Regulation of the macrophage cellular response by Clinacanthus nutans extracts in J774.2 macrophages

Abstract

Clinacanthus nutans is a medicinal plant from the Acanthaceae family, and it is well-known as 'Belalai gajah' and Sabah Snake Grass in Malaysia. C. nutans extracts have been reported to exhibit various bioactivities but reports about its immunomodulatory role are limited. Macrophage activation is lacking in certain macrophage-related immunodeficiency disorders, but it occurs constitutively in certain inflammatory diseases. Thus, the authors investigated the potential role of C. nutans extracts in regulating the macrophage cellular response in J774.2 mouse macrophages. J774.2 macrophages were treated with different concentrations (0–1000 µg/mL) of C. nutans ethanol, ethanol-aqueous, and aqueous extracts for 24, 48, and 72 h and the cytotoxicity effect was assayed using PrestoBlue assay. Then, the macrophages were incubated with 32 µg/mL of ethanol, 500 µg/mL of ethanol-aqueous, and 32 µg/mL of aqueous extracts of C. nutans for 48 h. Cytokine secretion, phagocytic activity, and expression of M1 and M2 activation markers were assessed using a multiplexed cytokine bead-based assay, fluorescence microscopy, and flow cytometry. All extracts did not show any significant cytotoxic effect towards J774.2 macrophages within the extract concentration range tested (cell viability >50%). The ethanol-aqueous extract showed the greatest potential for downregulating lipopolysaccharide-induced pro-inflammatory cytokine (IL-1β, IL-6, and IL-12p40) production and CD86 (M1 activation marker) expression level and for stimulating the phagocytic function of J774.2 macrophages.