Ros kit

The Fe²⁺ levels were measured from mouse plasma using the Iron Assay Kit (JaICA/CFE-005) according to the manufacturer’s instructions. GSH levels were measured using the GSH kit (Nanjing Jiancheng Bioengineering Institute, A005-1-2). The malondialdehyde (MDA) Kit (Nanjing Jiancheng Bioengineering Institute, A003-1-2) was used to measure the levels of the lipid peroxidation product MDA, while the reactive oxygen species (ROS) Kit (Nanjing Jiancheng Bioengineering Institute, E004) was used to measure the intracellular lipid ROS level.

DCFH-DA working solution was diluted in serum-free medium according to the ROS Kit instructions (Nanjing Jiancheng E004). The intracellular ROS release level was measured by flow cytometry, microplate reader, and fluorescence microscope, respectively. For the mirocplate reader detection, the Ex of 485 nm and Em of 525 nm were used.

ROS expression was determined by a ROS kit (Jiancheng Bioengineering Institute, Nanjing, China). A single-cell suspension was prepared from the mammary gland samples. The cell precipitates were suspended by diluted DCFH-DA in sample tubes, which were incubated at 37 °C for 60 min. The excitation wavelength was set at 500 nm and the emission wavelength was set at 525 nm for fluorescence detection. The corresponding fluorescence values were read.

C57BL/6 mice were acquired from Vital River Laboratories (Beijing, China) and primary mouse lung fibroblasts (MLF), MIC-CELL-0040 from PriCells (Hubei, China). RNA extraction kit was purchased from Tiangen Biotech (Beijing, China), reverse transcription kit and fluorescent quantitation PCR kit from Takara (Tokyo, Japan). Nrf2, Bach1, HO-1, GPx1 rabbit anti-mouse primary antibodies were purchased from Abcam (Cambridge, MA, USA). Protein Extraction Kit was obtained from Sigma-Aldrich (St. Louis, MO, USA) and ROS kit were purchased from Jiancheng Bioengineering Institute (Nanjing, China). Enzyme linked immunosorbent assay (ELISA) Kit was obtained from R&D (R&D Systems, Minneapolis, MN, USA) and Cloud-Clone Corp (USCN Life Science Inc, Wuhan, China). Primers were synthesized by Sangon (Shanghai, China). Hematoxylin staining kit and Masson trichrome staining kit were purchased from Solarbio (Beijing, China). TGF-β1 recombinant protein was obtained from R&D, and PFD was obtained from Shionogi & Co., Ltd (Osaka, Japan).

Mouse bone marrow neutrophils were cultured in DMEM containing 10% FBS at 37 °C and 5% CO₂ until the cell density reached 85–90%. The cells were harvested by centrifugation at 1000 rpm for 5 min and subcultured in fresh medium. Next, adherent cells in the logarithmic growth phase were selected, LPS at a final concentration of 1 µg/mL was added, and the reaction mixture was incubated for 5 h. mPEG-SPA-MDSPI16 (200 ng/mL and 400 ng/mL), MDSPI16 (200 ng/mL), or Sivelestat was added, and the resulting mixture was incubated for 12 h. Subsequently, cell precipitates were collected, and cytokine (IL-10, IL-6, IL-1β, TNF-α, COX-2, and iNOS) expression levels in mouse bone marrow neutrophils were assessed by qRT–PCR. Reactive oxygen species (ROS) levels in neutrophils treated with mPEG-SPA-MDSPI16 were determined by an ROS kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Neutrophil supernatants were collected, and cytokine expression levels (IL-10, IL-6, IL-1β, and TNF-α) in neutrophil supernatants were measured using ELISA kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. Neutrophil elastase activity in neutrophil supernatants was determined with a mouse neutrophil elastase detection kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

ROS level was assessed using a ROS kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) based on the manufacturer’s protocol. Briefly, SH-SY5Y cells were treated with DCFH-DA (5 μM) for 30 min in darkness. The fluorescence intensity of SH-SY5Y cells was determined with a FACScan flow cytometer.

The immunofluorescence was measured as described previously with some minor modifications [28 (link)]. Briefly, after IEC-6 was stimulated with corresponding treatment, the cells were fixed by 4% paraformaldehyde for 20min, ruptured by 0.3% Triton-100 for 9min, and blocked by goat serum (Beyotime, China), and ZO-1 (CST, America) primary antibody, secondary antibody (Thermo, America), and DAPI (Thermo, America) were incubated. ROS, Tunel, mitochondria, and JC-1 (ΔΨm) were stained according to the instruction of each kit. ROS kit was purchased from Jiancheng (China), Tunel kit was purchased from Roche (Germany), Mitochondrial kit was purchased from Roche (Germany), and JC -1 kit was purchased from Beyotime (China). Confocal imaging was performed by Leica SP5-II confocal microscope (Germany).