MiR-146/TNF-α/IL-6/osteocalcin crosstalk in anti-rheumatic potential of Galleria mellonella hemolymph from computational molecular modeling to in-vivo validation.

Rheumatoid arthritis (RA) is an inflammatory autoimmune illness that persistently and recurrently affects joints. In RA, miR-146a functions as a key regulator, modulating inflammation by targeting and downregulating cytokines that promote inflammation, such as TNF-α and IL-6, whereas osteocalcin, a bone metabolism marker, plays a role in bone remodeling and joint health. The interplay between these molecules significantly influences RA progression and severity by balancing inflammation and bone integrity. Conventional antirheumatic drugs often cause varying levels of side effects. As sustainable development initiatives grow, insects are gaining interest as sustainable food sources and potential medicinal agents. Notably, the increasing Galleria mellonella (G. mellonella) population has raised concerns about the spread of honeybee viruses, affects bee products and food security, and drives economic losses in the therapeutic market. Accordingly, hemolymph has crucial defensive and immunological effects in insects and has recently been investigated as an immunomodulatory agent in parasitic in-vitro and in-vivo rat models. This work was designed to elucidate the potential immunomodulatory impact of G. mellonella hemolymph on the crosstalk between miR-146a, IL-6, TNF-α, and osteocalcin in the context of RA, utilizing both computational molecular modeling and in-vivo validation. Computer-aided molecular simulation for immune and RA mediators is applied through specific cell annotation, targeted pathways, and in-silico protein‒protein and gene‒gene interactions with a gene relative-tissue expression heatmap, which is based on gas chromatographic‒mass spectrometric analysis of hemolymph. Our study is the first to adapt a preliminary test to optimize hemolymph dosing and toxicity. The rats were subsequently divided into four groups: healthy control, Freund's adjuvant-induced arthritis (utilized as a model that mimics human RA), methotrexate-treated arthritis, and hemolymph-treated arthritis groups. Our findings indicate that hemolymph contains valuable active compounds that have anti-inflammatory and antioxidant potential, increasing the impact of recovery on diseased joints in comparison with the arthritic and methotrexate groups. This is the first report investigating the maximum inhibition rate of G. mellonella hemolymph as an immunomodulatory and anti-inflammatory agent in an arthritic model.