Electroacupuncture (EA) treatment has proved to significantly decrease nociception in inflammatory nociception model by suppressing the phosphorylation of p38 mitogen-activated protein kinase (p38MAPK). However, repeated EA treatment results in gradual attenuation of its analgesic effects, which was defined as "EA tolerance." Recent studies have shown that let-7b-5p microRNA (miRNA) contributes to the EA tolerance. The present study aimed to explore the function of let-7b-5p in p38MAPK pathway and the development of EA tolerance in the inflammatory nociception. Dual luciferase reporter gene experiments were used in cortical neurons to determine the target gene locus of let-7b-5p. The threshold of nociception was assessed by tail flick latency (TFL) and paw withdrawal threshold (PWT). Western blots were used to measure the expression of mitogen-activated protein kinase phosphatase 1 (MKP-1) and phosphorylation level of p38MAPK after intracerebroventricular (ICV) injections of let-7b-5p agomir, antagomir, and controls. In vitro dual luciferase experiments demonstrated that the MKP-1-3′ untranslated region (UTR) is a target of let-7b-5p. In vivo experiment, rat with repeated EA treatment exhibits gradual decrease in TFL and PWT, which showed formation of EA tolerance. This trend was delayed after IVC injection of let-7b-5p antagomir and facilitated after IVC injection of let-7b-5p agomir. The protein levels of MKP-1 in the EA+let-7b-5p antagomir group were significantly higher than in the EA + let-7b-5p agomir group. However, P-p38MAPK in the EA+let-7b-5p antagomir group was significantly lower than in the EA+let-7b-5p agomir group. By upregulating the p38MAPK pathway through the inactivation of the MKP-1 gene, let-7b-5p contributes to EA tolerance in complete Freund's adjuvant (CFA)-induced inflammatory nociception rats. Our work revealed the mechanism of EA tolerance and indicated that let-7b-5p could be targeted to improve the long-term effects of EA.

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