Objective: To explore the expression characteristics of complement C3 in childhood asthma and its mechanism of regulating macrophage migration and polarization in immune inflammatory responses. Methods: (1) Clinical research section:a total of 200 children hospitalized and diagnosed with acute asthma exacerbation at Children's Hospital of Soochow University from January 2020 to December 2023 were retrospectively enrolled, including 135 males and 65 females with a mean age of (7.0±3.0) years. According to clinical symptoms on admission, patients were divided into mild group (n=89), moderate group (n=71) and severe group (n=40). Meanwhile, 100 healthy children receiving physical examination during the same period were recruited as healthy control group, consisting of 64 males and 36 females with a mean age of (6.6±3.5) years. Clinical characteristics were compared between the asthma group and healthy control group as well as among asthma subgroups of different severities. Multivariate logistic regression model was applied to analyze relevant factors associated with asthma severity. (2) Animal experiment section:Female C3 knockout (C3-/-) mice aged 6 to 8 weeks and weighing (20±2) g were selected. Female wild-type C57BL/6J mice aged 6 to 8 weeks with a body weight of (20±2) g served as the control group. There were 10 mice in each group. All mice were randomly divided into 4 groups using a random number table method: wild-type control group, wild-type asthma group, C3-/- control group and C3-/- asthma group, with 5 mice in each group. Asthma mouse models were established by airway instillation sensitization and challenge with cockroach extract, while phosphate buffered saline (PBS) was administered in control groups. All mice were anesthetized and sacrificed on day 14. Bronchoalveolar lavage fluid (BALF) was collected for classification and counting of inflammatory cells. Enzyme-linked immunosorbent assay was used to detect levels of Th2 cytokines including interleukin-4 (IL-4) and interleukin-5 (IL-5) in BALF, as well as serum total immunoglobulin E (IgE) and immunoglobulin G1 (IgG1). Lung tissues were harvested, and quantitative real-time polymerase chain reaction (qRT-PCR) was performed to assess the distribution and polarization of M1-type (iNOS⁺) and M2-type (Arg-1⁺) macrophages. (3) Cell experiment section: murine macrophage cell line RAW264.7 was cultured in vitro and treated with different concentrations of recombinant C3 protein to observe macrophage migration ability. Cells were divided into PBS group, recombinant C3 protein group, and recombinant C3 protein combined with SB290157 (C3a receptor antagonist) group according to interventions. Cells were stimulated with PBS, IL-4 and lipopolysaccharide (LPS), respectively. The polarization of M1 (iNOS⁺) and M2 (Arg-1⁺) macrophages was detected via qRT-PCR. Results: (1) Clinical sample results: the level of complement C3 in the asthma group was significantly higher than that in the healthy control group [(1.20±0.20) vs (0.98±0.18) g/L, P<0.001]. Children with moderate and severe asthma had elevated complement C3 levels compared with those with mild asthma [(1.27±0.19) vs (1.13±0.20) g/L, (1.22±0.16) vs (1.13±0.20) g/L, both P<0.05]. Multivariate logistic regression analysis