IntroductionTemporomandibular joint (TMJ) disorders, a group of conditions that cause pain and dysfunction of jaw muscles and TMJ, present significant challenges in clinical settings due to their complexity and the limitations of current treatments.1,2,3 Therefore, understanding the underlying mechanisms and identifying new treatment options are critical for improving pain management in patients with such disorders.More and more evidence has demonstrated complex interactions between gut microbiome and orofacial pain including TMJ pain.4 Gut microbiome refers to diverse community of microorganisms, which is involved in maintaining the overall health of the host and has been implicated in different neurological disorders.5 In our recent study6 using a complete Freund’s adjuvant (CFA)-induced inflammatory TMJ pain mouse model, intra-TMJ injection of CFA not only causes persistent TMJ pain but also reduces short-chain fatty acids (SCFAs) levels and decreases SCFA-producing bacteria in the gut. Following the CFA injection, butyrate shows the most pronounced reduction among the three major SCFAs.6 Similarly, other studies have also shown that butyrate plays crucial roles in modulating inflammation and pain.7,8 SCFAs, produced through the fermentation of dietary fibers by gut bacteria, have various beneficial effects, including reducing inflammation and producing analgesia.9,10,11 Despite these promising findings, the exact role of butyrate in TMJ pain and its underlying mechanisms remain largely unknown.In this study, we explored the therapeutic potential of butyrate in alleviating inflammatory TMJ pain using a mouse model. Tributyrin, a prodrug that releases butyrate in the gut, was used for long-term butyrate supplementation. To reveal cell-specific gene regulation that contributes to TMJ pain and its modulation by butyrate, we conducted single-cell multi-omics sequencing, including single-nucleus RNA sequencing (snRNA-seq) and single-nucleus Assay for Targeting Accessible-Chromatin sequencing (snATAC-seq), to analyze the cellular composition and gene expression/activity changes in the spinal trigeminal nucleus caudalis (Sp5C), a crucial relay center for trigeminal pain processing.12,13 Our comprehensive sequencing identified 12 distinct cell types in the Sp5C, and our differential gene analysis revealed significant changes in gene expression and gene activity patterns in response to CFA-induced inflammatory TMJ pain and tributyrin treatment. Together, our study provides novel insights into the role of butyrate in modulating inflammatory TMJ pain, highlighting its potential as a therapeutic agent in TMJ pain management. By elucidating the cellular and molecular changes associated with butyrate treatment, our findings pave the way for future research into targeted interventions for TMJ pain and enhance our understanding of the complex interactions between gut microbiome and pain modulation. Moreover, this study suggests that leveraging gut microbiome metabolites could be applied as a novel approach for treating other inflammatory pain conditions as well.ResultsTributyrin treatment inhibits CFA-induced inflammatory TMJ pain and recovers butyrate levels in miceUsing the CFA-induced inflammatory TMJ pain mouse model, we observed that 10-day oral administration of tributyrin (5 g/kg per day, Fig. 1a) significantly increased head withdrawal thresholds on the ipsilateral side of trigeminal nerve V3 branch-innervated facial skin areas (Fig. 1b) but had no effect on the contralateral thresholds of the mice (Fig. 1c), indicating that the tributyrin treatment inhibits CFA-induced TMJ pain. To examine how butyrate levels are altered during the TMJ pain condition and whether the tributyrin treatment can return butyrate back to normal levels, we collected mouse feces, blood, and Sp5C tissues on day 5 post-CFA when the mice showed the lowest withdrawal threshold. Using an ELISA assay, we measured butyrate concentrations in these samples. Our results showed that intra-TMJ injection of CFA significantly decreased butyrate levels in the feces, plasma, and Sp5C of the mice (Fig. 1d–f). More importantly, we found that the 10-day tributyrin treatment completely recovered the decreased butyrate levels in the mice (Fig. 1d–f), suggesting that the analgesic effect of tributyrin could be mediated through butyrate recovery in the mice.Fig. 1The alt text for this image may have been generated using AI.Full size imageTributyrin treatment inhibits CFA-induced inflammatory TMJ pain and recovers butyrate levels in mice. a Timeline of the experiment. Our von Frey test showed that 10-day tributyrin treatment via oral gavage significantly increased head withdrawal thresholds at days 5, 7, 9 post-CFA on the ipsilateral side of trigeminal nerve V3 branch-innervated facial skin areas (b) but had no effect on head withdrawal thresholds of the contralateral side (c). n = 6–12 mice per group, *P