Ml385

The RAW264.7 macrophage M1 model was created with 1 μg/mL LPS. It has been shown that AKBA can promote the expression of Nrf2, so the group with the addition of Nrf2 inhibitor (ML385) (5 μm)
³⁰ (link) (MedChemExpress, Monmouth Junction, NJ, USA) was designed. Cells were divided into the control group, control + AKBA group, control + ML385 group, LPS group, LPS + AKBA group, and LPS + AKBA + ML385 group. After passing the cells and waiting for 6 h to adhere to the wall, the serum‐free medium was replaced with starvation culture for 6 h, followed by LPS, AKBA and ML385 pretreatment for 12 h, and cells were collected for WB after 12 h of action (3 replicates in each group).
The Rma‐bm macrophage M1 model was created with 1 μg/mL LPS. It has been shown that AKBA can promote the expression of Nrf2, so the group with the addition of Nrf2 inhibitor (ML385) (5 μm) (MedChemExpress, Monmouth Junction, NJ, USA) was designed. Cells were divided into the control group, control + ML385 group, control + AKBA group, control + AKBA + ML385 group, LPS group, LPS + AKBA group, LPS + ML385 group, and LPS + AKBA + ML385 group. The optimal concentration of the AKBA group is 2.5 μm. After passing the cells and followed by LPS, AKBA and ML385 pretreatment for 12 h, and cells were collected for WB after 12 h of action (3 replicates in each group).

The MLT group was injected with 40 mg/kg MLT (MedChemExpress, New Jersey, United States) intraperitoneally within 7 days after surgery (Gurlek et al., 2004 (link); Venegas et al., 2012 (link); Kerem et al., 2014 (link)). The control group was treated with an equal concentration of saline. Additionally, in the MLT and ML385 (MedChemExpress, New Jersey, United States) cotreatment group, the rats were injected with 40 mg/kg MLT and 30 mg/kg ML385 (Gou et al., 2020 (link)).

For nasal administration, the Apelin-13 (0.2 mg/kg, 1 mg/kg) solution (10 μL) was pipetted bilaterally on the rhinarium, i.e., the glabrous skin around the nostrils, and allowed to diffuse in the squamous epithelium. After the drip, the mouse was fixed for 5–10 s to ensure the liquid was fully inhaled. Donepezil (Sigma-Aldrich, St. Louis, MO, USA) were dissolved in 0.9% saline. A freshly prepared solution of Donepezil was administered orally daily at a dose of 5 mg/kg [16 (link)]. ML385 (Medchem Express, South Brunswick, NJ, USA) is a specific inhibitor of Nrf2. Before each administration of Apelin-13, the mice received an intraperitoneal injection of 30 mg/kg ML385 [17 (link)], dissolved in saline containing 50% PEG300, with a 30 min interval. Zn(II)-protoporphyrin IX (ZnPP, Medchem Express, South Brunswick, NJ, USA) is an inhibitor of HO-1. Mice were injected intraperitoneally with 25 mg/kg ZnPP [18 (link)] (dissolved in saline) 30 min before each administration of Apelin-13.