CB2 receptor agonist AM1241 regulating the polarization of microglia reduces morphine tolerance through IL-4/STAT6 pathway.

Background: Pain seriously impacts patients' life quality. The use of morphine for pain is common, but tolerance limits its application in clinic. However, there is no exact mechanism for tolerance. In this study, we explored how microglial polarization and IL (interleukin)-4,along with a CB2 receptor agonist, affect reducing morphine tolerance.

Method: The cells cultivated with morphine or combined CB2R agonist AM1241, CB2R antagonist AM630, CB1R antagonist AM281, and IL-4 inhibitor (IL-4I).Mice were injected with these drugs for 7 days, and hot plate behavioral tests were performed 30 min after administration respectively. Mice received a single morphine injection on day 8.Samples were taken post-tests. The expression of iNOS, SOCS3, IL-4 and STAT6 mRNA were detected by qPCR; the expression of iNOS, SOCS3, p-STAT6 and STAT6 protein were detected by Western blot. Inflammatory cytokines were detected with Elisa kit.

Results: The M1 marker iNOS increased, the M2 marker SOCS3 decreased, p-STAT6 protein did not change, and the cytokines increased after morphine treatment. The paw withdrawal latency (PWL) value, IL-4 mRNA and p-STAT6 protein increased after AM1241 treatment, iNOS decreased and SOCS3 increased after AM1241 treatment, AM1241 decreased the pro-inflammatory cytokines, increased IL-4, IL-10 secretion. AM630 and IL-4I reversed the effect of AM1241 on PWL, M1 M2 markers.

Conclusion: The polarization of microglia in the direction of M1 caused morphine tolerance, AM1241 increased the IL-4 mRNA and induced the phosphorylation protein of STAT6 to reduce the tolerance, and AM1241 induced microglia to polarization in the direction of M2. AM1241 regulated microglia polarization through IL-4/STAT6 pathway, thereby reducing tolerance.