Cells were subjected to immunofluorescence analysis using an antibody directed against AIF1 (green). Nuclei were counterstained with DAPI (blue). The fluorescence was visualized and imaged under a fluorescence microscope. C: control group; M: morphine group; M + M: morphine+methadone group; M + I: morphine+isorhynchophylline; M + MH: morphine+minocycline hydrochloride.

qPCR analysis of Il1b, Tlr4, Itgam, Nos2, Oprm1, Oprd1, Drd2, Htr2a, Gad1, Gad2, Bdnf, and Ntrk2 expression in BV2 cells (n = 3). *p < 0.05, **p < 0.01 vs the C group; #p < 0.05, ##p < 0.01 vs the M group. C: control group; M: morphine group; M + M: morphine + methadone group; M + I: morphine + isorhynchophylline group; M + MH: morphine + minocycline hydrochloride group.

Isorhynchophylline can inhibit morphine-induced CPP responses. (A) Road maps of zebrafish in the CPP compartment. (B) Time spent in the drug-paired box post-CPP (n = 8). *p < 0.05, **p < 0.01 vs the C group; ^#p < 0.05, ^##p < 0.01 vs the M group. C: control group; M: morphine group; M + M: morphine + methadone group; M + I: morphine + isorhynchophylline group.

Comparison of community structure among the fecal microbiota of the C, M, M + M, and M + I groups. (A) Venn diagram illustrating the overlap of the operational taxonomic units (OTUs) identified in the fecal microbiota of the four groups. (B) Shannon curves among the four groups. (C) Alpha diversity of the fecal microbiome among the four groups according to Sobs, Chao, ACE, and Shannon indices. (D) Beta diversity of the fecal microbiome among the four groups based on principal coordinate analysis (PCoA) and nonmetric multidimensional scaling (NMDS). *p < 0.05, **p < 0.01 vs the C group; ^#p < 0.05, ^##p < 0.01 vs the M group. C: control group; M: morphine group; M + M: morphine + methadone group; M + I: morphine + isorhynchophylline group.

Comparison of species composition among the fecal microbiota of the C, M, M + M, and M + I groups at the phylum level. (A) Relative abundances of the phyla present in samples from the four groups. Percent abundance of (B) Actinobacteria (C) Bacteroidetes (D) Firmicutes (E) Fusobacteria, and (F) Proteobacteria at the phylum level among the four groups. (G) The Bacteroidetes/Firmicutes (B/F) ratio of the four groups. *p < 0.05, **p < 0.01 vs the C group; ^#p < 0.05, ^##p < 0.01 vs the M group. C: control group; M: morphine group; M + M: morphine + methadone group; M + I: morphine + isorhynchophylline group.

Differentially regulated functional pathways based on KEGG enrichment analysis inferred from 16S rRNA gene sequences using PICRUSt. *p < 0.05, **p < 0.01 vs the control (C) group; ^#p < 0.05, ^##p < 0.01 vs the M group.

qPCR analysis of il1b, tlr4b, itgam, oprm1, oprd1, oprd1b, gad2, drd2a, drd2b, htr1aa, htr2aa, bdnf, and ntrk2 expression in the zebrafish brain (n = 3). *p < 0.05, **p < 0.01 vs the C group; ^#p < 0.05, ^##p < 0.01 vs the M group. C: control group; M: morphine group; M + M: morphine + methadone group; M + I: morphine + isorhynchophylline group.

qPCR analysis of il1b, tlr4b, itgam, oprm1, oprd1, oprd1b, gad2, drd2a, drd2b, htr1aa, htr2aa, bdnf, and ntrk2 expression in the zebrafish gut (n = 3). *p < 0.05, **p < 0.01 vs the C group; ^#p < 0.05, ^##p < 0.01 vs the M group. C: control group; M: morphine group; M + M: morphine + methadone group; M + I: morphine + isorhynchophylline group.

