Effects of Wuyi Yinqiao Fangyi Prescription on lung mucosal immunity and related factors in mice with chronic LPS-induced lung injury
吴医银翘防疫方对慢性脂多糖诱导肺损伤小鼠肺黏膜免疫及其相关因子的影响

摘要

目的 研究吴医银翘防疫方对脂多糖（LPS）诱导的慢性肺损伤小鼠肺黏膜免疫及其相关细胞因子变化的影响。方法 采用从鼻腔向呼吸道内反复（第１天、29天和57天）滴入LPS（30μg/6μL）的方法建立慢性肺损伤小鼠动物模型，将小鼠随机分为对照组、模型组、吴医银翘防疫方低（5.0g生药/kg）、中（10.0g生药/kg）、高（20.0g生药/kg）剂量组。造模成功后各组对应给予授试物3周后麻醉处死动物，摘取肺组织和肺黏膜组织；HE染色观察肺组织病理改变；ELISA 法测定肺黏膜组织 IL-4、IL-6、TGF-β水平和IFN-γ、TNF-α水平；Western blot法和免疫组化法分别检测肺黏膜组织中IgA、pIgR蛋白表达和Ang II、ACE和ACE2蛋白分布与强度变化。结果 与对照组相比，模型组小鼠的肺脏组织肺泡萎缩、间隔变宽和充血水肿，以及伴随大量的炎性浸润情况等，且病理组织学损伤评分明显升高（P＜0.05）；模型组小鼠肺脏黏膜组织 IgA、pIgR 蛋白表达明显升高（P＜0.05），IgA 相关细胞因子 IL-4、IL-6、TGF-β水平及 pIgR 相关细胞因子 IFN-γ、TNF-α水平均显著高于对照组（P＜0.05），且模型组小鼠肺黏膜组织Ang II、ACE、ACE2 蛋白染色分布平均光密度强度值（IOD/area）均显著性高于对照组（P＜0.05）；与模型组相比，吴医银翘防疫方以剂量依赖性减轻了肺损伤小鼠肺组织破坏程度（P＜0.05），各剂量组明显抑制肺损伤小鼠肺黏膜组织中IgA、pIgR蛋白表达下降和IL-6、IFN-γ水平的升高（P均＜0.05）；其中吴医银翘防疫方的高剂量能明显抑制肺损伤小鼠肺黏膜组织中IL-4、TGF-β和TNF-α水平的升高（P均＜0.05）。另外发现吴医银翘防疫方以剂量依赖性抑制肺损伤小鼠肺黏膜组织中Ang II、ACE和ACE2蛋白染色分布增强及其平均光密度强度升高，与模型组相比，中、高剂量组具有显著性差异（P均＜0.05）。结论 吴医银翘防疫方通过调控肺组织损伤黏膜免疫屏障低下相关 IgA和pIgR的表达及其相关的炎症因子IL-4、IL-6、TGF-β和IFN-γ、TNF-α水平，抑制肺黏膜中 Ang II、ACE、ACE2活性过度表达，从而调节肺黏膜免疫功能改善LPS诱导的肺黏膜损伤程度。

Abstract

Objective To study the effects of Wuyiyinqiao Jianguo Prescription on lung mucosal immunity and related cytokines in mice with LPS induced chronic lung injury.
Methods A mouse model of chronic lung injury was established by repeated infusion of LPS (30μg/6μL) from nasal cavity to respiratory tract (day 1, day 29 and day 57). The mice were randomly divided into control group, model group, low (5.0g crude drug /kg), medium (10.0g crude drug /kg) and high (20.0g crude drug /kg) dose groups. After successful modeling, the animals were anesthetized and killed after 3 weeks after receiving the test material, and the lung tissue and pulmonary mucosal tissue were extracted. HE staining was used to observe the pathological changes of lung tissue. The levels of IL-4, IL-6, TGF-β, IFN-γ and TNF-α in lung mucosa were determined by ELISA. The expression of IgA and pIgR proteins and the distribution and intensity of Ang II, ACE and ACE2 proteins in lung mucosa were detected by Western blot and immunohistochemistry, respectively.
Results Compared with the control group, the pulmonary alveolar atrophy, septum widening, hyperemia and edema, and a large number of inflammatory infiltrates were observed in the model group, and the histopathological injury score was significantly increased (P < 0.05). The expressions of IgA and pIgR proteins in lung mucosal tissues of mice in model group were significantly increased (P < 0.05), and the levels of IgA related cytokines IL-4, IL-6, TGF-β and PIGr-related cytokines IFN-γ and TNF-α were significantly higher than those in control group (P < 0.05). The mean optical density intensity (IOD/area) of Ang II, ACE and ACE2 protein staining distribution in lung mucosal tissue of mice in model group was significantly higher than that in control group (P < 0.05). Compared with model group, Wuyiyinqiao Jiangjian formula reduced the damage degree of lung tissue in mice with lung injury in a dose-dependent manner (P < 0.05), and inhibited the decreased expression of IgA and pIgR protein and the increased levels of IL-6 and IFN-γ in lung mucosal tissue of mice with lung injury significantly in each dose group (P < 0.05). The elevated levels of IL-4, TGF-β and TNF-α in lung mucosal tissue of mice with lung injury were significantly inhibited by the high dose of WuyiYinjujian prescription (P < 0.05). In addition, it was found that Wuyiyinqiao Jianguo prescription inhibited the enhancement of Ang II, ACE and ACE2 protein staining distribution and the increase of average optical density intensity in lung mucosal tissues of mice with lung injury in a dose-dependent way. Compared with model group, there were significant differences between medium and high dose groups (P < 0.05).
Conclusion   By regulating the expression of IgA and pIgR, as well as the levels of IL-4, IL-6, TGF-β, IFN-γ and TNF-α, WuyiYinjujian prescription can inhibit the overexpression of Ang II, ACE and ACE2 activity in lung mucosa. In this way, the lung mucosal immune function can be regulated to improve the degree of lung mucosal injury induced by LPS.