The Alkaloid Caulerpin Exhibits Potent and Selective Anti-Inflammatory Activity Through Interaction with the Glucocorticoid Receptor

Abstract

Inflammation plays a central role in various pathological conditions, necessitating the search for safer and more effective anti-inflammatory agents. This study investigates the anti-inflammatory activity of caulerpin, a bisindolic alkaloid isolated from Caulerpa racemosa. In vitro assays demonstrated that caulerpin significantly reduced nitric oxide, TNF-α, IL-6, and IL-12 levels in macrophages stimulated with LPS + IFN-γ, without affecting cell viability. In silico toxicity predictions using Protox 3.0 reinforce a favorable safety profile of caulerpin. Molecular docking and molecular dynamics simulations revealed its high-affinity binding to the glucocorticoid receptor ligand-binding domain (GR-LBD), suggesting a mechanism of action similar to dexamethasone. The involvement of the glucocorticoid receptor was confirmed by the partial reversal of caulerpin's effects upon RU486 treatment. In vivo, caulerpin exhibited a favorable safety profile, with no signs of acute toxicity at an oral dose of 100 mg/kg. Moreover, in a mouse model of endotoxic shock, caulerpin administration significantly improved survival rates in a dose-dependent manner, providing complete protection at 4 mg/kg. These findings highlight caulerpin as a promising candidate for the development of novel anti-inflammatory therapies. Further studies are warranted to explore its pharmacokinetics, optimize its structure, and evaluate its efficacy in chronic inflammatory diseases.

Keywords: anti-inflammatory activity; caulerpin; glucocorticoid receptor; macrophages.

Figure 1

Chemical structure of caulerpin.

Figure 2

Prediction of caulerpin interaction with cytochrome P450 enzymes. The chart shows the enzymes predicted as active (red) or inactive (blue) in the metabolism of the compound. Values above 0.7 indicate that caulerpin may activate or inhibit certain CYP450 enzymes. A probability threshold of 0.7 was set for significance.

Figure 3

Effects of caulerpin on macrophages in vitro. Mouse peritoneal exudate macrophages stimulated or not with LPS + IFN-γ were cultured in the absence or presence of caulerpin (10, 20, or 40 µM) or dexamethasone (Dexa; 10 µM). (A) Cell viability was determined by the Alamar Blue method. Cell-free supernatants were collected for nitrite (B), TNF-α (C), IL-6 (D), IL-10 (E), and IL-12 (F) quantification. “−” refers to the group of untreated and unstimulated cells. C– refers to the group of untreated cells stimulated with LPS + IFN-γ. Data are expressed as the mean ± standard deviation (S.D.) of nine replicates obtained from three independent experiments. * p < 0.05 compared to stimulated and untreated cells; # p < 0.05 compared to unstimulated and untreated cells; $ p < 0.05 compared to dexamethasone-treated cells.

Figure 4

Molecular dynamics simulation analysis. (A) Radius of gyration (Rg); (B) root mean square deviation (RMSD). Molecular dynamics data for the free GR-LBD are represented by gray lines, the dexamethasone–GR-LBD complex by brown lines, and the caulerpin–GR-LBD complex by black lines.

Figure 5

Molecular dynamics simulation analysis. (A) Root mean square fluctuation (RMSF); (B) hydrogen bond (H-bond) frequency. Molecular dynamics data for the free GR-LBD are represented by gray lines, the dexamethasone–GR-LBD complex by brown lines, and the caulerpin–GR-LBD complex by black lines.

Figure 6

Cluster analysis of GR-LBD complexes with dexamethasone and caulerpin. (A) RMSD distribution for the dexamethasone–GR-LBD complex; (B) RMSD distribution for the caulerpin–GR-LBD complex.

Figure 7

Representative structure of the dexamethasone–GR-LBD complex (cluster 1). (A) 3D representation; (B) 2D representation of dexamethasone–GR-LBD interactions.

Figure 8

Representative structures of the caulerpin–GR-LBD complex (clusters 1 and 2). (A) 3D representation and (B) 2D representation of caulerpin–GR-LBD interactions for the representative structure from cluster 1; (C) 3D representation and (D) 2D representation of caulerpin–GR-LBD interactions for the representative structure from cluster 2.

Figure 9

Involvement of glucocorticoid receptor in the immunomodulatory effect of caulerpin. (A) Nitrite concentrations, (B) IL-6 levels, and (C) TNF-α levels were measured in macrophages stimulated with LPS + IFN-γ. Cells were treated with caulerpin (40 µM) or dexamethasone (Dexa; 10 µM). In some cultures, cells were treated with these compounds in the presence of RU486 (glucocorticoid receptor antagonist, RU; 10 µM). “−” refers to the group of untreated and unstimulated cells. C– refers to the group of untreated cells stimulated with LPS + IFN-γ. Veh = Vehicle. Data are expressed as the mean ± S.D. of nine replicates obtained from three independent experiments. * p < 0.05 compared to stimulated and untreated cells; # p < 0.05 compared to stimulated and treated with dexamethasone-treated cells; $ p < 0.05 compared to caulerpin-treated cells.

Figure 10

Survival curve of mice treated with caulerpin and subjected to endotoxic shock. Mice were orally treated with caulerpin (2.5, 5, or 10 mg/kg) or dexamethasone (2.5 mg/kg). Animals in the vehicle-treated group received saline solution containing 5% DMSO. Survival was monitored for four days following LPS challenge. Data represent results from two independent experiments. * p < 0.05, ** p < 0.01 compared to the vehicle-treated group. Statistical analysis was performed using the log-rank (Mantel–Cox) test.