Supplementary Material for: Alanyl-glutamine inhibits the epithelial-mesenchymal transition of airway epithelial cells in asthmatic mice via DPP4-Sirt1 pathway

Introduction: Alanyl-glutamine (Ala-Gln) is a compound known for its protective effects in various tissue injuries. However, its role in asthma-related lung injuries remains underexplored. This study investigates the mechanisms by which Ala-Gln modulates sDPP4-induced airway epithelial-mesenchymal transition and Ovalbumin (OVA)-induced asthma in a mouse model. Methods: An asthma model was established in female C57BL/6J mice by using OVA. CD4+ T cells and bronchial epithelial cells (BECs) were isolated from the spleen and bronchi of the mice, respectively. Interventions included recombinant sCD26/sDPP4 protein, Ala-Gln, and EX527 (a SIRT1 inhibitor). Flow cytometry was used to assess Th17 and Treg cell populations. Mice were treated with Ala-Gln, EX527, and budesonide (BUD). Histopathological changes in lung tissues were evaluated using hematoxylin-eosin and Masson staining. White blood cell counts were measured with a hematology analyzer. The expression levels of DPP4, IL-17, SIRT1, SMAD2/3, N-cadherin, E-cadherin, MMP9, and α-SMA proteins were analyzed. Results: Treatment with recombinant sCD26/sDPP4 resulted in decreased E-cadherin expression in BECs and increased levels of α-SMA, MMP9, and N-cadherin, effects that were mitigated by Ala-Gln. Ala-Gln also prevented the reduction in SIRT1 expression in BECs and the increase in Th17 cell differentiation induced by recombinant sCD26/sDPP4. EX527 administration alongside Ala-Gln reversed these changes and enhanced the phosphorylation of SMAD2/3 through SIRT1 signaling. BUD alone reduced inflammation and fibrosis in bronchial tissue and lowered the Th17/Treg ratio in peribronchial lymph nodes. The therapeutic effect of BUD was further improved with concurrent Ala-Gln treatment. Conclusion: Ala-Gln can inhibit BEC fibrosis and Th17 cell differentiation mediated by recombinant sCD26/sDPP4 through the SIRT1 pathway. Combined with BUD, Ala-Gln enhanced therapeutic efficacy in OVA-induced asthma in mice, which could offer improved outcomes for asthmatic patients with elevated DPP4 levels.引言：丙氨酰谷氨酰胺（Alanyl-glutamine, Ala-Gln）是一种在多种组织损伤中展现出保护效应的化合物，但其在哮喘相关肺损伤中的作用仍未得到充分探索。本研究旨在探讨Ala-Gln调节可溶性DPP4（sDPP4）诱导的气道上皮间质转化，以及在小鼠模型中干预卵清蛋白（Ovalbumin, OVA）诱导哮喘的作用机制。 方法：本研究采用OVA诱导构建雌性C57BL/6J小鼠哮喘模型。分别从小鼠脾脏及支气管中分离CD4+ T细胞与支气管上皮细胞（bronchial epithelial cells, BECs）。干预试剂包括重组sCD26/sDPP4蛋白、Ala-Gln以及SIRT1抑制剂EX527。采用流式细胞术检测Th17与Treg细胞的群体比例。对小鼠分别给予Ala-Gln、EX527与布地奈德（budesonide, BUD）处理。通过苏木精-伊红染色与Masson染色评估肺组织的病理变化。使用血液分析仪检测白细胞计数。分析DPP4、IL-17、SIRT1、SMAD2/3、N-钙粘蛋白、E-钙粘蛋白、MMP9以及α-平滑肌肌动蛋白（α-SMA）的蛋白表达水平。 结果：重组sCD26/sDPP4处理可降低BECs中E-钙粘蛋白的表达，并上调α-SMA、MMP9与N-钙粘蛋白的水平，上述效应可被Ala-Gln所缓解。Ala-Gln同时可逆转重组sCD26/sDPP4诱导的BECs中SIRT1表达下调以及Th17细胞分化增加。联合EX527与Ala-Gln处理可逆转上述变化，并通过SIRT1信号通路增强SMAD2/3的磷酸化水平。单独使用布地奈德可减轻支气管组织的炎症与纤维化，并降低支气管周围淋巴结中Th17/Treg的比值。联合Ala-Gln治疗可进一步提升布地奈德的治疗效果。 结论：Ala-Gln可通过SIRT1通路抑制重组sCD26/sDPP4介导的BECs纤维化与Th17细胞分化。联合布地奈德使用时，Ala-Gln可增强小鼠OVA诱导哮喘模型的治疗效能，可为DPP4水平升高的哮喘患者提供更优的治疗方案。