Psychiatric disorders such as anxiety and depression precipitated by substance use occurred during both use and withdrawal. Exosomes play significant roles in biological functions and regulate numerous physiological and pathological processes in various diseases, in particular substance use disorders (SUDs) and other psychiatric disorders. To better understand the role of exosomal miRNAs in the pathology of symptoms of anxiety and depression in patients with SUDs, we first isolated circulating exosomes from heroin-dependent patients (HDPs) and methamphetamine-dependent patients (MDPs) and identified exosomal miRNAs that were differentially expressed between patients and healthy controls (HCs). Furthermore, the correlations between exosomal DE-miRNAs and symptoms of anxiety and depression which were measured using Hamilton-Anxiety (HAM-A)/Hamilton-Depression (HAM-D) Rating Scales in the participants. Notably, the expression level of exosomal hsa-miR-16-5p, hsa-miR-129-5p, hsa-miR-363-3p, and hsa-miR-92a-3p showed significantly negative correlations with HAM-A scores in both HDPs and MDPs. But all of the 4 DE-miRNAs lost significant correlations with HAM-D scores in HDPs. Functional annotation analyses showed that the target genes of the DE-miRNAs were mainly enriched for “synapse”, “cell adhesion”, “focal adhesion” and “MHC class II protein complex”. Our study suggests that a set of circulating exosomal miRNAs were associated with anxiety and depression in SUD patients and may have clinical utility as diagnostic and prognostic biomarkers.

Metformin exhibits therapeutic potential in behavioural deficits induced by methamphetamine (METH) in rats. Emerging studies suggest gut microbiota may impact psychiatric symptoms, but there is no direct evidence supporting metformin's participation in the pathophysiology of withdrawal symptoms via modulation of gut microbiota. In order to define the functional impacts of gut microbiota and metformin to the behavioural deficits during METH withdrawal, we utilized a combination of fecal microbiota transplantation (FMT), high-throughput sequencing, and untargeted metabolomics technologies. First, METH addicts exhibited higher α diversity and distinct microbial structures compared to healthy controls. In particular, the relative abundance of Rikenellaceae was positively correlated with the severity of anxiety and depression. Second, both human-to-mouse and mouse-to-mouse FMTs confirmed that METH-altered-microbiota transplantation is sufficient to promote anxiety and depression-like behaviours in recipient germ-free mice, and these behavioural disturbances could be ameliorated by metformin. In-depth analysis revealed that METH significantly altered the bacterial composition and structure as well as relative abundance of several bacterial taxa and metabolites, including Rikenellaceae and inosine, respectively, whereas add-on metformin could remodel these alterations. Finally, the inosine complementation successfully restored METH-induced anxiety and depression-like behaviours in mice. This study demonstrates that METH withdrawal-induced anxiety and depression-like behaviours are reversible and transmissible via gut microbiota in a mouse model. The therapeutic effects of metformin on psychiatric manifestations are associated with microbiota-derived metabolites, highlighting the role of the gut microbiota in substance use disorders and the pathophysiology of withdrawal symptoms.

Heroin addiction and withdrawal influence multiple physiological functions, including immune responses, but the mechanism remains largely elusive. The objective of this study was to investigate the molecular inflammatory interactome, particularly the cytokines and transcriptome regulatory network in heroin addicts undergoing withdrawal, compared to healthy controls (HCs). Twenty-seven cytokines were simultaneously assessed in 41 heroin addicts, including 20 at the acute withdrawal (AW) stage and 21 at the protracted withdrawal (PW) stage, and 38 age- and gender-matched HCs. Disturbed T-helper(T_h)1/T_h2, T_h1/T_h17, and T_h2/T_h17 balances, characterized by reduced interleukin (IL)-2, elevated IL-4, IL-10, and IL-17A, but normal TNF-α, were present in the AW subjects. These imbalances were mostly restored to the baseline at the PW stage. However, the cytokines TNF-α, IL-2, IL-7, IL-10, and IL-17A remained dysregulated. This study also profiled exosomal long non-coding RNA (lncRNA) and mRNA in the plasma of heroin addicts, constructed co-expression gene regulation networks, and identified lncRNA-mRNA-pathway pairs specifically associated with alterations in cytokine profiles and T_h1/T_h2/T_h17 imbalances. Altogether, a large amount of cytokine and exosomal lncRNA/mRNA expression profiling data relating to heroin withdrawal was obtained, providing a useful experimental and theoretical basis for further understanding of the pathogenic mechanisms of withdrawal symptoms in heroin addicts.

Despite decades of research in the field of substance withdrawal, molecular biomarkers and related mechanistic study have generally been lacking. In addition to known neurotransmitters, circulating miRNAs are found in small vesicles known as exosomes within blood that have diagnostic potential and are known to contribute to psychiatric disorders. The aim of this work was to characterize the changes in neurotransmitter and exosomal miRNA profiles during heroin and methamphetamine withdrawal using a cross-sectional study design, and to determine their associations to psychiatric comorbidities in a large group of patients with substance use disorders (SUDs). Using weighted gene co-expression network analysis, a series of known, conserved, and novel exosomal miRNAs were identified as being associated with the severity of anxiety and depression, as well as the concentrations of neurotransmitters GABA, choline, and serotonin. Bioinformatics analyses established that the differences in the miRNA profile target signaling pathways are significantly associated with developmental and intellectual abnormalities. Notably, a set of dysregulated miRNA signatures including hsa-mia-451a and hsa-mir-21a resulted in an AUC of 0.966 and 0.861, respectively, for predicting the patients with SUDs. Furthermore, hsa-miR-744a-5p was positively correlated with serotonin, and its important role in maintaining neuronal development and function was revealed using an in vitro human induced pluripotent stem cells derived neuronal model. Our results suggest that the miRNA content of circulating exosomes represent a biomolecular "fingerprint" of the progression of substance withdrawal and may uncover the putative mechanism of how these exosomal miRNAs contribute to psychiatric symptoms.

The gut microbiota is believed to play a significant role in psychological and gastrointestinal symptoms in heroin addicts. However, the underlying mechanism remains largely unknown. We show here that heroin addicts had a decrease in body mass index (BMI) and abnormal serum D-lactic acid (DLA), endotoxin (ET) and diamine oxidase (DAO) levels during their withdrawal stage, suggesting a potential intestinal injury. The gut microbial profiles in the mouse model with heroin dependence showed slightly decreased alpha diversity, as well as higher levels of Bifidobacterium and Sutterella and a decrease in Akkermansia at genus level compared to the control group. Fecal microbiota transplantation (FMT) further confirmed that the microbiota altered by heroin dependence was sufficient to impair body weight and intestinal mucosal barrier integrity in recipient mice. Moreover, short-chain fatty acids (SCFAs) profiling revealed that microbiota-derived propionic acid significantly decreased in heroin dependent mice compared to controls. Overall, our study shows that heroin dependence significantly altered gut microbiota and impaired intestinal mucosal barrier integrity in mice, highlighting the role of the gut microbiota in substance use disorders and the pathophysiology of withdrawal symptoms.