Bojungikki-tang enhances M1-like macrophage activation and promotes antitumor responses through IFN-γ co-stimulation

Bojungikki-tang (BJIKT), a traditional East-Asian multi-herbal prescription known for enhancing immune function, treating chronic fatigue, and improving general vitality, is often prescribed for conditions related to immune deficiency or cancer-related fatigue. However, its cellular mechanisms remain poorly defined.

To determine whether BJIKT promotes M1-like macrophage activation and exerts antitumor effects, particularly with interferon-gamma (IFN-γ), and elucidate the underlying signaling mechanisms.

THP-1 monocytes were differentiated into M0 macrophages using PMA and treated with BJIKT alone or with IFN-γ. Expression of M1/M2 markers and pro-inflammatory cytokines was assessed using RT-qPCR and ELISA. TLR4 signaling involvement was evaluated using the selective inhibitor TAK-242. Conditioned media (CM) from treated macrophages were applied to A549 lung cancer cells, followed by MTT, LDH, and Annexin V/7-AAD assays to assess antitumor activity. Individual herbs and BJIKT phytochemicals were also tested for cytokine-inducing potential.

BJIKT alone increased the secretion of pro-inflammatory cytokines (IL-6 and TNF-α) but did not induce full M1 polarization-co-treatment with IFN-γ markedly upregulated M1 genes, including CD80 and TNF-α. TAK-242 inhibited IFN-γ/LPS-induced cytokine and suppressed IFN-γ/BJIKT-induced effects, suggesting partial TLR4 dependence. CM from BJIKT/IFN-γ-treated macrophages reduced A549 cell viability, increased cytotoxicity, and induced apoptosis. Notably, Ginseng Radix and Glycyrrhizae Radix et Rhizoma partially mimicked BJIKT, while major phytochemicals showed no comparable activity.

BJIKT synergistically promotes M1-like macrophage activation through partial TLR4 dependence and elicits antitumor effects via macrophage-derived cytokines. These findings provide insight into how a traditional multi-herbal formula can influence macrophage activation and cytokine-mediated antitumor responses.