S100A8/S100A9–toll-like receptor 4 signaling identified using single-cell RNA sequencing in ST36 acupoint mediating acupuncture analgesia

Pain is a primary indication for acupuncture, with ST36 shown to effectively treat various types and locations of pain. Acupoints serve as pivotal starting points for acupuncture's therapeutic effects. However, how acupuncture stimulation alters the microenvironment at the ST36 acupoint to treat different pain conditions remains unclear. We investigated the analgesic effects of ST36 acupuncture in mice with adjuvant arthritis, identifying fibroblast and macrophage clusters as the main responsive populations at ST36 through single-cell sequencing analysis. These populations expressed high levels of S100 calcium-binding protein a8 (S100a8), S100a9, and Toll-like receptor 4 (TLR4) on both cells and afferent fibers. Furthermore, acupuncture at ST36 activated A and C afferent nerves. Intriguingly, acupoint injection of an S100a8/S100a9-TLR4 inhibitor reversed the excitation of A and C afferent nerves and diminished the analgesic effect of acupuncture. These findings offer novel insights into the ST36 acupoint microenvironment modulated by acupuncture and underscore the critical role of S100a8/S100a9 in initiating acupuncture-induced analgesia at ST36. The study identifies S100a8/S100a9 as a potential regulatory target for modulating acupuncture effects at acupoints, providing a molecular framework for standardized acupuncture interventions and their clinical applications. Overall design: After AIA induction, ACU was performed at ST36 on both hindlimbs, each ACU session lasted 30 min, with needles rotated intermittently. ACU was performed once daily. Mice were anesthetized and tissues from the ST36 acupoint were excised and stored in cold 1640 medium. Then, used single-cell sequencing technology and bioinformatics to reveal the cellular heterogeneity of the ST36 acupoint cell map and microenvironment.