Fig 2: Nerve injury causes a delayed and sustained increase in CB2 expression in the DRG.A–C, representative gel images and quantification of CB2 protein level in the rat DRG 5 (A), 10 (B), and 21 (C) days after sham or SNL surgery. D, representative gel images and quantification of CB2 protein level in the rat dorsal spinal cord 21 days after sham or SNL surgery. Data are shown as means ± SEM (n = 9 rats per group). GAPDH was used as endogenous loading control, and the mean value in the sham group was normalized to 1. *p < 0.05, **p < 0.01, compared with the sham group (Two-tailed Student’s t test). CB2, Type-2 cannabinoid receptors; DRG, dorsal root ganglion; SNL, spinal nerve ligation.

Fig 3: Nerve injury increases CB2 expression in DRG neurons.A, representative low- and high-magnification images of RNAscope in situ hybridization show colabeling of NeuN (green) and CB2 mRNA (red) in DRG tissue sections from sham and SNL rats 10 days after surgery. B–D, quantification of CB2 mRNA punctate signals in the DRG tissue section (B), the abundance of CB2 mRNA signals in DRG neurons (C), and amount of CB2 mRNA signals in small-, medium-, and large-diameter DRG neurons (D) in sham and SNL rats. Data are shown as means ± SEM (n = 9 images per group). *p < 0.05, ***p < 0.001 compared with the sham group (Two-tailed Student’s t test). CB2, Type-2 cannabinoid receptors; DRG, dorsal root ganglion; SNL, spinal nerve ligation.

Fig 4: Alterations in protein expression of ECS components in knee joints associated with the progression of MIA-induced OA rat model.Notes: Protein expression of CB1 and CB2 receptors (A, B), NAPE-PLD, FAAH enzymes (C, D), 15LOX2, COX2 enzymes (E, F) levels were determined by immunoblotting. Samples were collected before OA induction and 2, 7, 14, 21 and 28 days after MIA injection. The results are presented as fold of change (mean ± SEM) in comparison to the intact group (n=5). Representative bands from each group are presented below the charts. Statistical analysis was performed using one-way analysis of variance followed by Bonferroni post hoc test; values with p<0.05 were considered significant. *Denotes significant differences vs int (before OA induction) at p<0.05, **denotes significant differences vs int (before OA induction) at p<0.01, ***denotes significant differences vs int (before OA induction) at p<0.005, ****denotes significant differences vs int (before OA induction) at p<0.001, #denotes significant differences vs day 2 post-MIA injection at p<0.05, ##denotes significant differences vs day 2 post-MIA injection at p<0.01, ###denotes significant differences vs day 2 post-MIA injection at p<0.001, $denotes significant differences vs indicated bar at p<0.05, $$denotes significant differences vs indicated bar at p<0.01.Abbreviations: ECS, endocannabinoid system; MIA, monoiodoacetate; OA, osteoarthritis; FAAH, fatty acid amide hydrolase; NAPE-PLD, N-acyl phosphatidylethanolamine phospholipase D; SEM, standard error of the mean; int, intact animals.

Fig 5: Combined-acupoint rather than single-acupoint EA activated the endogenous cannabinoid (eCB) system. ELISA assay revealed that combined-acupoint EA but not single-acupoint EA increased the release of AEA (a) and 2-AG (b) after 6 days' EA treatment. ELISA assay demonstrated that the expression of CB1 (c) and CB2 (d) receptor was only upregulated in combined-acupoint EA-treated group. The western blot analysis confirmed combined-acupoint EA but not single-acupoint EA increased the expression of CB1 (e, g) and CB2 (f, h) in the spinal cord. Student's unpaired t-test. All data are mean ± SEM, *P < 0.05, **P < 0.01, **P < 0.01 vs. combined; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. combined; n.s.: not significant; N = 6 in each group. CCI: chronic constriction injury; EA: electroacupuncture; AEA: anandamide; 2-AG: 2-arachidonoyl glycerol; CB1: cannabinoid receptor 1; CB2: cannabinoid receptor 2.