L8880

LPS (L8880, Solarbio, Beijing, China) combined with IFN-γ (P00106, Solarbio, Beijing, China) or IL-4 (HZ-1004, Proteintech, Wuhan, China) was used to construct the model of M1 and M2 polarization in RAW264.7 cells [32 (link),33 (link),34 (link)]. Briefly, RAW264.7 cells were inoculated in 24-well plates at a density of 50 × 10⁴ per well. In the control group, no treatment was labeled as M0 macrophages. In the model of the M1 polarization group, LPS 100 ng/mL combined with IFN-γ 20 ng/mL was given, while IL-4 20 ng/mL was given in the M2 polarization group. RNA from the cells were extracted after 12 h treatment, and then they were converted into cDNA. The levels of TGF-β, ARG-1, TNF-α, and iNOS of RAW264.7 were assessed using qRT-PCR.β-Actin as the housekeeping gene.

For the construction of in vitro cell model of SCI, primary spinal neurons were stimulated with LPS. In this work, the concentration of LPS (L8880, Solarbio) was 100 ng/mL [22 (link)].

All experiments were approved by the Institutional Animal Care and Use Committee at the Shanxi Medical University. Sixty female C57BL/6 mice (22–26 g in weight) were used due to a higher incidence of IC/BPS in females than males in humans and the easier feasibility of intravesical surgery in females.
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³⁰ (link) Mice were anesthetized by intraperitoneal injection with 0.3 ~ 0.4 mL of 2% pentobarbital sodium solution. The bladder was then catheterized urethrally with a polyethylene catheter (PE‐50), instilled with 1 mL of protamine (10 mg/mL; Sigma‐Aldrich), and retained for 45 min. After draining the instilled liquid, 1 mL of LPS (750 μg/mL; Solarbio; L8880) was instilled and kept for 30 min. Repeat the preceding steps for three consecutive days. At day 14 after cystitis induction, mice were analyzed for phenotypic and functional changes or treated with MSC‐EVs or TAK‐242 followed by phenotypic and functional analyses.

Primary Th17 cells, bone marrow‐derived macrophages (BMDM), and the RAW264.7 cell line were employed to investigate the findings. They were all cultured in an incubator at 37°C with 5% CO₂. Th17 and BMDM were cultured in RPMI‐1640 containing 10% FBS and 1% Penicillin–Streptomycin Solution. RAW264.7 cells were cultured in a high‐glucose DMEM medium supplemented with 10% FBS and 1% Penicillin–Streptomycin Solution. Th17 and BMDM are non‐proliferative or have low proliferation rates and were collected before each experiment. RAW264.7 has the ability to proliferate and was passaged every 2 days at a ratio of 1:5. The RAW264.7 cells were frozen every other generation. The freezing medium consisted of high‐glucose DMEM culture medium, FBS and DMSO in a ratio of 5:4.5:0.5. After placing the cells in a programmed freezing container, they were initially stored in a − 80°C freezer overnight before being transferred to liquid nitrogen.
BMDM and RAW264.7 cells required drug treatment in a serum‐free culture medium before the experiments. They were incubated with IL‐17A (100 ng/mL; Peprotech, 210–17) for 12 h, followed by media replacement with a serum‐free culture medium. After that, they were incubated with LPS (100 ng/mL; Solarbio, L8880) for 12 h and either total protein or total RNA was extracted.

Pleural and peritoneal cells were isolated from mice and cultured in a 12-well plate at 37 °C in the presence of 5% CO₂ for 3 h. Then, cell polarization into M1 or M2 was performed by stimulation with IFN-γ (50 ng/ml, Peprotech, 315-05) plus LPS (10 ng/ml, Solarbio, L8880) or IL4 (20 ng/ml, Solarbio, P00021), respectively. After 24 h culture, cells were prepared for RNA extraction and qRT-PCR was done to measure the related gene expression level.

Firstly, rats were fasted for 12 h and had free access to drink. LPS (5 mg/kg, cat. no. L8880, Solarbio, Beijing, China) was slowly injected into the trachea of each rat to induce ALI [26 (link)]. 24 h after administration, rats were anesthetized by 2% pentobarbital sodium and then sacrificed by bloodletting from the abdominal aorta. The serum was separated from the blood. One portion of lung tissue was fixed in 10% formaldehyde and the other part was preserved at −80°C.
In the in vitro study, HPMECs in the LPS group were added with LPS (100 μg/mL) firstly, cultured at 37°C under 5% CO₂ for 24 h, and then cells were harvested. After LPS treatment for 1 hour, cells in the HRS group were added with HRS 10 μL/mL for further cultivation. In the HRSC group, HPMECs without LPS treatment were added with HRS 10 μL/mL and cultivated for 24 h. Then cells were collected.