Isorhynchophylline cannot improve morphine-induced CPP responses following antibiotic treatment. (A) Road maps of zebrafish in the CPP box. (B) Time spent in the drug-paired compartment post-CPP (n = 8). *p < 0.05, **p < 0.01 vs the C group; ^#p < 0.05, ^##p < 0.01 vs the A + M group; ^▲p < 0.05, ^▲▲p < 0.01 vs the A group.

Comparison of community structure among the fecal microbiota of the C, A, A + M, A + M + M, and A + M + I groups. (A) Venn diagram illustrating the overlap of the OTUs identified in the fecal microbiota of the five groups. (B) Shannon curves among the five groups. (C) The alpha diversity of the fecal microbiome among the five groups based on Sobs, Chao, ACE, and Shannon index values. (D) The beta diversity of the fecal microbiome among the five groups based on principal coordinate analysis (PCoA) and nonmetric multidimensional scaling (NMDS). *p < 0.05, **p < 0.01 vs the C group; ^#p < 0.05, ^##p < 0.01 vs the A + M group; ^▲p < 0.05, ^▲▲p < 0.01 vs the A group. C: control group; A: antibiotic group; A + M: antibiotic + morphine group; A + M + M: antibiotic + morphine + methadone group; A + M + I: antibiotic + morphine + isorhynchophylline group.

Comparison of species composition among the fecal microbiota of the C, A, A + M, A + M + M, and A + M + I groups at the phylum level. (A) Relative abundances of the phyla present in samples from the five groups. Percent of (B) Actinobacteria abundance (C) Bacteroidetes abundance (D) Firmicutes abundance (E) Fusobacteria abundance, and (F) Proteobacteria abundance at the phylum level among the five groups. (G) The Bacteroidetes/Firmicutes (B/F) ratio of the five groups. *p < 0.05, **p < 0.01 vs the C group; ^#p < 0.05, ^##p < 0.01 vs the A + M group; ^▲p < 0.05, ^▲▲p < 0.01 vs the A group. C: control group; A: antibiotic group; A + M: antibiotic + morphine group; A + M + M: antibiotic + morphine + methadone group; A + M + I: antibiotic + morphine + isorhynchophylline group.

Differentially regulated functional pathways based on KEGG enrichment analysis inferred from 16S rRNA gene sequencing using PICRUSt. *p < 0.05, **p < 0.01 vs the control (C) group; ^#p < 0.05, ^##p < 0.01 vs the A + M treatment group; ^▲p < 0.05, ^▲▲p < 0.01 vs the antibiotic (A) treatment group.

qPCR analysis of il1b, tlr4b, itgam, oprm1, oprd1, oprd1b, gad2, drd2a, drd2b, htr1aa, htr2aa, bdnf, and ntrk2 expression in the zebrafish brain (n = 3). *p < 0.05, **p < 0.01 vs the C group; ^#p < 0.05, ^##p < 0.01 vs the A + M group; ^▲p < 0.05, ^▲▲p < 0.01 vs the A group. C: control group; A: antibiotic group; A + M: antibiotic + morphine group; A + M + M: antibiotic + morphine + methadone group; A + M + I: antibiotic + morphine + isorhynchophylline group.

qPCR analysis of il1b, tlr4b, itgam, oprm1, oprd1, oprd1b, gad2, drd2a, drd2b, htr1aa, htr2aa, bdnf, and ntrk2 expression in the zebrafish gut (n = 3). *p < 0.05, **p < 0.01 vs the control C group; ^#p < 0.05, ^##p < 0.01 vs the A + M group; ^▲p < 0.05, ^▲▲p < 0.01 vs the A group. C: control group; A: antibiotic group; A + M: antibiotic + morphine group; A + M + M: antibiotic + morphine + methadone group; A + M + I: antibiotic + morphine + isorhynchophylline group.

Schematic of the experimental design. (A) Schematic of the experimental design for analysis of morphine-induced activation of BV2 cells. (B) Schematic of the experimental design for the CPP test. (C) Schematic of the experimental design for antibiotic treatment combined with the CPP test.