Molecular Docking Identifies 1,8-Cineole (Eucalyptol) as A Novel PPARγ Agonist That Alleviates Colon Inflammation

Abstract

Inflammatory bowel disease, comprising Crohn's disease (CD) and ulcerative colitis (UC), is often debilitating. The disease etiology is multifactorial, involving genetic susceptibility, microbial dysregulation, abnormal immune activation, and environmental factors. Currently, available drug therapies are associated with adverse effects when used long-term. Therefore, the search for new drug candidates to treat IBD is imperative. The peroxisome proliferator-activated receptor-γ (PPARγ) is highly expressed in the colon. PPARγ plays a vital role in regulating colonic inflammation. 1,8-cineole, also known as eucalyptol, is a monoterpene oxide present in various aromatic plants which possess potent anti-inflammatory activity. Molecular docking and dynamics studies revealed that 1,8-cineole binds to PPARγ and if it were an agonist, that would explain the anti-inflammatory effects of 1,8-cineole. Therefore, we investigated the role of 1,8-cineole in colonic inflammation, using both in vivo and in vitro experimental approaches. Dextran sodium sulfate (DSS)-induced colitis was used as the in vivo model, and tumor necrosis factor-α (TNFα)-stimulated HT-29 cells as the in vitro model. 1,8-cineole treatment significantly decreased the inflammatory response in DSS-induced colitis mice. 1,8-cineole treatment also increased nuclear factor erythroid 2-related factor 2 (Nrf2) translocation into the nucleus to induce potent antioxidant effects. 1,8-cineole also increased colonic PPARγ protein expression. Similarly, 1,8-cineole decreased proinflammatory chemokine production and increased PPARγ protein expression in TNFα-stimulated HT-29 cells. 1,8-cineole also increased PPARγ promoter activity time-dependently. Because of its potent anti-inflammatory effects, 1,8-cineole may be valuable in treating IBD.

Keywords: 1,8-cineole (eucalyptol); DSS-colitis; IBD; PPARγ.

Figure 1

Molecular docking and dynamics of 1,8-cineole with PPARγ. The docked pose and the interactions at the binding site of PPARγ. (a) Interactions of amorfrutin B and (b) interactions of 1,8-cineole. The analysis of the MD simulation (c) RMSD in PPARγ backbone atoms, (d) RMSD in 1,8-cineole atoms and amorfrutin B atoms, (e) RMSF in PPARγ residues, (f) hydrogen bond analysis for 1,8-cineole and amorfrutin B, (g) The analysis of trajectories extracted at different time intervals (50–150 ns) of the MD simulations.

Figure 2

Effect of 1,8-cineole on the disease activity index (DAI), colonic length, and myeloperoxidase (MPO) activity. DSS administration significantly (a) increased the DAI, (b,c) decreased colonic length, and (d) increased MPO activity. 1,8-cineole treatment significantly prevented (a) the increase in DAI, (b,c) the decrease in colonic length, and (d) the increase in MPO enzyme activity. Data were obtained from n = 8 animals in each group. The results are expressed as the means ± SEM. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001.

Figure 3

Effect of 1,8-cineole on colon histology. (a,b) DSS administration significantly increased the colonic inflammation score and crypt aberration. 1,8-cineole treatment significantly prevented the increase in the colonic inflammation score and protected against crypt aberration. Data were obtained from n = 6 animals per group. The results are expressed as the means ± SEM. **** p ≤ 0.0001.

Figure 4

Effect of 1,8-cineole on proinflammatory cytokine protein and mRNA expression. (a–d) DSS administration significantly increased colonic proinflammatory cytokine levels. 1,8-cineole treatment significantly prevented the increase in proinflammatory cytokines. (e–h) Similarly, DSS administration significantly increased the expression of proinflammatory cytokine mRNA levels. 1,8-cineole treatment significantly prevented this increase in expression of proinflammatory cytokine mRNA. Data were obtained from n = 8 animals per group. The results are expressed as the means ± SEM. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, and ns indicates results that were not significant.

Figure 5

Effect of 1,8-cineole on COX2 and iNOS protein and mRNA expression as well as tissue nitrite levels. (a,c) The upper panel shows COX2 and iNOS protein expression by Western blot. A representative of protein bands that were normalized to GAPDH protein as an internal control is shown. Densitometry analysis shows that DSS administration significantly increased (a) COX2, (c) iNOS protein, and (b,d) mRNA expression. In contrast, 1,8-cineole treatment significantly prevented the increases in protein and mRNA expression. (a–d) DSS treatment increased tissue nitrite levels, while (e) 1,8-cineole treatment prevented this. Data were obtained from n = 4 animals/group. The results are expressed as the means ± SEM. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, and ns indicates results that were not significant.

Figure 6

Effect of 1,8-cineole on Keap1, Nrf2, HO1, NQO1, superoxide dismutase (SOD), and catalase (CAT) activity. The upper panel shows (a) Keap1 and (b,c) cytosolic Nrf2 and nuclear Nrf2 protein expression. Representative protein bands were normalized to GAPDH and lamin proteins as the internal controls as shown. Densitometric analysis showed that DSS administration increased Keap1 protein expression while 1,8-cineole did not further increase Keap1 protein expression. Nuclear Nrf2 protein expression was markedly increased by 1,8-cineole treatment compared to the DSS-administered group. (c) DSS administration significantly decreased (d) NQO1, (f) HO1 protein, and (e,g) mRNA expression, while 1,8-cineole treatment prevented these decreases. DSS administration significantly decreased (h) colon tissue SOD and (i) CAT enzyme activity, while 1,8-cineole treatment restored their activities toward the control levels. Data were obtained from n = 4 for protein, n = 6 for mRNA, and n = 8 animals for SOD and CAT enzyme activity; the results are expressed as the means ± SEM. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, and ns indicates results that were not significant.

Figure 7

Effect of 1,8-cineole on PPARγ, PPARα, PPARδ, and pNFκB/NFκB protein expression. (a) The side panel shows PPARγ, PPARα, PPARδ, and pNFκB/NFκB protein expression. Protein bands were normalized to the GAPDH protein internal control as shown. DSS administration significantly (b) decreased PPARγ expression and 1,8 cineole at the higher concentration (b) increased it above the control. DSS administration and 1,8-cineole treatment (c,d) did not alter PPARα and PPARδ expression. DSS administration markedly increased the phosphorylation of NFκB, while 1,8-cineole treatment at the higher concentration prevented this. Data were obtained from n = 4 animals. The results are expressed as the means ± SEM. ** p ≤ 0.01, and ns indicates results that were not significant.

Figure 8

Effect of 1,8-cineole on HT-29 cell viability, proinflammatory chemokine mRNA expression, PPARγ expression, and PPARγ promoter assay. (a) 1,8-cineole treatment did not affect HT-29 cell viability up to a 200 μm concentration. (b,c) 1,8-cineole significantly downregulated IL8 and CXCL1 mRNA expression in TNF-α-challenged HT-29 cells. (d,e) 1,8-cineole significantly increased PPARγ protein and mRNA expression. (f,g) PPARγ siRNA significantly downregulated PPARγ protein and mRNA expression. (h) PPARγ siRNA transfection downregulated IL8 mRNA expression, and 1,8-cineole treatment partially prevented this. (i) siRNA transfection did not affect CXCL1 mRNA expression in the presence of 1,8-cineole. (j,k) 1,8-cineole treatment significantly enhanced PPARγ promoter activation time-dependently (12 and 24 h). The PPARγ activator (GW1929) and PPARγ inhibitor (GW9662) worked as expected. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, ****p ≤ 0.0001, and ns indicate results that were not significant.