[Electroacupuncture alleviates ferroptosis in lung tissue by regulating Keap1/Nrf2 pathway in asthmatic mice]

To observe the effect of electroacupuncture (EA) on the activities of Kelch-like epichlorohydrin-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, pulmonary inflammation, and ferroptosis in house dust mite (HDM)-induced asthma model mice, so as to explore its underlying mechanisms in ameliorating asthma.

Female C57BL/6 mice were randomly allocated to 4 groups:control, model, EA, and dexamethasone (DEX) groups (n=6 per group). The asthma mouse model was established by repeated intranasal instillation of HDM 50 μg (in PBS) on day 0, 7 and 14 (for immunization), and 25 μg (in PBS) on day 21, 22 and 23 (for sensitization). EA (1 mA, 2 Hz/15 Hz) was applied to "Dazhui" (GV14), "Feishu" (BL13), and "Zusanli" (ST36) for 30 min, once every other day for 5 times. Mice of the DEX group received intraperitoneal injection of dexamethasone (1 mg/kg) once every other day for 5 times after modeling. The mice's general conditions were recorded and their asthma behavioral scores (0 to 9 points) were assessed according to the severity of symptoms of nasal grabbing, itch-scratching, and asthmatic attack. The mice's pulmonary function including forced expiratory volume in 0.1 second (FEV_0.1), forced vital capacity (FVC), the ratio of FEV_0.1/FVC, and peak expiratory flow (PEF) were measured using a pulmonary function detector. Additionally, the histopathological changes, inflammation scores and airway wall thickness (total bronchial wall area [Wat] / bronchial basement membrane perimeter [Pbm]) in the lung tissues were assessed using H.E. staining, periodic acid-Schiff (PAS) staining, and Masson staining, respectively. The contents of immunoglobulin E (IgE), tumor necrosis factor-alpha (TNF-α) in serum, interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) in bronchoalveolar lavage fluid, and malondialdehyde (MDA), glutathione (GSH), and iron in the serum and lung homogenates were detected using ELISA and bicinchoninic acid (BCA), separately. The protein and mRNA expression levels of Keap1, Nrf2, solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase 4 (GPX4), acyl-CoA synthetase long-chain family member 4 (ACSL4), and transferrin receptor 1 (TFR1) in the lung tissue were detected by Western blot and PCR, separately. The positive expressions of SLC7A11, GPX4, ACSL4, and TFR1 in the lung tissue were detected using immunohistochemical staining.

Compared to the control group, the asthma behavioral score, inflammation score, Wat/Pbm, contents of TNF-α, IgE, MDA and iron in the serum, contents of IL-1β and IL-6 in the bronchoalveolar lavage fluid, and contents of MDA and iron in the lung tissue, expression levels of Keap1, ACSL4 and TFR1 proteins and mRNAs in the lung tissue, as well as the immunoactivity of pulmonary ACSL4 and TFR1 were significantly increased (P<0.01), while the FEV_0.1, ratio of FEV_0.1/FVC, and the PEF, and serum and pulmonary GSH contents, expression levels of pulmonary Nrf2, SLC7A11 and GPX4 proteins and mRNAs, and immunoactivity of SLC7A11 and GPX4 significantly decreased in (P<0.01) the model group. In comparison with the model group, all the above indicators of mice in both EA and DEX groups were reversed (P<0.01, P<0.05). The therapeutic effects of EA were strikingly superior to those of DEX in raising PEF, and serum and pulmonary GSH (P<0.05, P<0.01), and in down-regulating serum IgE and iron, pulmonary iron, and expression levels of pulmonary ACSL4 mRNA (P<0.05, P<0.01), but striking inferior to those of DEX in raising expression levels of pulmonary Nrf2, SLC7A11 and GPX4 proteins and mRNAs, and SLC7A11 and GPX4 immunoactivity (P<0.05, P<0.01), and in lowering IL-6 of bronchoalveolar lavage fluid, serum and pulmonary MDA contents, and the expression of pulmonary Keap1 protein and mRNA (P<0.05, P<0.01).

EA can alleviate HDM-induced pulmonary inflammation and decreased pulmonary function in mice with asthma, which may be related to its function in modulating ferroptosis mediated by the Keap1/Nrf2 pathway.