Herbacetin as a natural GPR35 agonist for allergic asthma relief via dual regulation of PI3K/Akt/mTOR and MAPK signaling.

Abstract

Allergic asthma is a chronic inflammatory airway disease with complex pathogenesis and limited therapeutic options. G protein-coupled receptor 35 (GPR35) is increasingly recognized as an important regulator of immune and inflammatory responses and represents a potential therapeutic target for asthma. Herbacetin (HBN), a natural flavonoid with anti-inflammatory activity, has shown potential anti-asthmatic effects; however, its direct molecular targets and underlying mechanisms remain unclear. This study investigated whether the anti-allergic effects of HBN are mediated through GPR35 activation and explored the associated downstream signaling pathways. Agonist activity was evaluated using dynamic mass redistribution assays in CHO-K1-hGPR35 and CHO-K1-mGPR35 cells. Molecular docking and molecular dynamics simulations were employed to analyze HBN-GPR35 binding. GPR35 internalization was assessed in HT-29 cells by immunofluorescence. Changes in PI3K/Akt/mTOR and MAPK pathway phosphorylation were examined by Western blotting, with the GPR35 antagonist ML145 used for validation. The results showed that HBN acted as a potent GPR35 agonist, with EC₅₀ values of 7.45 μM for hGPR35 and 1.39 μM for mGPR35, and induced receptor internalization, which was blocked by ML145. Molecular docking revealed a strong binding affinity between HBN and GPR35 (-8.249 kcal/mol), and molecular dynamics simulations confirmed the stability of the complex. Mechanistically, HBN inhibited PI3K/Akt/mTOR phosphorylation while promoting activation of MAPK signaling pathways, including ERK, p38, and JNK; these effects were reversed by ML145. In conclusion, herbacetin functions as a GPR35 agonist and exerts anti-allergic effects in asthma through GPR35-dependent modulation of PI3K/Akt/mTOR and MAPK signaling pathways, supporting GPR35 as a promising therapeutic target for allergic asthma.