Modulation of autophagy by natural bioactive compounds in macrophages during mycobacterium infection

Abstract

Tuberculosis (TB) remains a global health challenge, exacerbated by the emergence of drug-resistant strains and the limitations of current therapeutic strategies. Host-directed therapy (HDT), particularly through the modulation of autophagy and inflammatory responses, has emerged as a promising alternative. This study aimed to screen bioactive compounds derived from Antrodia cinnamomea-specifically Antcin B, Antcin H, Versisponic acid D, Dehydroeburicoic acid (DEA), and Zhankuic acid B (ZAB)-for their potential to induce autophagy and modulate inflammation in the context of mycobacterial infection. Using Mycobacterium smegmatis as a model organism and RAW 264.7 munne macrophages as host cells, the antibacterial and immunomodulatory effects of the selected compounds were evaluated. Bacterial clearance was assessed through both extracellular and intracellular colony-forming unit (CFU) assays. Western blotting was performed to detect the expression of autophagy markers ATG5, Beclin- 1, LC3B, and p62. In parallel, qRT-PCR analysis was conducted to quantify transcript levels ofIL-l~, ATG5, LC3B, NLRP3, and iNOS. Among the tested molecules, Antcin H showed the most potent dual action, significantly reducing bacterial load in both extracellular and intracellular environments while upregulating autophagy markers (ATG5, Beclin-l, LC3B) and inflammatory genes (IL-l~, NLRP3), and downregulating p62 and iNOS. Antcin B also promoted bacterial clearance and selectively upregulated A TG5 while suppressing Beclin-1, LC3B, and p62 at the protein level, suggesting a Beclin-independent autophagy pathway.