Art v 4 sensitization (OR = 2.58, 95% CI: 1.27–5.25, P = 0.009) was an independent risk factor for comorbid eczema, and patients with eczema had a significantly higher sensitization rate to Art v 4 than the patients without eczema (45.7% vs. 24.7%, X² = 7.232, P = 0.007). Similarly, Art an 7 (OR = 2.40, 95% CI: 1.21–4.74, P = 0.012) was an independent risk factor for comorbid chronic urticaria, with the patients with chronic urticaria showing a significantly higher sensitization rate to Art an 7 than the patients without chronic urticaria (39.0% vs. 20.8%, X² = 6.994, P = 0.008). Furthermore, the number of sensitized Artemisia components was positively associated with the risk of allergic comorbidities: each additional sensitized component increased the risk of asthma by 21% (OR = 1.21, 95% CI: 1.03–1.41, P = 0.021), food allergy by 29% (OR = 1.29, 95% CI: 1.07–1.55, P = 0.007), and chronic urticaria by 42% (OR = 1.42, 95% CI: 1.12–1.79, P = 0.004). In vitro, Artemisia pollen stimulation caused morphological changes in A549 cells, including cell shrinkage and decreased intercellular adhesion, but no significant apoptosis was observed. At the transcriptomic level, HMOX1 expression was upregulated following stimulation; however, protein-level validation showed that Artemisia pollen did not significantly increase HO-1 protein expression, nor did it downregulate key epithelial junction proteins (ZO-1, E-cadherin, Occludin).

Conclusion CRD can be applied to evaluate the sensitization patterns to individual Artemisia pollen allergen components. The presence of sIgE against multiple Artemisia components was significantly associated with common allergic comorbidities, including asthma, food allergy, anaphylaxis, eczema, and chronic urticaria. Moreover, the sIgE positivity rates and levels of various Artemisia components differed significantly between patients with and without these comorbid allergic conditions. However, this study did not confirm that Artemisia pollen induces allergic asthma through disruption of epithelial junction proteins or by triggering ferroptosis in airway epithelial cells.