Calcarea carbonica treatment rescues lipopolysaccharide-induced inflammatory response in human mononuclear cells via downregulation of inducible cyclooxygenase pathway

Objective: Prolonged use of nonsteroidal anti-inflammatory drugs is associated with severe side effects and toxicity. Therefore, we studied the anti-inflammatory role of Calcarea carbonica which had minimal toxicity at the low doses.Methods: THP-1 human mononuclear cells were treated with C. carbonica to evaluate the 50% cytotoxicity concentration(CC 50 ) and 50% effective concentration(EC 50 ). Cell survival was evaluated in lipopolysaccharide-stimulated C. carbonica-treated cells. Nitric oxide（NO） and tumor necrosis factor-a（TNF-a） were measured to evaluate the anti-inflammatory activity of C. carbonica. Cyclooxygenase-2（COX-2） protein expression was determined by Western blotting analysis, and the interaction of C. carbonica with the COX-2 protein was evaluated using molecular docking simulation.Results: The CC 50 and EC 50 of C. carbonica were found to be 43.26 and 11.99 mg/mL, respectively. The cell survival assay showed a 1.192-fold（P = 0.0129）, 1.443-fold（P = 0.0009） and 1.605-fold（P = 0.0004）increase in cell survival at 24, 48 and 72 h after initiating C. carbonica treatment, respectively. C. carbonica-treated cells showed a reduction in NO levels by 2.355 folds（P = 0.0001）, 2.181 folds（P = 0.0001） and 2.071 folds（P = 0.0001） at 24, 48 and 72 h, respectively. The treated cells also showed a reduction in TNFa levels by 1.395 folds（P = 0.0013）, 1.541 folds（P = 0.0005） and 1.550 folds（P = 0.0005） at 24, 48 and72 h, respectively. In addition, a 1.193-fold reduction（P = 0.0126） in COX-2 protein expression was found in C. carbonica-treated cells. The molecular docking showed interaction of C. carbonica with the phenylalanine 367 residue present in active site of Cox-2.Conclusion: C. carbonica exhibited anti-inflammatory properties in lipopolysaccharide-stimulated cells by significantly reducing NO production and TNF-a level through downregulation of the COX-2 protein. This effect is probably mediated through interaction of C. carbonica with the phenylalanine 367 residue present in active site of Cox-2.