At rvd1

The NP model was established via unilateral L5-6 spinal nerve ligation (SNL) according to the method suggested by Ye and Savelieva (2015) (Ye et al., 2015 (link)). Briefly, the SD rats were anesthetized intraperitoneally with 1% sodium pentobarbital (Nembutal, 50 mg/kg), the left L5-6 nerve was exposed by blunt dissection, and transected distal to the ligation using a 5–0 silk thread. To examine the therapeutic and mechanistic effect of AT-RvD1 on the established spinal never injury, the animals were randomly administrated to different doses of AT-RvD1 (Cayman Chemical, 10 or 100 ng per rat per day, intrathecally) and 3-methyladenine (3-MA; MedChemExpress, HY-19312, 15 mg/kg/d, intraperitoneally) divided into different treatment groups after SNL surgery. The rats were injected with different doses of AT-RvD1 or 3-MA for the first three consecutive days after surgery according to our previous studies (Wang et al., 2020 (link); Wang et al., 2021 (link)). On Day 7 after SNL, cervical dislocation was performed when the rats were lightly anesthetized with pentobarbitone (120 mg/kg, intraperitoneally). The ipsilateral dorsal horn of spinal cord for each rat was removed and collected for further experimental analysis.

The pulmonary fibrosis model was induced by intratracheal instillation (i.t.) of BLM [Sigma Aldrich, Saint Louis, MO, USA; 0.06 U/mouse in a final volume of 30 μl of saline (SAL)] as previously described (22 (link), 23 (link)). Mice were divided into three groups: 1) SAL group = mice received i.t. administration of SAL and i.v. administration of ethanol 0.1% and 0.01% (diluted in SAL) on days 7 and 10, respectively; 2) BLM group = mice received BLM i.t. injection on day 0 and i.v. administration of SAL on days 7 and 10, respectively; and 3) BLM + ATRvD1 group = mice received BLM i.t. injection on day 0 and i.v. administration of ATRvD1 (Cayman Chemicals, Ann Arbor, MI, USA) 2.5 μg/kg in 0.1% ethanol and 0.5 μg/kg in 0.02% ethanol on days 7 and 10, respectively. On day 14, bronchoalveolar lavage (BAL) was performed. The lungs were immediately removed postmortem and used for histological analysis, cytokine quantification by ELISA, and detection of primary cell-derived EVs.

LXB₄, RvE1, RvD2, and AT-RvD1, purchased from Cayman Chemicals, were validated by assessing the characteristic UV chromophores for each of these mediators as well as their physical properties in LC-MS/MS, including characteristic MS/MS spectra (24 (link)). Lipid mediator concentrations were determined by using the extinction coefficient characteristic for each of the conjugated-double-bond systems as well as measuring maximal UV absorbance. Endotoxin content was assessed using ToxinSensor Chromogenic LAL Endotoxin Assay Kit (Antibodies Online), which was found to be below limits of detection in all preparations.

A quantity of 100 μl of growth factor‐reduced Matrigel (GFR‐MG) (8 mg/mL; 2:1 GFR‐MG: DMEM‐Ham's F12 [1:1] medium; Becton Dickinson, Franklin Lakes, NJ) was allowed to solidify in a 37°C incubator for 1 h in eight‐well chambers mounted on No. 1.5 German borosilicate coverglass (Nalge Nunc International, Naperville, IL). Then, Par‐C10 cells (15,000 cells/well; passages 30–60) were plated on the GFR‐MG in DMEM‐Ham's F12 (1:1) medium with supplements and treated with TNF‐α (100 ng/mL; Becton Dickinson), then incubated for 1 h or 6 h at 37°C. Cells were then treated with AT‐RvD1 (100 ng/mL; Cayman Chemical, Ann‐Arbor, MI), and varying doses of DEX (25–100 ng/mL). Three‐dimensional Par‐C10 cell clusters were used for assays after incubation at 37°C with 95% air and 5% CO₂ for 72 h.

The 2,2′ azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS+), 2,4,6-Tripyridyl-s-triazine (TPTZ), 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB), bisacrylamide, hexadecyl trimethyl ammonium bromide (HTAB), brilliant blue stain, phenanthroline, phenylmethylsulfonyl fluoride, reduced glutathione (GSH), nitroblue tetrazolium (NBT), o-dianisidine dihydrochloride, and Trolox were obtained from Sigma-Aldrich (St. Louis, MO, USA). Tris was obtained from Amresco (Solon, OH, USA). Enzyme-linked immunosorbent assay (ELISA) kits were obtained from eBioscience (San Diego, CA, USA). Acrylamide sodium dodecyl sulfate (SDS), Superscript1III, Oligo(dT)12-18 primers, Platinum SYBRGreen I, and primers were obtained from Invitrogen (Carlsbad, CA, USA). AT-RvD1 was purchased from Cayman Chemicals (Ann Arbor, MI, USA). All other reagents used were of pharmaceutical grade.