Department of Respiratory and Critical Care Medicine, The Second Hospital of Hebei Medical University, Shijiazhuang, Hebei 050000; Department of Respiratory Medicine, The Third Hospital of Hebei Medical University, Shijiazhuang, Hebei 050051, China.
Department of Respiratory and Critical Care Medicine, The Second Hospital of Hebei Medical University, Shijiazhuang, Hebei 050000, China.
Chin Med J (Engl). 2018 Oct 20;131(20):2461-2473. doi: 10.4103/0366-6999.243551.
Fine particulate matter (PM) exacerbates airway inflammation and hyperreactivity in patients with asthma, but the mechanism remains unclear. The aim of this study was to observe the effects of prolonged exposure to high concentrations of PMon the pathology and airway hyperresponsiveness (AHR) of BALB/c mice undergoing sensitization and challenge with ovalbumin (OVA) and to observe the effects of apoptosis and T-cell immunoglobulin and mucin domain 1 (TIM-1) in this process.
Forty female BALB/c mice were divided into four groups: control group, OVA group, OVA/PM group, and PM group (n = 10 in each group). Mice in the control group were exposed to filtered clean air. Mice in the OVA group were sensitized and challenged with OVA. Mice in the OVA/PM group were sensitized and challenged as in the OVA group and then exposed to PMfor 4 h per day and 5 days per week for a total of 8 weeks using a nose-only "PMonline enrichment system" in The Second Hospital of Hebei Medical University. Mice in the PM group were exposed to the PM online enrichment system only. AHR was detected. Bronchoalveolar lavage fluid (BALF) was collected for cell classification. The levels of interleukin-4 (IL-4), IL-5, and IL-33 in BALF were measured using enzyme-linked immunosorbent assay. Changes in histological structures were examined by light microscopy, and changes in ultramicrostructures were detected by electron microscopy. Apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay in the lung tissues. Western blotting and immunohistochemistry were utilized to analyze the expression of Bcl-2, Bax, and TIM-1 in the lungs.
The results showed that AHR in the OVA/PM group was significantly more severe than that in the OVA and PM groups (P < 0.05). AHR in the PM group was also considerably more severe than that in the control group (P < 0.05). The BALF of OVA/PM group (28.00 ± 6.08 vs. 12.33 ± 4.51, t = 4.631, P = 0.002) and PM group (29.00 ± 3.00 vs. 12.33 ± 4.51, t = 4.927, P = 0.001) had more lymphocytes than the BALF of the control group. The number of neutrophils in the BALF of the OVA/PM group (6.67 ± 1.53 vs. 3.33 ± 1.53, t = 2.886, P = 0.020) and PM group (6.67 ± 1.53 vs. 3.33 ± 1.53, t = 2.886, P = 0.020) was much higher than those in the BALF of OVA group (P < 0.05). TUNEL assays showed that the number of apoptotic cells in the OVA/PM group was significantly higher than that in the OVA group (Tunel immunohistochemical scores [IHS%], 1.20 ± 0.18 vs. 0.51 ± 0.03, t = 8.094, P < 0.001) and PM group (Tunel IHS%, 1.20 ± 0.18 vs. 0.51 ± 0.09, t = 8.094, P < 0.001), and that the number of apoptotic cells in the PM group was significantly higher than that in the control group (Tunel IHS%, 0.51 ± 0.09 vs. 0.26 ± 0.03, t = 2.894, P = 0.020). The concentrations of IL-4 (77.44 ± 11.19 vs. 48.02 ± 10.02 pg/ml, t = 4.595, P = 0.002) and IL-5 (15.65 ± 1.19 vs. 12.35 ± 0.95 pg/ml, t = 3.806, P = 0.005) and the Bax/Bcl-2 ratio (1.51 ± 0.18 vs. 0.48 ± 0.10, t = 9.654, P < 0.001) and TIM-1/β-actin ratio (0.78 ± 0.11 vs. 0.40 ± 0.06, t = 6.818, P < 0.001) in the OVA/PM group were increased compared to those in the OVA group. The concentrations of IL-4 (77.44 ± 11.19 vs. 41.47 ± 3.40 pg/ml, t = 5.617, P = 0.001) and IL-5 (15.65 ± 1.19 vs. 10.99 ± 1.40 pg/ml, t = 5.374, P = 0.001) and the Bax/Bcl-2 ratio (1.51 ± 0.18 vs. 0.97 ± 0.16, t = 5.000, P = 0.001) and TIM-1/β-actin ratio (0.78 ± 0.11 vs. 0.31 ± 0.06, t = 8.545, P < 0.001) in the OVA/PM group were increased compared to those in the PM group. The concentration of IL-4 (41.47 ± 3.40 vs. 25.46 ± 2.98 pg/ml, t = 2.501, P = 0.037) and the Bax/Bcl-2 ratio (0.97 ± 0.16 vs. 0.18 ± 0.03, t = 7.439, P < 0.001) and TIM-1/β-actin ratio (0.31 ± 0.06 vs. 0.02 ± 0.01, t = 5.109, P = 0.001) in the PM group were also higher than those in the control group.
