Table 2.
SPM profiles in IL-1β stimulated diseased tendon stromal cells incubated in MaR1 and inhibitors of 15-PGDH.
| DHA bioactive metabolome | Q1 | Q3 | Tendon stromal cells Lipid mediators levels (pg/incubation) | |||||
|---|---|---|---|---|---|---|---|---|
| Disease + IL-1β | Disease + IL-1β + MaR1 | Disease + IL-1β + Indo | Disease + IL-1β + MaR1 + Indo | Disease + IL-1β + SW033291 | Disease + IL-1β + MaR1 + SW033291 | |||
| RvD1 | 375 | 141 | 0.2 ± 0.2 | 0.0 ± 0.0 | — | — | — | — |
| RvD2 | 375 | 141 | 0.6 ± 0.4 | 0.9 ± 0.3 | — | — | 0.1 ± 0.1 | 0.2 ± 0.2 |
| RvD3 | 375 | 147 | 0.3 ± 0.3 | 0.4 ± 0.4 | — | — | — | — |
| RvD4 | 375 | 101 | 1.3 ± 1.3 | 0.5 ± 0.3 | — | — | — | — |
| RvD5 | 359 | 199 | 34.0 ± 3.8 | 32.9 ± 7.1 | 1.3 ± 1.3** | 0.3 ± 0.3** | 8.6 ± 1.2** | 7.1 ± 1.6 |
| RvD6 | 359 | 101 | 14.9 ± 2.5 | 13.5 ± 2.3 | 4.1 ± 0.5** | 7.4 ± 1.1** | 3.1 ± 1.3** | 2.0 ± 0.4 |
| 17R-RvD1 | 375 | 141 | 1.2 ± 0.7 | 0.6 ± 0.2 | — | 0.2 ± 0.2 | — | 0.1 ± 0.1 |
| 17R-RvD3 | 375 | 147 | 2.5 ± 1.5 | 2.4 ± 0.9 | — * | — | — | 0.0 ± 0.0 |
| PD1 | 359 | 153 | 7.0 ± 1.1 | 3.5 ± 0.8* | 1.5 ± 1.0 * | 0.1 ± 0.1 | 1.4 ± 0.7** | 0.2 ± 0.2 |
| 17R-PD1 | 359 | 153 | 0.2 ± 0.1 | 0.2 ± 0.2 | — | 0.0 ± 0.0 | 0.1 ± 0.1 | 0.6 ± 0.3 |
| 10 S,17S-diHDHA | 359 | 153 | 34.6 ± 5.2 | 46.8 ± 4.7 | 0.6 ± 0.6* | 1.7 ± 1.7 | 0.9 ± 0.9 ** | 0.8 ± 0.8 |
| MaR1 | 359 | 221 | 0.9 ± 0.9 | 560.3 ± 64.4 ** | 0.0 ± 0.0 | 723.4 ± 42.5**$ | 0.7 ± 0.7 | 662.3 ± 59.5 **## |
| MaR2 | 359 | 191 | 0.5 ± 0.3 | — | 0.7 ± 0.3 | 1.9 ± 0.2*$ | 2.1 ± 0.5* | — # |
| 7 S,14S-diHDHA | 359 | 221 | 32.0 ± 5.2 | 34.9 ± 1.4 | — | 28.8 ± 1.9$ | —** | — |
| 4 S,14S-diHDHA | 359 | 101 | — | — | — | — | — | — |
| 14-oxo-MaR1 | 357 | 248 | 0.3 ± 0.2 | 18.1 ± 2.9 ** | 0.3 ± 0.2 | 11.0 ± 0.9 ** $ | 0.2 ± 0.2 | 7.6 ± 0.8 **# |
| n-3 DPA bioactive metabolome | ||||||||
| RvD1n-3 DPA | 377 | 143 | 3.2 ± 1.9 | 3.4 ± 0.8 | 0.1 ± 0.1 | 1.2 ± 0.6 | — | 0.5 ± 0.5 |
| RvD2n-3 DPA | 377 | 261 | 0.4 ± 0.3 | 0.3 ± 0.3 | — | 0.0 ± 0.0 | — | — |
| RvD5n-3 DPA | 361 | 263 | 25.4 ± 1.7 | 13.4 ± 2.5 | — ** | 0.7 ± 0.7 ** | — ** | — ** |
| PD1n-3 DPA | 361 | 183 | 0.9 ± 0.5 | 1.3 ± 0.3 | 0.4 ± 0.4 | 1.5 ± 0.1 | 4.2 ± 1.8 | 0.7 ± 0.4 |
| 10 S,17S-diHDPA | 361 | 183 | 16.8 ± 1.7 | 16.7 ± 2.3 | 5.2 ± 1.3 * | 5.1 ± 1.3 | 3.3 ± 0.5 ** | 3.5 ± 0.7 ** |
