Ghrelin Induces the Production of Hypothalamic NPY Through the AMPK-mTOR Pathway to Alleviate Cancer-induced Bone Pain

Abstract

Background/aim: Cancer-induced bone pain (CIBP) is one of the most common symptoms of bone metastasis of tumor cells. The hypothalamus may play a pivotal role in the regulation of CIBP. However, little is known about the exact mechanisms.

Materials and methods: First, we established a CIBP model to explore the relationship among hypothalamic ghrelin, NPY and CIBP. Then, we exogenously administered NPY and NPY receptor antagonists to investigate whether hypothalamic NPY exerted an antinociceptive effect through binding to NPY receptors. Finally, we exogenously administered ghrelin to investigate whether ghrelin alleviated CIBP by inducing the production of hypothalamic NPY through the AMPK-mTOR pathway. Body weight, food intake and behavioral indicators of CIBP were measured every 3 days. Hypothalamic ghrelin, NPY and the AMPK-mTOR pathway were also measured.

Results: The expression of hypothalamic ghrelin and NPY was simultaneously decreased in cancer-bearing rats, which was accompanied by CIBP. Intracerebroventricular (i.c.v.) administration of NPY significantly alleviated CIBP in the short term. The antinociceptive effect of NPY was reversed with the i.c.v. administration of the Y1R and Y2R antagonists. The administration of ghrelin activated the AMPK-mTOR pathway and induced hypothalamic NPY production to alleviate CIBP. This effect of ghrelin on NPY and antinociception was reversed with the administration of a GHS-R1α antagonist.

Conclusion: Ghrelin could induce the production of hypothalamic NPY through the AMPK-mTOR pathway to alleviate CIBP, which can provide a novel therapeutic mechanism for CIBP.

Keywords: AMPK-mTOR pathway; Cancer-induced bone pain; Ghrelin; NPY; SHZ-88.

Figure 1. Effects of SHZ-88 cells on the percentage of change in body weight (BW), daily food intake, behavioral tests, and bone destruction. Schematic diagram of the experimental procedures (A). The percentage of change in BW (B). Daily food intake (C). PWT was measured using von Frey filaments (D). PWL was measured using a hot plate experiment (E). Limb use score (F). The weight bearing ratio was measured using a dual channel weight averager machine (G). Bone destruction was measured by X-ray (H) and hematoxylin & eosin staining (I). Scale bars, 200 μm. The data are presented as the mean±standard error of the mean (SEM). The independent sample t-test was used for comparisons between the sham and CIBP groups. *p-Value <0.05. CIBP, Cancer-induced bone pain; PWT, paw withdrawal threshold; PWL, paw withdrawal latency.

Figure 2. Effects of SHZ-88 cells on the expression of hypothalamic ghrelin, NPY and the AMPK-mTOR pathway. The expression of hypothalamic ghrelin and NPY was measured by immunofluorescence (A) and western blotting (B, C). The expression of CaMKKβ, p-AMPK/AMPK and pmTOR/mTOR was measured by western blotting (D-E). β-Actin was used as control. Scale bars, 400 μm. The data are presented as the mean±standard error of the mean (SEM). The independent sample t-test was used for comparisons between the sham and CIBP groups. *p-Value <0.05. CIBP, Cancer-induced bone pain.

Figure 3. Hypothalamic NPY exerted an antinociceptive effect by binding to Y1R and Y2R in cancer-bearing rats. PWT was measured using von Frey filaments (A). PWL was measured using a hot plate experiment (B). Limb use score (C). The weight bearing ratio was measured using a dual channel weight averager machine (D). The data are presented as the mean±standard error of the mean (SEM). One-way analysis of variance followed by Tukey’s test was used for comparisons among multiple groups. *p-Value <0.05. CIBP, Cancer-induced bone pain; PWT, paw withdrawal threshold; PWL, paw withdrawal latency.

Figure 4. Effects of exogenous ghrelin on the percentage of change in body weight, daily food intake and behavior tests in cancer-bearing rats. Schematic diagram of the experimental procedures (A). The percentage of change in BW (B). Daily food intake (C). PWT was measured using von Frey filaments (D). PWL was measured using a hot plate experiment (E). Limb use score (F). The weight bearing ratio was measured using a dual channel weight averager machine (G). The data are presented as the mean±standard error of the mean (SEM). One-way analysis of variance followed by Tukey’s test was used for comparisons among multiple groups. *p-Value <0.05 vs. the sham group. ^#p-Value <0.05 vs. the CIBP group. ^p-Value <0.05 vs. the CIBP+ghrelin group. CIBP, Cancer-induced bone pain; i.c.v., intracerebroventricular; BL, base line; D-lys, [D-Lys3]-GHRP-6; BW, body weight; PWT, paw withdrawal threshold; PWL, paw withdrawal latency.

Figure 5. Effects of exogenous ghrelin on the expression of hypothalamic NPY and the AMPK-mTOR pathway. The expression of hypothalamic ghrelin and NPY was measured by immunofluorescence (A) and western blotting (B, C). The expression of CaMKKβ, p-AMPK/AMPK and pmTOR/mTOR was measured by western blotting (D, E). β-Actin was used as control. Scale bars, 400 μm. The data are presented as the mean±standard error of the mean (SEM). One-way analysis of variance followed by Tukey’s test was used for comparisons among multiple groups. *p-Value <0.05. CIBP, Cancer-induced bone pain.