目的:探究人β-防御素-3(hBD3)对呼吸道合胞病毒(RSV)感染诱发哮喘幼鼠气道重塑与白细胞激活的影响及其机制。方法:采用卵清白蛋白(OVA)合并RSV诱发建立幼鼠哮喘模型,期间予100 μg·kg-1·d-1 hBD3溶液灌胃;制备肺泡灌洗液(BALF),瑞氏染色测定BALF细胞总数与各细胞计数,ELISA法检测BALF和血清中免疫球蛋白IgE、白细胞介素(IL)-4、IL-5、肿瘤坏死因子-α(TNF-α)水平;HE染色、PAS染色和Masson染色观察肺组织病理学变化情况,观察切片测定各项气道重塑指标,免疫组化染色检测肺组织α-平滑肌肌动蛋白(α-SMA)表达,蛋白质印迹法检测肺组织生长因子与自噬相关蛋白的表达。结果:相较于模型组,经hBD3作用的幼鼠BALF中细胞总数以及嗜酸粒细胞、中性粒细胞、淋巴细胞的比例均降低,BALF和血清中IgE、IL-4、IL-5、TNF-α水平均下降,肺组织损伤明显缓解,炎症细胞浸润减轻,杯状细胞减少,肺组织胶原纤维面积较小,管壁面积/气管内周长(WAt/Pi)、支气管平滑肌面积/气管内周长(WAm/Pi)及支气管平滑肌细胞核数目/气管内周长(N/Pi)均下降,α-SMA阳性表达率下降,同时肺组织中血管内皮生长因子(VEGF)、转化生长因子-β1(TGF-β1)、胰岛素样生长因子-1(IGF-1)以及自噬相关蛋白LC3-Ⅱ与Beclin-1的蛋白表达水平均下调,P62蛋白表达水平则上调,差异具有统计学意义(P＜0.05)。结论:hBD3能够抑制RSV感染诱发哮喘幼鼠模型的气道炎症和气道重塑过程,介导白细胞失活,该作用可能与调控自噬水平相关。
Objective: To explore the effect and mechanism of human β-defensin-3(hBD3) on airway remodeling and white blood cell activation in young asthmatic rats induced by respiratory syncytial virus(RSV) infection. Methods: Ovalbumin(OVA) combined with RSV was used to induce an asthma model in young rats, during which 100 μg·kg-1·d-1 hBD3 solution was given by gavage. Lavage fluid(BALF) was prepared, and the total number of BALF cells and cell counts were determined by Wright's staining. The levels of immunoglobulin IgE, interleukin-4(IL-4), IL-5, and tumor necrosis factor-α(TNF-α) in BALF and serum were detected by ELISA. HE staining, PAS staining and Masson staining were used to observe the pathological changes of lung tissue and various airway remodeling indexes observed by pathological sections. Immunohistochemical staining and Western blot were used to detect the lung tissue α-SMA expression and the expression of growth factors and autophagy-related proteins in lung tissue leukocytes respectively. Results: Compared with the model group, the total number of cells in BALF, the proportion of eosinophils, neutrophils and lymphocytes in the pups treated with hBD3 were reduced. The levels of IgE, IL-4, IL-5 and TNF-α in BALF and serum decreased, and the lung tissue damage was significantly alleviated. Inflammatory cell infiltration and goblet cells were reduced. The area of collagen fibers in lung tissue was smaller, WAt/Pi, WAm/Pi and N/Pi were all reduced, and the positive expression rate of α-SMA was decreased. At the same time, the expression protein levels of VEGF, TGF-β1, IGF-1, LC3-Ⅱ and Beclin-1 in the tissues were down-regulated and the expression protein levels of P62 were up-regulated, the difference being statistically significant(P＜0.05). Conclusion: hBD3 can inhibit the airway inflammation and airway remodeling process in young rats with asthma induced by RSV infection, and mediate leukocyte inactivation, which may be related to the regulation of autophagy.
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