Supplementary Material for: Demethylzeylasteral (T-96) Alleviates Allergic Asthma via Inhibiting MAPK/ERK and NF-κB Pathway

Introduction: Demethylzeylasteral (T-96), a new extract of Tripterygium wilfordii Hook F (TWHF), exerted immunomodulatory properties in autoimmune diseases, but its effect on airway inflammatory diseases remains unclear. Our study aims to explore the protective effect and underlying mechanism of T-96 in allergic asthma. Methods: The OVA-induced asthmatic mice were administered by gavage with T-96 (0.1mg/10g, 0.3mg/10g or 0.6mg/10g) one hour before each challenge. The airway hyperresponsiveness was assessed, pathological changes were evaluated by HE and PAS staining and expressions of Th2 cytokines were determined by PCR and ELISA. The activation of MAPK/ERK and NF-κB pathway was assessed by western blot. Results: T-96 significantly relieved airway hyperresponsiveness in asthmatic mice, evidenced by reduced airway resistance (Raw) and increased lung compliance dynamic compliance (Cdyn). Also, enhanced inflammatory infiltration and mucus hypersecretion were ameliorated in lungs of asthmatic mice following increasing doses of T-96 treatment, accompanied by decreased eosinophils in bronchoalveolar lavage fluid (BALF), IgE and OVA specific-IgE levels in serum and downregulated IL-5 and IL-13 expressions in BALF and lung tissues as well. Notably, phosphorylation levels of p38 MAPK, ERK and p65 NF-κB were obviously increased in asthmatic mice compared with the control group, which were then abrogated upon T-96 treatment. Conclusion: This study first revealed that T-96 alleviated allergic airway inflammation and airway hyperresponsiveness via inhibiting MAPK/ERK and NF-κB pathway. Thus, T-96 could potentially act as a new anti-inflammatory agent in allergic asthma.

引言：去甲泽兰红素（Demethylzeylasteral，T-96）是雷公藤（Tripterygium wilfordii Hook F，TWHF）的新型提取物，其在自身免疫性疾病中展现出免疫调节特性，但针对气道炎症性疾病的作用尚不明确。本研究旨在探讨T-96对过敏性哮喘的保护作用及其潜在作用机制。
方法：本研究对卵清蛋白（OVA）诱导的哮喘小鼠，于每次激发前1小时予以T-96灌胃给药，剂量分别为0.1mg/10g、0.3mg/10g及0.6mg/10g。通过气道高反应性（airway hyperresponsiveness）检测评估气道功能，采用苏木精-伊红（hematoxylin-eosin，HE）染色与过碘酸雪夫（periodic acid-Schiff，PAS）染色评价肺部病理变化，利用聚合酶链式反应（PCR）与酶联免疫吸附试验（ELISA）测定辅助性T细胞2（Th2）细胞因子的表达水平；采用蛋白质印迹法（western blot）检测丝裂原活化蛋白激酶/细胞外调节蛋白激酶（MAPK/ERK）及核因子κB（NF-κB）信号通路的激活情况。
结果：T-96可显著缓解哮喘小鼠的气道高反应性，具体表现为气道阻力（Raw）降低与动态肺顺应性（dynamic compliance，Cdyn）升高。同时，随着T-96给药剂量升高，哮喘小鼠肺部的炎症浸润与黏液高分泌症状均得到改善，伴随支气管肺泡灌洗液（bronchoalveolar lavage fluid，BALF）中嗜酸性粒细胞数量减少，血清中免疫球蛋白E（IgE）及卵清蛋白特异性IgE（OVA specific-IgE）水平降低，支气管肺泡灌洗液与肺组织中白细胞介素5（IL-5）、白细胞介素13（IL-13）的表达亦显著下调。值得注意的是，与对照组相比，哮喘小鼠体内p38丝裂原活化蛋白激酶（p38 MAPK）、细胞外调节蛋白激酶（ERK）及p65核因子κB（p65 NF-κB）的磷酸化水平明显升高，而该升高效应可被T-96给药所阻断。
结论：本研究首次证实，T-96可通过抑制MAPK/ERK及NF-κB信号通路，减轻过敏性气道炎症与气道高反应性。因此，T-96有望成为过敏性哮喘的新型抗炎制剂。