Preparation of Pueraria lobata Root-Derived Exosome-Like Nanovesicles and Evaluation of Their Effects on Mitigating Alcoholic Intoxication and Promoting Alcohol Metabolism in Mice

Abstract

Purpose: Pueraria lobata (P. lobata), a dual-purpose food and medicine, displays limited efficacy in alcohol detoxification and liver protection, with previous research primarily focused on puerarin in its dried roots. In this study, we investigated the potential effects and mechanisms of fresh P. lobata root-derived exosome-like nanovesicles (P-ELNs) for mitigating alcoholic intoxication, promoting alcohol metabolism effects and protecting the liver in C57BL/6J mice.

Methods: We isolated P-ELNs from fresh P. lobata root using differential centrifugation and characterized them via transmission electron microscopy, nanoscale particle sizing, ζ potential analysis, and biochemical assays. In Acute Alcoholism (AAI) mice pre-treated with P-ELNs, we evaluated their effects on the timing and duration of the loss of the righting reflex (LORR), liver alcohol metabolism enzymes activity, liver and serum alcohol content, and ferroptosis-related markers.

Results: P-ELNs, enriched in proteins, lipids, and small RNAs, exhibited an ideal size (150.7 ± 82.8 nm) and negative surface charge (-31 mV). Pre-treatment with 10 mg/(kg.bw) P-ELNs in both male and female mice significantly prolonged ebriety time, shortened sobriety time, enhanced acetaldehyde dehydrogenase (ALDH) activity while concurrently inhibited alcohol dehydrogenase (ADH) activity, and reduced alcohol content in the liver and serum. Notably, P-ELNs demonstrated more efficacy compared to P-ELNs supernatant fluid (abundant puerarin content), suggesting alternative active components beyond puerarin. Additionally, P-ELNs prevented ferroptosis by inhibiting the reduction of glutathione peroxidase 4 (GPX4) and reduced glutathione (GSH), and suppressing acyl-CoA synthetase long-chain family member 4 (ACSL4) elevation, thereby mitigating pathological liver lipid accumulation.

Conclusion: P-ELNs exhibit distinct exosomal characteristics and effectively alleviate alcoholic intoxication, improve alcohol metabolism, suppress ferroptosis, and protect the liver from alcoholic injury. Consequently, P-ELNs hold promise as a therapeutic agent for detoxification, sobriety promotion, and prevention of alcoholic liver injury.

Keywords: Pueraria lobata root-derived exosome-like nanovesicles; acute alcoholism; ethanol metabolism; ferroptosis; loss of the righting reflex.

Figure 1

Characterization of P-ELNs, P-ELNs1, and P-ELNs2, Extract photos ((A), left), TEM images ((A), right), ζ potential (B), and NTA (C). TEM images include scale bars of 1 μm and 200 nm.

Figure 2

Analysis of the principal components of P-ELNs via protein BCA assay (A), Coomassie staining (B), RNA agarose gel (C), and TLC (D).

Figure 3

Evaluation of the optimal pre-oral dose of P-ELNs (0 [PBS], 2, 10, and 50 mg/(kg.bw)) in ethanol-exposed mice. Post-alcohol gavage, samples were collected at 60, 120, and 240 minutes for serum (A) and hepatic alcohol content (B), as well as hepatic ALDH (C) and ADH enzyme activity (D) analysis (n=5, *p<0.05, **p<0.01, ***p<0.001 vs PBS + EtOH).

Figure 4

Percentage of intoxicated mice following varying alcohol doses (3.6, 5.0, and 6.4 g/(kg.bw)) over different time intervals (<5, 5–10, and >10 min) in male and female mice (male in (A), female in (B), n=15); A schematic diagram of the P-ELNs oral administration in the AAI mice model (C) and the LORR experiment of the Ebriety and Sober-up time in AAI mice after oral administration of P-ELNs (male in (D), female in (E), n=6, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Figure 5

Biochemical markers of alcohol metabolism, including serum alcohol content and hepatic ALDH and ADH enzyme activity (male in (A), female in (B) in AAI mice by oral administration of P-ELNs (n=6, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Figure 6

Hepatic alcohol metabolism and ferroptosis protein biomarkers (male in (A), n=4), liver GSH content (male in (B), n=6), hepatic alcohol metabolism and ferroptosis protein biomarkers (female in (C), n=4), liver GSH content (female in (D), n=6) and changes in Oil Red O staining (E) in AAI mice by oral administration of P-ELNs (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).