Identification of the circRNA-miRNA-mRNA network for treating methamphetamine-induced relapse and behavioral sensitization with cannabidiol

Abstract

Aims: This study aims to investigate the pharmacological effects and the underlying mechanism of cannabidiol (CBD) on methamphetamine (METH)-induced relapse and behavioral sensitization in male mice.

Methods: The conditioned place preference (CPP) test with a biased paradigm and open-field test were used to assess the effects of CBD on METH-induced relapse and behavioral sensitization in male mice. RNA sequencing and bioinformatics analysis was employed to identify differential expressed (DE) circRNAs, miRNAs, and mRNAs in the nucleus accumbens (NAc) of mice, and the interaction among them was predicted using competing endogenous RNAs (ceRNAs) network analysis.

Results: Chronic administration of CBD (40 mg/kg) during the METH withdrawal phase alleviated METH (2 mg/kg)-induced CPP reinstatement and behavioral sensitization in mice, as well as mood and cognitive impairments following behavioral sensitization. Furthermore, 42 DEcircRNAs, 11 DEmiRNAs, and 40 DEmRNAs were identified in the NAc of mice. The circMeis2-miR-183-5p-Kcnj5 network in the NAc of mice is involved in the effects of CBD on METH-induced CPP reinstatement and behavioral sensitization.

Conclusions: This study constructed the ceRNAs network for the first time, revealing the potential mechanism of CBD in treating METH-induced CPP reinstatement and behavioral sensitization, thus advancing the application of CBD in METH use disorders.

Keywords: behavioral sensitization; cannabidiol; competing endogenous RNAs; methamphetamine; relapse.

FIGURE 1

Chronic administration of CBD inhibits METH‐induced CPP reinstatement in mice. (A) CPP test and RNA sequencing protocols. (B) Heat maps of mice's track in the CPP apparatus. (C) Administration of CBD (40 mg/kg, but not 20 mg/kg) during the 14‐day extinction phase inhibited METH‐induced CPP reinstatement in mice. *p < 0.05, ***p < 0.001 versus Pre‐conditioning test; ^## p < 0.01, ^### p < 0.001 versus Training test; ^& p < 0.05 versus METH group. Data were analyzed by two‐way ANOVA followed by Tukey's multiple comparison test, and all values are presented as the mean ± SEM (n = 7–8).

FIGURE 2

Chronic administration of CBD reduces METH‐induced locomotor sensitization, anxiety‐like behavior, and cognitive impairments in mice. (A) The schedule of locomotor sensitization, mood, and cognitive function tests. (B) The tracks of mice in the open‐field apparatus. (C) Chronic administration of CBD (40 mg/kg, but not 20 mg/kg) reduced METH‐induced locomotor sensitization in mice. ***p < 0.001 versus saline group on the same day; ^## p < 0.01, ^### p < 0.001 versus same group on day 7; ^&& p < 0.01 versus METH group on day 22. (D) Chronic administration of CBD alleviated METH‐induced anxiety‐like behavior in mice. *p < 0.05, ***p < 0.001; ^# p < 0.05. (E) The protocol of NOR test. (F) Heat maps of mice's track in the NOR test. (G) Chronic administration of CBD (40 mg/kg, but not 20 mg/kg) improved the reduction of recognition index (%) induced by METH after 2 h of training. **p < 0.01; ^# p < 0.05. (H) Chronic administration of CBD (20 mg/kg, but not 40 mg/kg) improved the reduction of recognition index (%) induced by METH after 24 h of training. *p < 0.05; ^### p < 0.001. The data were analyzed by two‐way ANOVA (C–D) or one‐way ANOVA (G–H) followed by Tukey's multiple comparison test; all values are presented as the mean ± SEM (n = 6–8).

