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. 2023 Jun 20;13(10):1135–1152. doi: 10.1016/j.jpha.2023.06.008

Fig. 6.

Fig. 6

3A5C7 monoclonal antibody (mAb) attenuates morphine antinociceptive tolerance and physical dependence in mice. (A) Western blots indicating the cross specificity of 3A5C7 mAb against mu-opioid receptor (MOR) in the cortex, cerebellum, and hippocampus from C57/B6 mice. (B) Experiment flowchart for testing the effects of chronically administered 3A5C7 mAb on morphine tolerance and dependence. (C) The effects of chronically administered 3A5C7 mAb on the antinociceptive effects of morphine in mice measured by hotplate test. (D) The body weight changes of morphine-tolerant mice. Two-way analysis of variance with Bonferroni's post hoc tests were used for statistical analysis (n = 6–7 mice in each group). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001, morphine + mAb group vs. morphine group. #P < 0.05, ##P < 0.01, and ###P < 0.001, morphine + mAb group vs. morphine + normal IgG (NIg) group. (E) Representative immunofluorescence staining of MOR and Rab5 in the mouse dorsal root ganglions (DRGs) after chronic 3A5C7 treatment (n = 3 mice in each group). (F) Cyclic adenosine monophosphate (cAMP) levels of cortex in mice from each treatment group (n = 3 mice in each group). (G) The level of cAMP of hippocampus in mice from each treatment group (n = 3 mice in each group). (H) Immunoblots showing the protein levels of protein kinase A (PKA) and G protein-coupled receptor kinase 2 (GRK2) in hippocampus of mice from each treatment (n = 3 mice in each group). (I) Quantification of the levels of PKA in Fig. 6H. (J) Quantification of the levels of GRK2 in Fig. 6H. (K) Chronic 3A5C7 mAb administration diminished naloxone-precipitated withdrawal jumping in mice (n = 5–8 mice in each group). (L) Experiment flowchart determining the acute effects of 3A5C7 mAb on naloxone-precipitated withdrawal jumping in morphine-tolerant mice. (M) Acute mAb administration diminished morphine withdrawal (n = 4–7 mice in each group). (N) cAMP concentrations of striatum in mice from each treatment (n = 3 mice in each group). One-way analysis of variance with Bonferroni's post hoc tests were used for statistical analysis in Figs. 6F, G, I–K, M, and N. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001. Data were presented as mean ± standard error of mean. GAPDH: glyceraldehyde 3-phosphate dehydrogenase; MPE: maximum possible effect; NS: normal saline.