Neuroprotective effects of melatonin and celecoxib against ethanol-induced neurodegeneration: a computational and pharmacological approach

Abstract

Purpose: Melatonin and celecoxib are antioxidants and anti-inflammatory agents that exert protective effects in different experimental models. In this study, the neuroprotective effects of melatonin and celecoxib were demonstrated against ethanol-induced neuronal injury by in silico, morphological, and biochemical approaches.

Methods: For the in silico study, 3-D structures were constructed and docking analysis performed. For in vivo studies, rats were treated with ethanol, melatonin, and celecoxib. Brain samples were collected for biochemical and morphological analysis.

Results: Homology modeling was performed to build 3-D structures for IL1β), TNFα, TLR4, and inducible nitric oxide synthase. Structural refinement was achieved via molecular dynamic simulation and processed for docking and postdocking analysis. Further in vivo experiments showed that ethanol induced marked neuronal injury characterized by downregulated glutathione, glutathione S-transferase, and upregulated inducible nitric oxide synthase. Additionally, ethanol increased the expression of TNFα and IL1β. Finally, neuronal apoptosis was demonstrated in ethanol-intoxicated animals using caspase 3 and activated JNK staining. On the other hand, melatonin and celecoxib treatment ameliorated the biochemical and immunohistochemical alterations induced by ethanol.

Conclusion: These results demonstrated that ethanol induced neurodegeneration by activating inflammatory and apoptotic proteins in rat brain, while melatonin and celecoxib may protect rat brain by downregulating inflammatory and apoptotic markers.

Keywords: cortex; docking; ethanol; hippocampus; neurodegeneration; simulation.

Figure 1

Computational analysis of IL1 β, TNFα, TLR4, iNOS residues. Notes: (A) Tertiary structures of iNOS, TNFα, IL1β, and TLR4. (B) Homology-modeling and tertiary-structure validation. Ramachandran plots for iNOS, TNFα, IL1β, and TLR4. (C) ProSA findings of iNOS, TNFα, IL1β, and TLR4. (D) Tertiary structures of COX2, Nrf2, HO1. (E) Sequence alignment of COX2 (rat accession number P35355 [PDB 1PXX]) and HO1 (rat accession number P06762 [PDB 1DVE]) by Clustal Omega. (F) Melatonin- and celecoxib-ligand structures drawn in ChemSketch and converted to PDB format by pymol.hgcghdc. Abbreviation: iNOS, inducible nitric oxide synthase.

Figure 1

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Figure 2

Docking results show the best pose of melatonin that fitted to iNOS, IL1β, TNFα, COX2, Nrf2, TLR4, and HO1.P Notes: Postdocking analyses visualized by Discovery Studio Visualizer in both 2-D and 3-D poses. Interaction between melatonin and iNOS (A, B), IL1β (D, E), TNFα (G, H), COX2 (J, K), Nrf2 (M, N), TLR4 (P, Q), and HO1 (S, T). 3-D poses (A, D, G, J, M, P, S) and 2-D (B, E, H, K, N, Q, T). (C, F, I, L, O, R, U) represents the best pose of melatonin that fitted to iNOS, IL1β, TNFα, COX2, Nrf2, TLR4, and HO1, respectively. Abbreviation: iNOS, inducible nitric oxide synthase.

Figure 3

Docking results showed the best pose of celecoxib that fitted to iNOS, IL1-β, TNFα, COX2, Nrf2, TLR4, HO1. Post-docking analysis were visualized by Discovery Studio Visualizer in both 2-D and 3-D poses. Interaction between celecoxib and iNOS were shown by panel (A, ), IL1β by panel (D, E), TNFα by panel (G, H), COX2 by panel (J, K), Nrf2 by panel (M, N), TLR4 by panel (P, Q), and HO1 by panel (S, T). 3-D poses were shown by panel (A, D, G, J, M, P, S) and 2-D by panel (B, E, H, K, N, Q, T). Panel (C, F, I, L, O, R, U) represents the best pose of celecoxib that fits in iNOS, IL1β, TNFα, COX2, Nrf2, TLR4, HO1 respectively. Abbreviation: iNOS, inducible nitric oxide synthase.

Figure 4

Effect of melatonin and celecoxib on neuronal cell death. Notes: (A) Regions of interest analyzed; (B) H&E staining showing the extent of surviving neurons in the cortex and hippocampus. Bar 50 μm, magnification 20×, n=5/group. Surviving neurons were marked by cytoplasmic swelling, scalloped neurons with intense cytoplasmic eosinophilia, and nuclear basophilia. These changes resulted from neuronal necrosis. Some cells had a shrunken appearance, along with pyknotic nuclei. Intensive neuropil vacuolation can be seen in the ethanol-only group. *Significant difference relative to control; ^#significant difference relative to ethanol group; ^θsignificant difference relative to ethanol + melatonin. Data presented as means ± SEM. Data analyzed by one-way ANOVA followed by post hoc Bonferroni multiple comparison using GraphPad Prism 5 software. ***P<0.001; ^#/θP<0.05; ^##/θθP<0.01.

Figure 5

Effect of melatonin and celecoxib on apoptotic markers. Notes: (A) Immunohistochemistry results for pJNK. Bar 20 μm, magnification 40×. (B) Immunohistochemistry results for caspase 3 in frontal, partial, and pyriform cortices. Bar μm, magnification 20×, n=5/group. Both pJNK and caspase 3 exhibited cytoplasmic localization in the three performed. Histograms show comparatively higher expression of pJNK and caspase 3 in various segments of the ethanol-only group. *Significant difference relative to control; ^#significant difference relative to ethanol group; ^θsignificant difference relative to ethanol + melatonin. Data presented as means ± SEM. Data analyzed by one-way ANOVA followed by post hoc Bonferroni multiple comparison using GraphPad Prism 5 software. ***^/###P<0.001; ^#/θP<0.05; ^##/θθP<0.01.

Figure 6

Effect of melatonin and celecoxib on inflammatory cytokines. Notes: (A) Immunohistochemistry results for COX2; (B) immunohistochemistry results for TNFα in frontal, partial and pyriform cortices, Bar 20 μm, magnification 40×, n=5/group. Both COX2 and TNFα exhibited cytoplasmic localization in the three experiments performed. Histograms indicate comparative expression of COX2 and TNFα in various segments of the ethanol-only group. *Significant difference relative to control; ^#significant difference relative to the ethanol group; ^θsignificant difference relative to ethanol + melatonin group. Data presented as means ± SEM. Data analyzed by one-way ANOVA followed by post hoc Bonferroni multiple comparison using GraphPad Prism 5. **^/##/θθP<0.01; ^#/*P<0.05; ***^/###P<0.001. Arrows indicates neuronal cells, where COX2 and TNF expression were significantly modulated, further these arrowed cells are shown in high magnification in the same panel.