FIGURE 3

Chronic administration of CBD alleviates METH‐induced moods and cognitive impairments in mice. (A) Heat map of mice's track in the EPM apparatus. (B) Chronic administration of CBD increased the time spent in the open arms in METH‐treated mice. *p < 0.05; ^## p < 0.01. (C) Both CBD and METH treatment do not affect the number of entries into the open arms in mice. n.s: not significant. (D) Heat maps of mice's track in the three‐chamber apparatus. (E) Chronic administration of CBD improved the reduction of the social ability index (%) caused by METH in mice. ***p < 0.001; ^## p < 0.01. (F) Both CBD and METH treatment do not affect the social novelty index (%) in mice. n.s, not significant. (G) Heat map of mice's track in the Y maze apparatus. (H) Chronic administration of CBD (40 mg/kg, but not 20 mg/kg) rescued the reduction of the alteration triplet (%) induced by METH in mice. **p < 0.01; ^# p < 0.05. (I) Chronic administration of CBD (40 mg/kg, but not 20 mg/kg) decreased the immobility time of METH‐treated mice in the TST test. *p < 0.05; ^## p < 0.01. (J) Chronic administration of CBD decreased the immobility time of METH‐treated mice in the FST test. **p < 0.01; ^# p < 0.05, ^## p < 0.01. All data were analyzed by one‐way ANOVA followed by Tukey's multiple comparison test; all values are presented as the mean ± SEM; n = 6–8.

FIGURE 4

circRNAs profiling in the NAc of mice from saline, METH, and 40 mg/kg CBD + METH groups. (A) The number of identified circRNAs and their composition in terms of gene distribution. (B) The distribution of identified circRNAs on the mouse chromosomes. (C) The volcano plot of DEcircRNAs in the NAc between the saline‐treated mice and METH‐treated mice. (D) The volcano plot of DEcircRNAs in the NAc between the 40 mg/kg CBD + METH‐treated mice and METH‐treated mice. (E) The number of upregulated and downregulated circRNAs in METH‐treated mice compared to the saline‐treated mice. (F) The number of upregulated and downregulated circRNAs in 40 mg/kg CBD + METH‐treated mice compared to the METH‐treated mice. (G) There are a total of 42 shared DEcircRNAs among (E) and (F). (H) A heatmap displayed the expression of 42 shared circRNAs in three paired samples of NAc from saline‐treated mice, METH‐treated mice, and 40 mg/kg CBD + METH‐treated mice. (I) The composition of 42 shared DEcircRNAs in terms of gene distribution.

FIGURE 5

mRNAs and miRNAs profiling in the NAc of mice from saline, METH, and 40 mg/kg CBD + METH groups. (A) The volcano plot of DEmRNAs in the NAc between the saline‐treated mice and METH‐treated mice. (B) The volcano plot of DEmRNAs in the NAc between the 40 mg/kg CBD + METH‐treated mice and METH‐treated mice. (C) There is a total of 40 shared DEmRNAs among the three groups. (D) A heatmap displayed the expression of 40 shared mRNAs in three paired samples of NAc from saline‐treated mice, METH‐treated mice, and 40 mg/kg CBD + METH‐treated mice. (E) The top 15 terms in the GO enrichment analysis of 40 shared DEmRNAs. (F) The top 10 pathways in the KEGG pathway analysis of 40 shared DEmRNAs. (G) The volcano plot of DEmiRNAs in the NAc between the saline‐treated mice and METH‐treated mice. (H) The volcano plot of DEmiRNAs in the NAc between the 40 mg/kg CBD + METH‐treated mice and METH‐treated mice. (I) There are a total of 11 shared DEmiRNAs among the three groups. (J) A heatmap displayed the expression of 11 shared miRNAs in three paired samples of NAc from saline‐treated mice, METH‐treated mice, and 40 mg/kg CBD + METH‐treated mice. (K) ceRNA network that was constructed based on the dataset of shared 42 DEcircRNAs, 11 DEmiRNAs, and 40 DEmRNAs using miRanda.

FIGURE 6

circMeis2‐miR‐183‐5p‐Kcnj5 network involves the positive effects of CBD on treating METH‐induced CPP reinstatement and behavioral sensitization in mice. (A–D) circRNAs expression in the NAc of CPP mice. (E) miR‐183‐5p expression in the NAc of CPP mice. (F,G) Kcnj5 expression in the NAc of CPP mice. (H–K) circRNAs expression in the NAc of behavioral sensitized mice. (L) miR‐183‐5p expression in the NAc of behavioral sensitized mice. (M,N) Kcnj5 expression in the NAc of behavioral sensitized mice. *p < 0.05, **p < 0.01, ***p < 0.001; ^# p < 0.05, ^## p < 0.01. All data were analyzed by one‐way ANOVA followed by Tukey's multiple comparison test; all values are presented as the mean ± SEM (n = 5).