通鼻消涕颗粒对慢性鼻窦炎大鼠PACAP/巨噬细胞/ECP的调节作用

Regulatory effect of Tongbi Xiaoti Granules on PACAP/macrophage/ECP in rats with chronic rhinosinusitis

摘要:

目的 探讨通鼻消涕颗粒（TBXTG）对卵清蛋白诱导的慢性鼻窦炎（CRS）大鼠模型垂体腺苷酸环化酶激活肽（PACAP）/巨噬细胞/嗜酸性粒细胞阳离子蛋白（ECP）的调节作用。方法 采用超高效液相色谱-质谱法（UPLC-MS）分析TBXTG的化学成分，将60只SPF SD大鼠随机分为正常对照组、模型组、克拉霉素组和低、中、高剂量TBXTG组，每组各10只。通过卵清蛋白致敏和激发建立CRS大鼠模型，造模成功后，所有治疗组均予以灌胃给药治疗2周。通过HE染色评估鼻黏膜炎症；免疫荧光染色检测M1/M2巨噬细胞极化；免疫组织化学检测鼻黏膜中PACAP、白细胞介素（IL）-10、转化生长因子β(TGF-β)及ECP表达；ELISA法测定血清PACAP和细胞因子IL-10、TGF-β、肿瘤坏死因子-α（TNF-α）、IL-6水平。使用GraphPad Prism 9.5.0和Image Pro Plus 6.0软件对数据进行分析。结果 通过UPLC-MS从TBXTG中鉴定出31种化合物，主要为黄酮类、酚酸类和黄酮苷类。与正常对照组比较，模型组大鼠鼻黏膜呈现明显的慢性炎症改变，大量炎性细胞浸润，腺体增生；与模型组相比，TBXTG各剂量组鼻黏膜炎症细胞浸润、腺体增生等病理变化显著减轻；TBXTG能显著抑制M1型巨噬细胞标志物CD86的表达，并促进M2型巨噬细胞标志物CD206的表达；上调鼻黏膜和血清中的PACAP水平（P＜0.05）；提高抗炎细胞因子IL-10、TGF-β的表达，降低促炎细胞因子TNF-α、IL-6以及ECP的表达（P＜0.05）。结论 TBXTG具有一定的抗炎作用，可上调PACAP表达，促进巨噬细胞从M1型向M2型极化，调节促炎和抗炎细胞因子平衡，从而减轻CRS大鼠的鼻黏膜炎症反应。

Abstract:

Objective To investigate the regulatory effect of Tongbi Xiaoti Granules (TBXTG) on the pituitary adenylate cyclase-activating polypeptide (PACAP)/macrophage/eosinophil cationic protein (ECP) in a rat model of ovalbumin-induced chronic rhinosinusitis(CRS).Methods The chemical composition of TBXTG was analyzed using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). Sixty SPF SD rats were randomly divided into six groups: normal control group, model group, clarithromycin group, and low-, medium-, and high-dose TBXTG groups, with 10 rats in each group. A CRS rat model was established by ovalbumin sensitization and challenge. After successful modeling, all treatment groups received oral gavage for two weeks. Nasal mucosal inflammation was assessed by hematoxylin-eosin (HE) staining, M1/M2 macrophage polarization was detected by immunofluorescence staining, and the expressions of PACAP, interleukin (IL)-10, transforming growth factor-β (TGF-β), ECP in nasal mucosa were examined by immunohistochemistry. Serum levels of PACAP and cytokines [IL-10, TGF-β, tumor necrosis factor-α (TNF-α), IL-6] were determined by enzyme-linked immunosorbent assay (ELISA). Data were analyzed using GraphPad Prism 9.5.0 and Image-Pro Plus 6.0.Results A total of 31 compounds were identified from TBXTG, mainly flavonoids, phenolic acids, and flavonoid glycosides. Compared with the normal control group, rats in the model group exhibited marked chronic inflammatory changes in the nasal mucosa, characterized by substantial inflammatory cell infiltration and glandular hyperplasia. In contrast to the model group, the pathological changes such as inflammatory cell infiltration and glandular hyperplasia in the nasal mucosa of each dose group of TBXTG were significantly alleviated. TBXTG significantly inhibited the expression of the M1 macrophage marker CD86 and promoted the expression of the M2 macrophage marker CD206. Furthermore, it upregulated the levels of PACAP in mucosa and serum (P<0.05), increased anti-inflammatory cytokines (IL-10, TGF-β), and reduced pro-inflammatory cytokine TNF-α, IL-6 and ECP expression (P<0.05).Conclusions TBXTG exerts certain anti-inflammatory effects. It can upregulate the expression of PACAP, promote macrophage polarization from M1 type to M2 type, and regulate balance between pro-inflammatory and anti-inflammatory cytokines, thereby alleviating the nasal mucosal inflammatory responses in CRS rats.