五味沙棘散对卵清蛋白诱导的过敏性支气管哮喘大鼠的保护作用及机制

目的 探究五味沙棘散对卵清蛋白（ovalbumin，OVA）诱导的过敏性哮喘大鼠的保护作用及机制。方法 SD大鼠随机分为对照组、模型组、地塞米松（0.5 mg/kg）组和五味沙棘散（0.5、2.0 g/kg）组，建立OVA诱导的大鼠早期支气管哮喘模型，苏木素-伊红（HE）和Masson染色观察支气管病理情况及纤维化程度；免疫组化检测支气管间隙连接蛋白43（connexin 43，Cx43）的表达和分布；ELISA检测血清中免疫球蛋白E（immunoglobulin E，IgE）和白细胞介素-13（interleukin-13，IL-13）水平；亚甲基蓝法和WSP-1荧光探针法测定血清中硫化氢（hydrogen sulfide，H₂S）水平。建立CdCl₂诱导的支气管上皮BEAS-2B细胞损伤模型，MTT法检测细胞活性；Hoechest 33342染色法观察细胞核形态变化；MitoSOX染色检测细胞线粒体活性氧（mitochondrial reactive oxygen species，mtROS）水平；Western blotting检测半胱氨酸天冬氨酸蛋白酶-3（cystein-asparate protease-3，Caspase-3）、核因子红细胞系2相关因子（nuclear factor erythroid 2-related factor，Nrf2）和Kelch样ECH关联蛋白1（Kelch like ECH associated protein 1，Keap1）蛋白表达。结果 五味沙棘散能够减少支气管黏膜上皮细胞脱落和黏液分泌（P＜0.001），降低胶原纤维的沉积和Cx43的表达及分布（P＜0.05、0.01、0.001），降低血清中IgE、IL-13水平（P＜0.05），升高血清中H₂S水平（P＜0.05、0.01）。五味沙棘散含药血清呈剂量相关性地抑制CdCl₂诱导的BEAS-2B细胞凋亡及mtROS水平（P＜0.01、0.001），上调Caspase-3和Nrf2蛋白表达（P＜0.05、0.01），下调Keap1蛋白表达（P＜0.01）。结论 五味沙棘散对OVA诱导的哮喘大鼠和CdCl₂诱导的支气管上皮细胞凋亡均具有保护作用，其机制可能与调控Nrf2/Keap1信号通路有关。

Protective effect and mechanism of Wuwei Shaji Powder against ovalbumin-induced allergic bronchial asthma in rats

Objective To investigate the protective effect and mechanism of Wuwei Shaji Powder (五味沙棘散, WSP) on ovalbumin (OVA)-induced allergic bronchial asthma in rats. Methods SD rats were randomly divided into control group, model group, dexamethasone (0.5 mg/kg) group and WSP (0.5, 2.0 g/kg) groups, rats were sensitized with OVA to induce an early bronchial asthma model. Hematoxylin-eosin (HE) and Masson staining were used to observe the pathological changes and degree of fibrosis in the bronchi; Immunohistochemistry was performed to detect the expression and distribution of of connexin 43 (Cx43) in bronchi; ELISA was used to measure the levels of immunoglobulin E (IgE) and interleukin-13 (IL-13) in serum; Methylene blue method and WSP-1 fluorescence probe method were used to determine hydrogen sulfide (H₂S) level in serum. The CdCl₂-induced bronchial epithelial cell (BEAS-2B) injury model was established, cell viability was detected by MTT assay; Nuclear morphological changes were observed by Hoechest 33342 staining; Mitochondrial reactive oxygen species (mtROS) level in cell was detected using MitoSOX staining. The expression levels of cystein-asparate protease-3 (Caspase-3), nuclear factor erythroid 2-related factor (Nrf2) and Kelch like ECH associated protein 1 (Keap1) were analyzed by Western blotting. Results WSP could reduce the shedding of bronchial mucosal epithelial cells and mucus secretion (P < 0.001), decrease the deposition of collagen fibers and the expression and distribution of Cx43 (P < 0.05, 0.01, 0.001), lower the levels of IgE and IL-13 in serum (P < 0.05), elevated H₂S level in serum (P < 0.05, 0.01). WSP drug-containing serum showed dose-dependent inhibition of CdCl₂-induced BEAS-2B cell apoptosis and mtROS levels (P < 0.01, 0.001), up-regulation of Caspase-3 and Nrf2 protein expressions (P < 0.05, 0.01), and down-regulation of Keap1 protein expression (P < 0.01). Conclusion WSP has a protective effect on OVA-induced asthma rats and CdCl₂-induced apoptosis of bronchial epithelial cells, and its mechanism may be related to the regulation of Nrf2/Keap1 signaling pathway.