Single-Cell RNA Transcriptomics and Multi-omics Analyses Reveal the Clinical Effects of Acupuncture on Methadone Reduction.

Opioid use disorders (OUDs) pose a substantial global health burden, with methadone maintenance treatment (MMT) widely adopted as an intervention; however, MMT is associated with immunosuppression, metabolic disturbances, and dysbiosis of the gut microbiota. Despite the potential of acupuncture in reducing methadone dosages and opioid addiction, the underlying biological mechanisms remain unclear. Therefore, we aimed to integrate clinical trial data with multi-omics analysis, including single-cell sequencing, transcriptomics, metabolomics, and metagenomics, to evaluate the effects of acupuncture in patients undergoing MMT. We collected peripheral blood mononuclear cells, plasma, and fecal samples from 48 MMT participants in a randomized, placebo-controlled trial. Participants were divided into acupuncture (n = 25) and sham-acupuncture (n = 23) groups. After 8 weeks of intervention, 84% of patients in the acupuncture group achieved ≥20% reduction in methadone dosage, compared to 39% in the sham-acupuncture group (P < 0.01). Our findings revealed that acupuncture may activate the defense response to viruses, with altered immune cell functions in classical monocytes correlating with clinical responses to reduced methadone doses. Acupuncture might ameliorate intestinal microbial disruptions caused by OUD by up-regulating Bilophila and modulating bile acid metabolism. Furthermore, acupuncture up-regulated galectin-9 (LGALS9)-mediated intercellular communication between classical monocytes and other immune subsets. To further validate the mechanistic link between bile acid metabolism and immune regulation, we conducted in vitro experiments using THP-1 monocytes treated with cholic acid. The results showed that bile acid exposure suppressed galectin-9 and IFN-γ expression, while low-dose bile acid (simulating acupuncture effects) partially reversed this effect. These findings support a bile acid-galectin-interferon axis that may be modulated by acupuncture in OUD. Collectively, our results provide clinical and mechanistic evidence supporting acupuncture as a potential adjunct therapy to mitigate the adverse effects of long-term opioid use.