Buyang Huanwu Decoction stabilizes atherosclerotic vulnerable plaques by regulating intestinal flora, TLR4-NF-κB-NLRP3 inflammatory pathway and mitophagy

Abstract

Objective: This study explores the anti-atherosclerosis (AS) effects of Buyang Huanwu Decoction (BYHWD), focusing on its regulatory effects on the TLR4/NF-κB/NLRP3 inflammatory pathway, gut microbiota metabolites, and mitochondrial autophagy. Through the triple regulatory mechanisms of gut microbiota, the TLR4/NF-κB/NLRP3 inflammatory pathway, and mitochondrial autophagy, this study explores a novel strategy for stabilizing vulnerable AS plaques.

Materials and methods: The active components of Buyang Huanwu Decoction (BYHWD) were detected using LC-MS/MS. By feeding a high-fat diet (HFD) and adding 1.3 % choline chloride to the drinking water to induce ApoE^-/- mice gut microbiota dysbiosis, an AS mouse model with vulnerable plaques was established. The treatment groups were administered low, medium, and high doses of BYHWD, as well as broad-spectrum antibiotics. The effects of BYHWD on the vulnerable plaque area in the aorta, collagen content, macrophage and α-SMA protein expression, levels of inflammatory cytokines, reactive oxygen species (ROS), LC3 and NLRP3 expression, gut microbiota composition and abundance, serum trimethylamine-N-oxide (TMAO) levels, and the total bile acid content in the liver, serum, and gallbladder, as well as mitochondrial autophagy, were evaluated applying hematoxylin-eosin (HE) staining, Oil Red O staining, Sirius Red staining, immunohistochemistry, ELISA, immunofluorescence, 16S rRNA sequencing, biochemical analysis, and LC-MS detection. Western blot for TLR4, MyD88, ASC, pro-caspase-1, caspase-1, NLRP3, p-NF-κB/NF-κB, GPR41, GPR43, CYP7A1, CYP27A1, FMO3, FXR, TGR5, NIX, BNIP3, FUNDC1, PINK1, and Parkin proteins expression level.

Results: A total of 31 major active components were identified in Buyang Huanwu Decoction (BYHWD). BYHWD significantly reduced the vulnerable plaque area in the ApoE^-/- mouse model of AS, decreased the expression of inflammatory cytokines, inhibited the protein expression of TLR4, MyD88, p-NF-κB/NF-κB, ASC, pro-caspase-1, NLRP3, FMO3, NIX, BNIP3, FUNDC1, and PINK1/Parkin in aortic tissues, downregulated ROS levels and mitochondrial autophagy activity, regulated gut microbiota abundance, reduced serum TMAO levels, and up-regulated the expression of gut microbiota-related proteins, including GPR41, GPR43, CYP7A1, CYP27A1, FXR, and TGR5.

Conclusion: BYHWD exerts anti-AS effects through the inhibition of the TLR4/NF-κB/NLRP3 inflammatory pathway, modulating the gut microbiota, and stabilizing mitochondrial autophagy. The in-depth investigation of this mechanism effectively expands the therapeutic potential of BYHWD in the prevention and treatment of cardiovascular diseases and provides new theoretical insights and therapeutic targets for AS-related research.

Keywords: Atherosclerosis; Buyang Huanwu Decoction; Gut microbiota; Mitophagy; TLR4-NF-κB-NLRP3 inflammatory pathway.