Table 1.
PASS predicted activity scores of pentacyclic triterpenoids for NF-κB inhibitor activity.
Table 2.
Docking results of PTs on IKKβ based on glide dock score and number of hydrogen bond interactions (Schrodinger 9.0).
Fig 1.
Hydrogen bond interaction of CA (A), AA (B) and UA (C) at GLU 89 and SER 85 gatekeeper residues of NEMO/IKKβ association complex (PDB code: 3BRV).
Fig 2.
CA, AA and UA inhibited in vitro IKKβ kinase activity.
Dose response results of in vitro kinase assay for three compounds CA (■), AA (●) and UA (♦) against IKKβ. A) IC50 data of compounds against IKKβ. B) IC50 data of standard staurosporine against IKKβ. C) Chemical structures of CA, AA and UA. Data point represents averages of two independent replicates. Data exhibiting inhibition was fitted with a sigmoidal dose-response curve to derive IC50 values.
Fig 3.
CA, AA and UA affect viability of RAW 264.7 cells as evident in MTT assay.
Cytotoxicity was evaluated by MTT assay after exposure of RAW 264.7 cells to increasing concentrations of CA (■), AA (♦) and UA (●) (10 to 150 μM) for 24 h. Cells were plated in each well (10,000 cells/well) of the 96-well tissue-culture plates, after 70% confluence, cells were treated with different concentrations of CA (■), AA (♦) and UA (●) for 24 h. After drug treatment MTT assay was performed as described under materials and methods section. Data were plotted as percent viability (% control).
Fig 4.
CA inhibited Akt-IKKα/β-NF-κB pathway in RAW 264.7 cells.
Cells were pre-treated for 90 min with different concentrations of CA (20, 50, 70 μM) and then treated with LPS (1 μg/ml) for 20 min. Nuclear extracts were prepared as described in materials and methods section and determined for western blotting of NF-κB and Phospho-IKKα/β proteins using specific antibodies. GAPDH served as both an internal loading control and as a nuclear fractionation control. Density ratios versus GAPDH were determined by densitometry. Results are representative of three independent experiments.
Fig 5.
AA inhibited Akt-IKKα/β-NF-κB pathway in RAW 264.7 cells.
Cells were pre-treated for 90 min with different concentrations of AA (70, 90,120 μM) and then treated with LPS (1 μg/ml) for 20 min. Nuclear extracts were prepared as described in materials and methods section and determined for western blotting of NF-κB and Phospho-IKKα/β proteins using specific antibodies. GAPDH served as both an internal loading control and as a nuclear fractionation control. Density ratios versus GAPDH were determined by densitometry. Results are representative of three independent experiments.
Fig 6.
UA inhibited Akt-IKKα/β-NF-κB pathway in RAW 264.7 cells.
Cells were pre-treated for 90 min with different concentrations of UA (70,100, 120 μM) and then treated with LPS (1 μg/ml) for 20 min. Nuclear extracts were prepared as described in materials and methods section and determined for western blotting of NF-κB and Phospho-IKKα/β proteins using specific antibodies. GAPDH served as both an internal loading control and as a nuclear fractionation control. Density ratios versus GAPDH were determined by densitometry. Results are representative of three independent experiments.
Fig 7.
Effects of CA, AA and UA on LPS-induced NF-κB transcriptional activity.
Cells were transiently co-transfected with pNF-κB-luc reporter and then either left untreated (Control) or were pre-treated with IC50 concentrations of CA (50 μM), AA (90 μM) and UA (100 μM) for 90 min. LPS (1 μg/ml) was added and cells were further incubated for 4 h, then harvested and luciferase activities were determined using a luciferase assay system and a luminometer. Bars show means ± SD (n = 3). *P (ANOVA) < 0.05 versus LPS only-treated group.
Fig 8.
CA, AA and UA inhibited IFN-γ release from LPS stimulated RAW cells.
In vitro (supernatant) level of IFN-γ in LPS stimulated RAW cells treated with CA, AA and UA detected through indirect ELISA. Y axis represents the absorbance at 450 nm. Data represented as mean ± SD of three independent experiments. **P < 0.01 (ANOVA) as compared with LPS treated group.
Fig 9.
Proposed mechanism of action of pentacyclic triterpenoids on LPS induced NF-κB signaling.
Abrogation of NF-κB activation by pentacyclic triterpenoids may be resulted due to down regulated IKKβ phosphorylation.