The anti-inflammatory and immunomodulatory potential of braylin: Pharmacological properties and mechanisms by <i>in silico</i>, <i>in vitro</i> and <i>in vivo</i> approaches - Fig 5

<p>Docking solutions showing the main interactions for (A) RU486 (pink), (B) dexamethasone (orange) and (C) braylin (cyan) superimposed in GR (pdb 1NHZ).</p

Effects of braylin on tail flick and hot plate tests in mice.

<p>Panels representing the latency in seconds in the tail flick (panel A) and hot plate (panel B) tests, after ip injection of braylin (BRA; 100 mg/kg), vehicle (50% propylene glycol in saline; control group) or morphine (5 mg/kg; reference drug). Data are reported as means ± SEM; <i>n</i> = 6 mice per group. * Significantly different from the control group (<i>p</i> < 0.05). Two-way ANOVA followed by the Bonferroni’s test.</p

Effects of braylin on cytokines paw levels during CFA-induced inflammation.

<p>Mice were injected with braylin (BRA; 50 mg/kg), vehicle (50% propylene glycol in saline; control group) or dexamethasone (Dexa; 2 mg/kg; reference drug) by ip route 40 minutes before CFA (injected at time zero). The naïve group consists of mice that did not receive any experimental manipulation. Panels shows the paw levels of (A) interleukin-1β (IL-1β), (B) tumor necrosis factor-α (TNF-α), (C) interleukin-6 (IL-6), (D) interleukin-13 (IL-13), (E) interleukin-10 (IL-10) and (F) transforming growth factor-β (TGF-β), determined in skin tissues samples by ELISA, 3 hours after the CFA injection. The results are expressed as picograms of cytokine per milligram of protein. Data are expressed as means ± SEM; <i>n</i> = 6 mice per group. * Significantly different from the vehicle group in the same time (<i>p</i> < 0.05); ^# significantly different from the naive group (<i>p</i> < 0.05). ANOVA followed by Tukey´s multiple comparison test.</p

Best poses of the docking results to RU486 (pink), dexamethasone (orange) and braylin (cyan) superimposed in GR (pdb 1NHZ).

<p>Best poses of the docking results to RU486 (pink), dexamethasone (orange) and braylin (cyan) superimposed in GR (pdb 1NHZ).</p

Chemical structure of braylin.

<p>Chemical structure of braylin.</p

Cytotoxic effect of braylin and its modulation of nitric oxide production on macrophages.

<p>Panels A and C: J774 cells (A) or peritoneal exudate macrophages (C) were incubated with vehicle (50% propylene glycol in saline, Ct, control group) or different concentrations of braylin (BRA; 10, 20, 40 or 80 μM) for 72 hours and cell viability was determined by Alamar Blue assay. Gentian violet (GV) was used as positive control. Data are expressed as means ± SEM; <i>n</i> = 9 determinations per group. *Significantly different from the vehicle treated cultures (<i>p</i> < 0.05). ANOVA followed by Tukey´s multiple comparison test. Panels B and D: Concentrations of nitrite were determined in J774 macrophages (B) or peritoneal exudate macrophages (D) treated with vehicle (50% propylene glycol in saline, Ct+, control group), braylin (BRA; 10, 20 or 40 μM) or dexamethasone (Dexa; 40 μM) in the presence of LPS (500 ng/mL) + IFN-γ (5 ng/mL). Cell-free supernatants were collected 24 hours after treatments for nitrite quantification by the Griess method. Ct- shows concentrations of nitrite in unstimulated cells. Data are expressed as means ± SEM; <i>n</i> = 9 determinations per group. *Significantly different from the vehicle treated cultures stimulated with LPS + IFN-γ (<i>p</i>< 0.05). ANOVA followed by Tukey´s multiple comparison test.</p

Effect of braylin on cytokine production by activated macrophages.

<p>Concentrations of TNF-α, IL-1β and IL-6 were determined in cultures of J774 macrophages (panels A, C and E) or peritoneal exudate macrophages (panels B, D and F) treated with vehicle (50% propylene glycol in saline, Ct+, control group), braylin (BRA; 10, 20 or 40 μM) or dexamethasone (Dexa; 40 μM) in the presence of LPS (500 ng/mL) plus IFN-γ (5 ng/mL). Cell-free supernatants were collected 4 hours (for TNF-α measurement) and 24 hours (for IL-1β and IL-6) after treatments for ELISA assay. Ct- shows cytokine concentrations in unstimulated cells. Data are expressed as means ± SEM; <i>n</i> = 10 determinations per group. *Significantly different from the vehicle treated cultures stimulated with LPS + IFN-γ (<i>p</i> < 0.05). ANOVA followed by Tukey´s multiple comparison test.</p

Involvement of glucocorticoid receptors and NF-κB dependent transcriptional activity in the immunomodulatory effect of braylin.

<p>Panel A shows data from glucocorticoid receptor antagonism assay. Concentrations of TNF-α were determined in J774 macrophages treated with vehicle (50% propylene glycol in saline, Ct+, control group), braylin (BRA, 40 μM), RU486 (GR antagonist, 10 μM) + braylin 40 μM, dexamethasone (Dexa; 40 μM) or RU486 (10 μM) + dexamethasone (40 μM) in the presence of LPS (500 ng/mL) and IFN-γ (5 ng/mL). Cell-free supernatants were collected 4 hours after treatments for TNF-α measurement by ELISA. Ct- and RU-show concentrations of TNF-α in unstimulated cells, treated with vehicle and RU486, respectively. Data are expressed as means ± SEM; <i>n</i> = 10 determinations per group. ^$Significantly different from the vehicle treated cultures unstimulated (<i>p</i> < 0.05); *Significantly different from the vehicle treated cultures stimulated with LPS + IFN-γ (<i>p</i> < 0.05). ⁺Significantly different from the group untreated with antagonist (<i>p</i> < 0.05). Panel B shows the effect of braylin on the activation of NF-κB on RAW 264.7 Luc macrophages. Cells were pretreated with vehicle (50$</sup>Significantly different from the vehicle treated cultures unstimulated (<i>p</i> < 0.05); *Significantly different from the vehicle treated cultures stimulated with LPS + IFN-γ (<i>p</i> < 0.05). ^#Significantly different from the Dexa group (<i>p</i> < 0.05). ANOVA followed by Tukey´s multiple comparison test.</p