| MaR1n-3 DPA | 361 | 249 | 3.9 ± 2.1 | 0.5 ± 0.2 | 0.4 ± 0.4 | 1.5 ± 1.5 | 0.1 ± 0.1 | 0.1 ± 0.1 |
| EPA bioactive metabolome | ||||||||
| RvE1 | 349 | 195 | — | — | — | 0.2 ± 0.0 $ | —** | 0.2 ± 0.1 |
| RvE2 | 333 | 199 | 49.0 ± 11.7 | 31.8 ± 2.4 | 18.9 ± 2.0 * | 15.8 ± 3.5 | 23.6 ± 4.0 * | 12.5 ± 1.4 *# |
| RvE3 | 333 | 201 | 0.4 ± 0.3 | 0.5 ± 0.3 | — | — | — | — |
| AA bioactive metabolome | ||||||||
| LXA4 | 351 | 217 | — | — | — | — | — | — |
| LXB4 | 351 | 221 | 1.4 ± 1.4 | 17.2 ± 17.2 | — | 3.1 ± 3.1 | — | — |
| 5 S,15S-diHETE | 335 | 235 | 121.8 ± 14.8 | 750.1 ± 203.5* | —* | —* | —** | —** |
| 15epi-LXA4 | 351 | 217 | — | — | — | — | — | — |
| 15epi-LXB4 | 351 | 221 | 35.0 ± 7.3 | 69.8 ± 12.1* | 18.5 ± 3.6* | 33.8 ± 5.3$ | —** | —** |
| 15-oxo-LXA4 | 349 | 233 | — | 0.1 ± 0.1 | — | — | 0.1 ± 0.0 | 0.1 ± 0.1 |
| LTB4 | 335 | 195 | — | — | — | |||
| 5 S,12S-diHETE | 335 | 195 | 45.6 ± 45.6 | 36.4 ± 18.3 | — | — | — | |
| PGD2 | 351 | 189 | 50.0 ± 4.0 | 34.0 ± 9.5 | 0.7 ± 0.7** | 0.7 ± 0.7** | 1.2 ± 1.2** | 1.0 ± 1.0** |
| PGE2 | 351 | 189 | 1655.4 ± 108.7 | 1275.0 ± 95.0 * | 4.6 ± 3.6** | 8.9 ± 1.9** | 154.5 ± 14.8** | 103.0 ± 14.4**# |
| PGF2α | 353 | 193 | 258.1 ± 26.0 | 400.7 ± 138.5 | —** | ** | 2.4 ± 2.2** | 3.3 ± 1.9** |
| TxB2 | 369 | 169 | 374.8 ± 31.5 | 554.3 ± 191.6 | —** | —** | 3.3 ± 3.0** | 8.3 ± 0.6** |
Tendon stromal cells (60,000 cells per well) were derived from diseased supraspinatus tendons. Cells were incubated for 24 h (37 °C) with indomethacin (indo; 10 µM), SW033291 (25 µM) or vehicle (2 h, 37 °C), then with MaR1 (10 nM) or vehicle (24 h 37 °C) and with IL-1β (37 °C; 24 h). Incubations were quenched using ice-cold methanol containing deuterium labelled internal standards and lipid mediators (LM) were identified and quantified using LM-profiling (see methods for details). Q1, M-H (parent ion) and Q3, diagnostic ion in the MS-MS (daughter ion). Results are expressed as pg/incubation. Mean ± SEM of n = 3 per incubation. *P < 0.05, **P < 0.01 vs Disease + IL1β incubations. $P < 0.05, $ $P < 0.01 vs Disease + IL1β + Indo; #P < 0.05, ##P < 0.01 vs Disease + IL1β + SW. The detection limit was ~0.1 pg. -, Below levels found in media alone.