Exacerbated AHR associated with allergic asthma caused by PMis related to increased apoptosis and TIM-1 activation. These data might provide insights into therapeutic targets for the treatment of acute exacerbations of asthma induced by PM.
细颗粒物（PM）可加剧哮喘患者的气道炎症和高反应性，但机制尚不清楚。本研究旨在观察长时间暴露于高浓度 PM 对卵清蛋白（OVA）致敏和激发的 BALB/c 小鼠的病理学和气道高反应性（AHR）的影响，并观察细胞凋亡和 T 细胞免疫球蛋白和粘蛋白结构域 1（TIM-1）在此过程中的作用。
将 40 只雌性 BALB/c 小鼠分为对照组、OVA 组、OVA/PM 组和 PM 组（每组 10 只）。对照组小鼠暴露于过滤后的清洁空气中。OVA 组小鼠用 OVA 致敏和激发。OVA/PM 组小鼠如 OVA 组一样进行致敏和激发，然后每天暴露于 PM 中 4 小时，每周 5 天，使用河北医科大学第二医院的“PM 在线富集系统”共 8 周。PM 组小鼠仅暴露于 PM 在线富集系统中。检测 AHR。收集支气管肺泡灌洗液（BALF）进行细胞分类。采用酶联免疫吸附试验（ELISA）检测 BALF 中白细胞介素-4（IL-4）、白细胞介素-5（IL-5）和白细胞介素-33 的水平。用光镜观察组织学结构变化，用电镜检测超微结构变化。通过末端脱氧核苷酸转移酶介导的 dUTP 缺口末端标记（TUNEL）试验测定肺组织中的细胞凋亡。采用 Western blot 和免疫组化法分析肺组织中 Bcl-2、Bax 和 TIM-1 的表达。
与 OVA 组和 PM 组相比，OVA/PM 组的 AHR 明显更严重（P < 0.05）。PM 组的 AHR 也明显比对照组更严重（P < 0.05）。与对照组相比，OVA/PM 组（28.00 ± 6.08 比 12.33 ± 4.51，t = 4.631，P = 0.002）和 PM 组（29.00 ± 3.00 比 12.33 ± 4.51，t = 4.927，P = 0.001）的 BALF 中有更多的淋巴细胞。与 OVA 组相比，OVA/PM 组（6.67 ± 1.53 比 3.33 ± 1.53，t = 2.886，P = 0.020）和 PM 组（6.67 ± 1.53 比 3.33 ± 1.53，t = 2.886，P = 0.020）的 BALF 中中性粒细胞数量明显增加。与 OVA 组相比，OVA/PM 组的 TUNEL 检测凋亡细胞数量明显更高（Tunel 免疫组化评分[IHS%]，1.20 ± 0.18 比 0.51 ± 0.03，t = 8.094，P < 0.001）和 PM 组（Tunel IHS%，1.20 ± 0.18 比 0.51 ± 0.09，t = 8.094，P < 0.001），PM 组的凋亡细胞数量也明显高于对照组（Tunel IHS%，0.51 ± 0.09 比 0.26 ± 0.03，t = 2.894，P = 0.020）。与 OVA 组相比，OVA/PM 组的白细胞介素-4（IL-4）（77.44 ± 11.19 比 48.02 ± 10.02 pg/ml，t = 4.595，P = 0.002）和白细胞介素-5（IL-5）（15.65 ± 1.19 比 12.35 ± 0.95 pg/ml，t = 3.806，P = 0.005）浓度以及 Bax/Bcl-2 比值（1.51 ± 0.18 比 0.48 ± 0.10，t = 9.654，P < 0.001）和 TIM-1/β-actin 比值（0.78 ± 0.11 比 0.40 ± 0.06，t = 6.818，P < 0.001）升高。与 OVA 组相比，OVA/PM 组的白细胞介素-4（77.44 ± 11.19 比 41.47 ± 3.40 pg/ml，t = 5.617，P = 0.001）和白细胞介素-5（15.65 ± 1.19 比 10.99 ± 1.40 pg/ml，t = 5.374，P = 0.001）以及 Bax/Bcl-2 比值（1.51 ± 0.18 比 0.97 ± 0.16，t = 5.000，P = 0.001）和 TIM-1/β-actin 比值（0.78 ± 0.11 比 0.31 ± 0.06，t = 8.545，P < 0.001）升高。与对照组相比，OVA/PM 组的白细胞介素-4（41.47 ± 3.40 比 25.46 ± 2.98 pg/ml，t = 2.501，P = 0.037）和 Bax/Bcl-2 比值（0.97 ± 0.16 比 0.18 ± 0.03，t = 7.439，P < 0.001）以及 TIM-1/β-actin 比值（0.31 ± 0.06 比 0.02 ± 0.01，t = 5.109，P = 0.001）也更高。
PM 导致的过敏性哮喘加重的 AHR 与细胞凋亡和 TIM-1 激活有关。这些数据可能为治疗 PM 引起的哮喘急性加重提供治疗靶点。
