Figure 1

Effects of 13-(S)-HPOTrE and 13-(S)-HOTrE on cells viability, generation of NO and ROS in RAW 264.7 cells and mouse peritoneal macrophages.

(A) Effects of 13-(S)-HPOTrE and 13-(S)-HOTrE on the viability of RAW 264.7 cells were measured by MTT assay. Cells were treated with different concentrations (1, 10, 100 and 200 μM) of ALA metabolites for 24 h. The percent cell growth following treatment was calculated, in comparison with untreated control cells. The values represent mean ± SD of three independent experiments. *Indicates significance (p < 0.05) when compared with untreated control cells. (B) Nitrite levels in the culture medium of RAW 264.7 cells pre-incubated with different concentrations (1, 50 and 100 μM) of ALA metabolites or dexamethasone (10 μM) for 3 h and further stimulated with or without LPS (100 ng/ml) for the next 24 h. 13-(S)-HPOTrE showed more effective dose-dependent reduction in NO level as compared to 13-(S)-HOTrE. Dexamethasone, a steroidal anti-inflammatory drug that suppresses NO production, is used as positive control. *Indicates significance (p < 0.05) compared to LPS treated cells. (C) Intracellular ROS level in RAW 264.7 cells following pretreatment with 100 μM of ALA metabolites for 3 h then stimulated with LPS for 16 h. 13-(S)-HPOTrE reduced ROS level more efficiently compared to 13-(S)-HOTrE. N-Acetyl Cysteine (NAC, 5 mM), a ROS inhibitor was used as positive control. The values represent mean ± SD of three independent experiments. *Indicates significance (p < 0.05) compared to LPS treated cells. (D) Intracellular ROS level in peritoneal macrophages following pre-incubation with 100 nM ALA metabolites for 3 h then challenged with LPS for 16 h. Similar to above result, 13-(S)-HPOTrE reduced ROS level more efficiently compared to 13-(S)-HOTrE. This is representative FACS chromatogram of the three independent experiments.