Fig. 5: Nimbolide interacts with TNF-α and inhibits TNF-α regulated inflammatory signaling.

a Docking model of nimbolide in the active site of TNF-α (PDB ID: 2AZ5) and (b) its ligand-protein interactions in the binding site of TNF-α. The dark pink dashed lines represent hydrogen bonds. H-bond distances (in Å) between heteroatoms of ligand and amino acid residues are as follows: Ser60 (3.4Å) and Leu120 (3.2Å). The red line indicates arene-arene interaction with Tyr59. A549 cells were pre-treated with nimbolide for 24 h and stimulated by LPS (1 µg/ml) for 12 h. Animals were pre-treated with nimbolide (0.3, 1 and 3 mg/kg) for 5 days later LPS (50 μg) was administered. c TNF-α protein expression was analyzed by confocal microscope. d A 5-µm-sized sections of lung tissues were subjected to IHC to determine TNF-α expression and images were captured at ×400 magnification. Protein expressions of TNF-α, p-p38 MAPK, p-IKK-α/β, p-IκB-α, p-NF-κB, p-GSK-3β, and mTOR were determined by western blotting in (e) A549 cells and (f) lung tissues, respectively. The HDACs such as HDAC-1, 2, 3, and 4 protein expressions were studied in (g) A549 cells and (h) lung tissues. i Nimbolide (0.05, 0.1, 1, and 2.5 µM) upon HDAC levels were analyzed by HDAC fluorometric kit and HDAC inhibitory activity was compared with trichostatin A (2.5 µM). Data represented as mean ± SEM (n=3 independent experiments). *P < 0.05 and **P < 0.01 are significantly different from the trichostatin A group