Chrysosplenetin acts as a homeostasis stabilizer with dual-function in shattering Plasmodium berghei K173 resistance to artemisinin driven by both ABC transporters and heme-ROS/GSH axis.

BACKGROUND: Chrysosplenetin (CHR), a polymethoxy flavonol co-occurring with artemisinin (ART) in Artemisia annua L., reverses ART resistance in Plasmodium berghei K173 potentially by downregulating intestinal P-glycoprotein (P-gp, encoded by Mdr1a) expression. In the present study, we further elaborated on the mechanism by comparing differences in antimalarial activity and resistance-associated molecular expression profiles between ART alone and combination therapy in blood and tissues of Mdr1a wild-type (WT) and knockout (KO) mice infected with either sensitive or resistant malarial parasites. METHODS: We evaluated the effects of monotherapy and combination therapy in WT and KO mice infected with sensitive and resistant P. berghei K173 strains. The mRNA expressions of multi-resistance proteins (Mrp1, 2, 4, 5) and breast cancer resistance proteins (Bcrp) were detected. Hemoglobin levels, mRNA expressions of cytokines including tumor necrosis factor-α (IFN-α), interferon-α (IFN-α), and interleukin (IL-1β) in blood and tissues, and redox balance (ROS/GSH levels), as well as gene or protein expression of signaling pathway (PI3K/AKT-mTOR and MAPK) were investigated. RESULTS: In drug-resistant mice, combination therapy maintained the highest survival (100%) and inhibition (30%) rates and the lowest parasitaemia percentage (approximately 20.0%), irrespective of Mdr1a gene status. Furthermore, combination reshaped the spatial and ART resistance-phenotypic disparities in Mrps and Bcrp mRNA expressions (with a fold change ranging from 1.35 to 38.03), ROS/GSH balance (ranging from 1.02-fold to 10.18-fold), hemoglobin levels (ranging from 1.04-fold to 1.20-fold), and cytokine profiles (ranging from 1.14-fold to 37.79-fold) induced by ART alone, which were partially dysregulated by Mdr1a deficiency. Monotherapy and combination exert oppositely regulatory effects on the PI3K/AKT-mTOR pathway in a tissue-, Mdr1a genotype-, and parasite sensitivity/resistance-dependent manner (ranging from 1.52-fold to 84.00-fold). Specifically, CHR reversed ART-induced changes via PI3K/AKT protein inhibition (ranging from 1.20-fold to 63.00-fold), which was contingent on P-gp functionality. Finally, mitogen-activated protein kinase (MAPK) pathway was involved in the antagonistic regulation between ART alone and combination therapy in a P-gp-independent manner (ranging from 1.39-fold to 16.69-fold). CONCLUSIONS: The efflux pump function of P-gp is probably not a critical factor in the mechanism by which CHR reverses ART resistance. Instead, CHR acts as a homeostasis stabilizer with dual functions: it disrupts Plasmodium berghei K173 resistance to ART driven by both ABC transporters and the heme-ROS/GSH axis, in which the non-transport function of P-gp on ART is involved.