Hispidol Regulates Behavioral Responses to Ethanol through Modulation of BK Channels: A Novel Candidate for the Treatment of Alcohol Use Disorder

Abstract

Alcohol use disorder (AUD) is the most common substance use disorder and poses a significant global health challenge. Despite pharmacological advances, no single drug effectively treats all AUD patients. This study explores the protective potential of hispidol, a 6,4'-dihydroxyaurone, for AUD using the Caenorhabditis elegans model system. Our findings demonstrate that hispidol-fed worms exhibited more pronounced impairments in thrashes, locomotory speed, and bending amplitude, indicating that hispidol exacerbated the detrimental effects of acute ethanol exposure. However, hispidol significantly improved ethanol withdrawal behaviors, such as locomotory speed and chemotaxis performance. These beneficial effects were absent in slo-1 worms (the ortholog of mammalian α-subunit of BK channel) but were restored with the slo-1(+) or hslo(+) transgene, suggesting the involvement of BK channel activity. Additionally, hispidol increased fluorescence intensity and puncta in the motor neurons of slo-1::mCherry-tagged worms, indicating enhanced BK channel expression and clustering. Notably, hispidol did not alter internal ethanol concentrations, suggesting that its action is independent of ethanol metabolism. In the mouse models, hispidol treatment also demonstrated anxiolytic activity against ethanol withdrawal. Overall, these findings suggest hispidol as a promising candidate for targeting the BK channel in AUD treatment.

Keywords: BK channel; alcohol use disorder; ethanol intoxication; hispidol; withdrawal.

Figure 1

Effects of hispidol on ethanol-induced acute behaviors in C. elegans. (A) Chemical structure of hispidol; Age-synchronized worms were exposed to 300 mM ethanol for 10 min, and ethanol-induced behavioral phenotypes, including (B) thrashing, (C) locomotion speed, and (D) body amplitude were observed. Data are presented as the mean ± S.D., with results obtained from three independent experiments. Statistical significance is indicated as follows: ^### p < 0.001 compared with naïve animals; * p < 0.05, ** p < 0.01, *** p < 0.001 compared with ethanol-exposed animals.

Figure 2

Effects of hispidol on ethanol withdrawal-induced behaviors in C. elegans. (A) Age-synchronized worms were exposed to 150 mM ethanol for 24 h and subsequently transferred to fresh plates for a 1-h withdrawal period; (B) Locomotion speed of withdrawn worms was assessed under a dissecting microscope; (C) The chemotactic ability of withdrawn worms toward the attractant OP50 was monitored every 15 min for 1 h. Statistical significance is indicated as follows: ^### p < 0.001 compared with naïve animals; ** p < 0.01 and *** p < 0.001 compared with ethanol-exposed worms.

Figure 3

Involvement of BK channel modulation in hispidol-mediated alterations of ethanol-induced behaviors. (A) Age-synchronized slo-1(js379), slo-1(RNAi), slo-1(+/js379), and slo-1(hslo-1+/js379) worms were exposed to 300 mM ethanol for 10 min, and their locomotion speed was measured; (B) Locomotion speed of slo-1(js379), slo-1(RNAi), slo-1(+/js379), and slo-1(hslo-1+/js379) worms was assessed following ethanol withdrawal; (C) Internal ethanol concentration in wild-type worms was spectrophotometrically analyzed at three different time points: after 10 min of ethanol exposure (acute), after 24 h of ethanol exposure (chronic), and after an additional 1-h withdrawal following 24 h of ethanol exposure). Statistical significance is indicated as follows: *** p < 0.001 compared with hispidol-untreated worms; n.s. not significant.

Figure 4

Effects of hispidol on BK channel clustering. (A) Expression patterns of mCherry in JPS572 (vxEx345 [slo-1p::slo-1(+)::mCherry::unc-54 3′UTR + myo-2p::mCherry]) worms, captured at 100× magnification using fluorescence microscopy; (B) Quantification of slo-1 puncta, representing clustered BK channels in cholinergic neurons; (C) Carbofuran-induced paralysis monitored every 15 min in wild-type and slo-1(js379) worms; (D) Locomotion speed of ctn-1(RNAi) worms observed after 10 min of ethanol exposure and following withdrawal induction. Statistical significance is indicated as follows: ** p < 0.01, *** p < 0.001 compared with hispidol-untreated worms; n.s., not significant.

Figure 5

Effects of hispidol on ethanol-induced behaviors in mice. (A) Body weights of C57BL/6 mice monitored over seven days during a series of ethanol intoxications; (B) Motion trajectories recorded during the open field test; (C) Total distance traveled and (D) entries of center zone by mice in the open field test; (E) Motion trajectories recorded during the elevated plus maze test; (F) Time spent in the open arms in the elevated plus maze test. Statistical significance is indicated as follows: ^# p < 0.05 and ^### p < 0.001 compared with naïve animals; ** p < 0.01 and *** p < 0.001 compared with ethanol-exposed worms.