indicated that complement C3 was positively correlated with moderate-severe asthma (OR=12.373, 95%CI: 1.756-87.153). (2) Animal experimental results: the total number of inflammatory cells, counts of neutrophils, eosinophils, macrophages and lymphocytes, as well as levels of IL-4 and IL-5 in BALF were lower in the C3-/- asthma group than those in the wild-type asthma group. Serum total IgE and IgG1 levels were also decreased (all P<0.05). qRT-PCR results revealed that the relative mRNA expression of iNOS and Arg-1, together with the M2/M1 ratio in lung tissues were lower in the C3-/- asthma group (all P<0.05). (3) Cell experimental results: compared with the PBS group, recombinant C3 protein significantly enhanced macrophage migration ability, and the absorbance (A) increased with the elevation of recombinant C3 protein concentration (all P<0.05). After LPS stimulation, macrophage differentiation toward M1 phenotype was markedly higher in the recombinant C3 protein+SB290157 group than in the PBS group and recombinant C3 protein group (all P<0.001). Following treatment with PBS, IL-4 or LPS, M2 polarization level was higher in the recombinant C3 protein group relative to the PBS group, while the recombinant C3 protein+SB290157 group presented lower M2 polarization than the other two groups (all P<0.01). Conclusions: Complement C3 is markedly elevated in children with asthma and positively correlated with moderate and severe asthma. Complement C3 may enhance macrophage migration and promote M2 polarization, thereby participating in and aggravating immune inflammatory responses in asthma. 目的： 探讨补体C3在儿童哮喘中的表达特征及其对调控巨噬细胞迁移极化参与免疫炎症反应的机制。 方法： （1）临床研究部分：回顾性纳入2020年1月至2023年12月在苏州大学附属儿童医院住院确诊为哮喘急性发作的200例患儿，男135例，女65例，年龄（7.0±3.0）岁，根据患儿入院时临床症状将其分为轻度组（n=89）、中度组（n=71）、重度组（n=40）。同时选取该院同期健康体检儿童100名作为健康对照组，男64名，女36名，年龄（6.6±3.5）岁。比较哮喘组和健康对照组及不同严重程度哮喘组患儿临床特征的差异，采用多因素logistic回归模型分析影响患儿哮喘严重程度的相关因素。（2）动物实验部分：选取6~8周龄C3基因敲除（C3-/-）雌性小鼠，体重（20±2）g，选取6~8周龄C57BL/6J雌性小鼠作为野生对照组，体重（20±2）g，每组10只。采用随机数字法分组，将小鼠分为4组（野生对照组、野生哮喘组、C3-/-对照组及C3-/-哮喘组），每组各5只。哮喘组采用蟑螂提取物进行气道滴注致敏及激发，建立哮喘模型，对照组以磷酸缓冲液（PBS）替代。第14天麻醉后处死所有小鼠。收集支气管肺泡灌洗液（BALF），计算BALF中炎症细胞的分类及数量，并采用酶联免疫吸附试验法检测BALF中Th2型细胞因子［白细胞介素-4（IL-4）、白细胞介素-5（IL-5）等］及血清总免疫球蛋白E（IgE）和免疫球蛋白G1（IgG1）水平。收集肺组织，实时荧光定量聚合酶链式反应（qRT-PCR）检测分析肺组织M1型（iNOS+）和M2型（Arg-1+）巨噬细胞的分布与极化情况。（3）细胞实验部分：体外培养小鼠巨噬细胞系（Raw264.7），给予不同浓度C3重组蛋白，观察巨噬细胞的迁移能力。根据干预方式分为PBS组、C3重组蛋白组及C3重组蛋白组+SB290157（C3a受体拮抗剂）组。分别给予PBS、IL-4、脂多糖（LPS）干预，通过qRT-PCR方法分析肺组织M1型（iNOS+）和M2型（Arg-1+）巨噬细胞的极化情况。 结果： （1）临床样本结果：哮喘组患儿补体C3水平高于健康对照组［（1.20±0.20）比（0.98±0.18）g/L，P<0.001］，且中、重度哮喘组患儿补体C3水平均高于轻度组［（1.27±0.19）比（1.13±0.20）g/L、（1.22±0.16）比（1.13±0.20）g/L，均P<0.05］。多因素logistic回归模型分析显示，补体C3与中重度哮喘呈正相关（OR=12.373，95%CI：1.756~87.153）。（2）动物实验结果：C3-/-哮喘组BALF中炎症细胞总数、中性粒细胞计数、嗜酸性粒细胞、巨噬细胞计数、淋巴细胞计数、IL-4、IL-5均低于野生哮喘组，且血清总IgE和血清总IgG1亦低于野生哮喘组（均P<0.05）。qRT-PCR结果显示，C3⁻/⁻哮喘组小鼠肺组织中iNOS mRNA、Arg-1 mRNA相对表达量、M2/M1比值均低于野生哮喘组（均P<0.05）。（3）细胞实验结果：相比PBS组，C3重组蛋白能显著增强巨噬细胞的迁移能力，且C3重组蛋白的浓度越高吸光度（A）越高（均P<0.05）。C3重组蛋白+SB290157组使用LPS处理后巨噬细胞向M1型分化较PBS组及C3重组蛋白组高（均P<0.001）；PBS或IL-4或LPS干预后，C3重组蛋白组巨噬细胞向M2型极化均较PBS组高，且C3重组蛋白+SB290157组巨噬细胞向M2型极化均低于PBS组及C3重组蛋白组（均P<0.01）。 结论： 补体C3在儿童哮喘中显著升高，与中重度哮喘正相关。补体C3可能通过增强巨噬细胞迁移并促进其向M2型极化，从而参与并加重哮喘的免疫炎症反应。.
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arXiv · 2026-04-07
arXiv · 2026-04-26