Fig 1: (A) Representative images of GFAP immunofluorescence, a marker of reactive gliosis, in the ipsilateral and contralateral dorsal horn of the spinal cord. Images are from rats that received intra-articular injection of saline or MIA, and treatment with vehicle or the CB2 receptor agonist JWH133 (1 mg/kg, days 1–28) (scale bar = 5 µm). (B) Quantification of ipsilateral GFAP immunofluorescence expressed as a % of the immunofluorescence for the contralateral spinal cord for the three treatment groups (n = 6–7 sections per rat, n = 4 rats per treatment). Systemic administration of JWH-133 significantly attenuated increases in GFAP immunofluorescence, compared to the effects of vehicle, statistical analysis was conducted via a one-Way ANOVA with a Bonferroni post-hoc test,*p<0.05. (C) Example gel zymography of MMP-9 and MMP-2 activity in the spinal cord from the various treatment conditions, as well as positive controls for purified MMP 2 and MMP 9. (D) Quantification of MMP-9 and pro and active MMP-2 activity in the ipsilateral spinal cord in MIA-treated rats and saline-treated rats. Systemic administration of JWH-133 significantly attenuated increases in MMP-9, MMP-2 and active MMP-2 activity in the spinal cord in MIA-treated rats, compared to the effects of vehicle. Data are expressed as mean densitometry ± SEM (n = 4 rats per group), statistical analysis one-way ANOVA and Bonferroni post-hoc test,**p<0.01, ***p<0.001 vs. saline/Vehicle; #p<0.05, ###p<0.001 vs. MIA/Vehicle.
Fig 2: Catalytic efficiency of MMP-2 on fluorogenic probes and assessment of the ABN toxicity. (a) Schematic showing the catalytic efficiency of MMP-2 on TIAH–LH, immobilized on the surface of microplate wells and incubated with the 50 nM catalytic domain for 2 h at 37 °C. Initial cleavage velocity of the substrate conjugated onto the nanocarrier surface at (b) 32.5 Å and (c) 53.4 Å from the core, highlighting the catalytic efficiency. The lines represent the Michaelis–Menten model fit to the data. mean ± s.e.m. is denoted by a horizontal line with error bars; n = 2 wells per probe concentration; N = 3; R 2 = 0.87; R 2 = 0.99. K cat, catalytic constant; K M, Michaelis–Menten constant. (d–g) Blood levels of hepatotoxicity markers of the mice injected with the ABN were equal to the levels found in the controls. The mean body weight remained stable for 5 d after ABN injection. Each square represents one mouse, and the mean value is denoted by the red bar. The Shapiro–Wilk test is followed by the unpaired two-tailed t-test; *P > 0.05. ALT, alanine aminotransferase; AST, aspartate aminotransferase. (h, i) Viability of 4T1 and ID8 cells after incubation with the ABN at different concentrations for 24 h. mean ± s.e.m. is denoted by a horizontal line with error bars; n = 2–3 wells per condition; N = 3; one-way ANOVA with Bonferroni multiple comparisons; **P < 0.05. Ctrl, positive control: acetaminophen at 25 mM in cell culture media.
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