Figure 7. Treatment with isoprenaline abolished the anti-tumor effect of celastrol.

(A) Schematics of in vivo single-unit recordings in celiac-superior mesenteric ganglion (CG-SMG). L-368,899, the Oxt receptor (OTR) antagonist (OTR anta), and celastrol were applied through a guide cannula directed to third ventricle (3V). (B) A CG-SMG image was taken during the operation. (C) Example waveform of the single unit detected. (D) Normalized firing rate of recorded CG-SMG neurons in response to celastrol infusion in vehicle (top) and OTR antagonist (bottom) groups. Dashed line indicates the time point of celastrol delivery. i.c.v., intracerebroventricular injection. n = 68 cells (vehicle) from 7 mice or 44 cells (OTR antagonist) from 6 mice. (E) Statistics of average firing frequency of CG-SMG neurons in response to celastrol infusion in vehicle and OTR antagonist groups. Solid and dotted lines indicate medians and quartiles, respectively. n = 68 cells (vehicle) from 7 mice or 44 cells (OTR antagonist) from 6 mice. (F and G) Correlation of firing rate before and after celastrol infusion in vehicle (F) and OTR antagonist (G) groups. Green filled circles represent individual units with significantly lower firing frequency after celastrol infusion. Red triangles represent the units with higher firing rates. Gray squares indicate neurons without significant difference in firing rates. Inset: proportions of CG-SMG neurons with significantly decreased rates, increased rates, or no change in rates after celastrol infusion in vehicle (F) and OTR antagonist group (G). n = 68 cells (vehicle) from 7 mice or 44 cells (OTR antagonist) from 6 mice. (H and I) Colitis-associated cancer (CAC) was induced in male C57 BL/6 mice (2 months of age). These mice were then i.c.v. administered with vehicle (control) or celastrol every other day. In the meantime, the mice were i.p. injected with saline or isoprenaline (ISO) on a daily basis. These treatments were continued for 3 weeks (see also Figure 7—figure supplement 2A). Tumor number (H) and diameter (I) are shown. ns, not significant. n = 8 (saline) or 7 (ISO) mice per group. (J) The density of proliferating cell nuclear antigen (PCNA)-positive cells in tumor tissue. ns, not significant. n = 4 mice per group. (K) The density of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells in tumor tissue. ns, not significant. n = 4 mice per group. Data are presented as means ± SD (C) or means ± SEM (H–K). *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA with Bonferroni’s post hoc test (E, H–K).

Figure 7—figure supplement 1. The preganglionic nerve fiber is crucial for brain administered celastrol to suppress neuronal activities in celiac-superior mesenteric ganglion (CG-SMG).

(A) A schematic diagram of the experimental design. Male C57 BL/6 mice (2 months of age) were implanted with a guide cannula directed to the third ventricle. After 2 weeks of recovery, the preganglionic nerve fiber of CG-SMG was transected. The other groups of mice were administered with sham operations. Subsequently, these mice were i.c.v. administered with vehicle (control) or celastrol. Two hours later, CG-SMG was dissected and fixed in 4% paraformaldehyde (PFA). (B) Double immunofluorescence staining for c-Fos (red) and tyrosine hydroxylase (TH, in green) of the CG-SMG. Cell nuclei were counterstained with DAPI (blue). Scale bars, 50 μm. (C) The percentage of TH-positive cells expressing c-Fos in the CG-SMG. ns, not significant. n = 5 mice per group. Data are presented as means ± SEM. *p < 0.05, one-way ANOVA with Bonferroni’s post hoc test (C).

Figure 7—figure supplement 2. Activation of β2 adrenergic receptor abolishes the tumor suppression effect of centrally administered celastrol.

(A) A schematic diagram of the experimental design. The colitis-associated cancer was induced in the male C57 BL/6 mice (2 months of age) using azoxymethane (AOM) and dextran sodium sulfate (DSS). Subsequently, these mice were i.c.v. administered with vehicle (control) or celastrol (Cel) every other day. In the meanwhile, these mice were i.p. injected with saline or isoprenaline (ISO), a β2 adrenergic receptor agonist, on a daily basis. These treatments were continued for 3 weeks. (B and C) Body weight (B) and food intake (C) in mice throughout the experiment. n = 8 (saline) or 7 (ISO) mice per group. (D) Representative images of colorectal tissue. White eclipses outline the individual tumor. (E) Colorectal length. n = 8 (saline) or 7 (ISO) mice per group. (F) Immunohistochemical staining for proliferating cell nuclear antigen (PCNA) of tumor tissues. Scale bars, 50 μm. (G) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay of tumor tissues. TUNEL labeling is shown in red. Cell nuclei were counterstained with DAPI (blue). Scale bars, 20 μm. Data are presented as means ± SEM.