Verbascoside mitigated ferroptosis in asthma pathogenesis and suppressed macrophages chemotaxis and inflammation

House dust mites (HDM) and ovalbumin (OVA) are environmental allergens that can trigger allergic diseases like asthma. Research suggests a connection between ferroptosis and inflammation, with inhibiting ferroptosis potentially reducing inflammation and bodily harm. However, the role of ferroptosis in HDM and OVA-induced allergic airway inflammation in asthma is not well understood. Verbascoside is known for its anti-inflammatory, antioxidant, and antimicrobial properties, but its impact on ferroptosis is still uncertain.

Here, we assessed the role of ferroptosis in HDM and OVA exposure triggered allergic airway inflammation, and explored whether ferroptosis was a therapeutic strategy and verbascoside as a ferroptosis inhibitor for relieving allergic inflammation triggered by HDM and OVA exposure.

We established mice models of asthma using HDM and OVA, and administered the ferroptosis inhibitors Fer-1 and DFO alongside verbascoside. We then evaluated asthmatic airway inflammation by performing pathological staining of lung tissues, differential leukocyte counts in bronchoalveolar lavage fluid (BALF), and ELISA-based measurements of inflammatory cytokines in BALF. Alterations in ferroptosis were assessed through transmission electron microscopy of lung tissue, measurements of oxidative and antioxidant molecules, and lipid peroxidation assays.To further investigate the mechanism of verbascoside, we employed human airway epithelial cell lines (HBE and Beas-2b) in vitro, using the classical ferroptosis inducer erastin to establish a cellular model. Using qPCR, intracellular iron measurements, lipid peroxide detection, and cellular transmission electron microscopy, we evaluated verbascoside's inhibition of erastin-induced ferroptosis and associated inflammation.We then conducted exploratory validation studies in vivo and in vitro using CCR2 and CCR5 inhibitors to determine whether verbascoside alleviates allergic airway inflammation by inhibiting epithelial cell ferroptosis and subsequent macrophage recruitment.

Exposure to OVA or HDM induced iron overload, lipid peroxidation, and ferroptosis in mice lungs. Both Fer-1 and DFO treatments alleviated allergic airway inflammation triggered by OVA or HDM challenge, while also suppressing erastin-induced iron overload, lipid ROS accumulation, ferroptosis, and pro-inflammatory cytokine expression in airway epithelial cells. Verbascoside similarly reduced airway inflammation and ferroptosis both in vitro and in OVA-/HDM-induced murine asthma models. RNA sequencing revealed that verbascoside modulates ferroptosis-associated pathways in airway epithelial cells. In OVA-exposed mice lungs, protein levels of key ferroptosis regulators were altered: TFR1 and FTH increased, whereas SLC7A11, GPX4, and FPN decreased. Additionally, lipid-metabolizing enzymes-15-LO1, ACSL4, and LPCAT3-were upregulated. These abnormalities were reversed by verbascoside treatment. Moreover, verbascoside diminished the secretion of CCL2 and CCL5, thereby reducing macrophage chemotaxis toward ferroptotic airway epithelial cells. In vitro blockade of CCR2 and CCR5 limited this chemotactic response, while in vivo inhibition of these receptors mitigated allergic airway inflammation and lowered type 2 cytokine levels in BALF from OVA-challenged mice. Overall, verbascoside attenuated chemokine-driven macrophage recruitment and subsequent inflammatory cytokine expression in the lungs following OVA exposure.

These findings indicated that verbascoside attenuated ferroptosis and protected against OVA/HDM exposure induced allergic airway inflammation, demonstrating verbascoside as a ferroptosis inhibitor and therapeutic strategy for allergic damage induced by OVA and HDM exposure, and allergic inflammatory diseases related to ferroptosis.