Molecular Targets and Mechanisms of 6,7-Dihydroxy-2,4-dimethoxyphenanthrene from Chinese Yam Modulating NF-κB/COX-2 Signaling Pathway: The Application of Molecular Docking and Gene Silencing

Chinese yam (Dioscorea opposita) tuber has a significant effect of invigorating the intestine and improving the symptoms of long-term diarrhea according to the records of the Chinese Pharmacopoeia. Phenanthrene polyphenols from Chinese yam, with higher inhibition of cyclooxygenase-2 (COX-2) than anti-inflammatory drugs, are an important material basis in alleviating ulcerative colitis via nuclear factor kappa-B (NF-κB)/COX-2 pathway, based on our previous research. The present study further explored the target and molecular mechanisms of phenanthrenes’ modulation of the NF-κB/COX-2 signaling pathway by means of molecular docking and gene silencing. Firstly, interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α) expression of 6-hydroxy-2,4,7-trimethoxyphenanthrene (PC2)/6,7-dihydroxy-2,4-dimethoxyphe-nanthrene (PC4) were compared on TNF-α induced human colon adenocarcinoma (Caco-2) cells. Secondly, molecular docking and dynamics simulation were implemented for PC2/PC4 and COX-2. Finally, COX-2 silencing was performed on TNF-α induced Caco-2 cells to confirm the target of PC4 on NF-κB/COX-2 pathway. Lower expression of IL-8 and TNF-α in PC4 treated Caco-2 cells indicated that PC4 had stronger anti-inflammatory activity than PC2. The binding of PC4 and COX-2 was stronger due to the hydrogen bond between hydroxyl group and Tyr385. No significant differences were found in phosphorylation nuclear factor kappa-B inhibitor alpha (pIkBα), phosphorylation NF-κB (pNF-κB) and phosphorylation extracellular signal-regulated kinase 1/2 (pERK1/2) expression between control and PC4 group after silencing, while these protein expressions significantly decreased in PC4 group without silencing, which confirmed that COX-2 was the important target for PC4 in alleviating ulcerative colitis. These findings indicate that PC4 was supposed to have inhibited NF-κB pathway mediated inflammation via suppression of positive feedback targeting COX-2.