Reduced circ_lrrc49 in trigeminal ganglion contributes to neuropathic pain in mice by downregulating Ist1 and impairing autophagy

Orofacial neuropathic pain is a common symptom induced by orofacial nerve injury caused by a range of trauma or dental and maxillofacial procedures but lacks effective treatment. Circular RNAs (circRNAs) participate in the regulatory processes of neuropathic pain. Nevertheless, the biological roles of circRNAs in orofacial neuropathic pain remain unexplored. In this study, circRNA sequencing and Real-time quantitative polymerase chain reaction (RT-qPCR) were carried out. Notably, a novel circRNA named circ_lrrc49 was identified to be downregulated following chronic constriction injury of the infraorbital nerve (CCI-ION) in mice on day 14. Subsequent RNA Antisense Purification (RAP)-mass spectrometry and RNA immunoprecipitation found a direct interaction between circ_lrrc49 and increased sodium tolerance 1 homolog (Ist1). Western blot (WB) identified decreased expression of Ist1 on day 14 post-CCI-ION. Considering the known relationship between Ist1 and autophagy, LC3-II and p62 were detected to be upregulated, and an accumulation of autophagosomes were observed at the same time point. Besides, the knockdown of circ_lrrc49 by small interfering RNA (siRNA) reduced Ist1 expression, increased LC3-II, p62 levels and autophagosomes amount, and evoked orofacial mechanical hypersensitivity, which could be counteracted by the Ist1 overexpression. Similarly, the knockdown of Ist1 by siRNA also increased LC3-II and p62 levels and evoked orofacial mechanical hypersensitivity without influence on circ_lrrc49. Moreover, autophagy activation by rapamycin alleviated orofacial mechanical hypersensitivity evoked by CCI-ION or circ_lrrc49 knockdown. In conclusion, our data revealed the existence of a circ_lrrc49/Ist1/autophagy signaling axis contributing to the progression of orofacial neuropathic pain. These discoveries reveal the intricate molecular processes that drive orofacial neuropathic pain and identify circ_lrrc49 as a promising target for potential therapeutic interventions. Our study uncovers a circ_lrrc49/Ist1/autophagy axis in orofacial neuropathic pain. Key findings include: (1) circ_lrrc49 downregulation induces mechanical hypersensitivity, and its overexpression alleviates mechanical hypersensitivity caused by CCI-ION; (2) circ_lrrc49 interacts with Ist1, regulating its expression; (3) circ_lrrc49 knockdown impairs autophagy, and Ist1 overexpression counteracts this, reducing mechanical hypersensitivity; (4) autophagy activation by rapamycin relieves CCI-ION-induced mechanical hypersensitivity. These discoveries identify circ_lrrc49 as a promising therapeutic target for orofacial neuropathic pain. CCI-ION, chronic constriction injury of the infraorbital nerve; TG, trigeminal ganglion.image