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

doi:10.1016/j.joim.2020.06.001

Abstract

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 (CC50) and 50% effective concentration (EC50). Cell survival was evaluated in lipopolysaccharide-stimulated C. carbonica-treated cells. Nitric oxide (NO) and tumor necrosis factor-α (TNF-α) 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 CC50 and EC50 of C. carbonica were found to be 43.26 and 11.99 μg/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 TNF-α levels by 1.395 folds (P = 0.0013), 1.541 folds (P = 0.0005) and 1.550 folds (P = 0.0005) at 24, 48 and 72 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 simulated molecular docking showed interaction of C. carbonica with the phenylalanine 367 residue present in COX-2’s active site.

Conclusion

C. carbonica exhibited anti-inflammatory properties in lipopolysaccharide-stimulated cells by significantly reducing NO production and TNF-α 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 COX-2’s active site.

Key words: Inflammation, Calcarea carbonica, Cyclooxygenase-2, Nitric oxide, Tumor necrosis factor-α, Anti-inflammatory

Swatantra Kumar, Vimal K Maurya, Debadatta Nayak, Anil Khurana, Raj K Manchanda, Srinivasulu Gadugu, Madan LB Bhatt, Shailendra K Saxena. Calcarea carbonica treatment rescues lipopolysaccharide-induced inflammatory response in human mononuclear cells via downregulation of inducible cyclooxygenase pathway. Journal of Integrative Medicine, 2020, 18(5): 441-449.

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URL: http://www.jcimjournal.com/EN/10.1016/j.joim.2020.06.001

http://www.jcimjournal.com/EN/Y2020/V18/I5/441

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