Research ArticleExperimental Studies
Open Access

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

LONGJIE XU, LILI HOU, CHUN CAO and XIAOHUA LI
In Vivo May 2024, 38 (3) 1133-1142; DOI: https://doi.org/10.21873/invivo.13548

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.

Key Words:

Cancer-induced pain is a mixed type of pain that includes both neuropathic and inflammatory pain. Cancer-induced bone pain (CIBP) is the most common source of moderate to severe cancer pain, and approximately 75% of patients with advanced cancer experience CIBP (1). CIBP is derived from several cancers, including breast, prostate, and lung cancer, and is also an important cause of anxiety, depression, decreased quality of life and performance status (2). At present, the treatment methods for CIBP usually include surgery, radiotherapy, and drug therapy (3). However, only 50% of patients achieve temporary pain alleviation from conventional treatments (1). Moreover, radiotherapy could result in severe marrow depression, and drugs, such as bisphosphonates, could cause hypocalcemia, osteonecrosis of the jaw and acute kidney injury, which indicate the need for a new mechanism of CIBP (2, 4).

The arcuate nucleus of the hypothalamus, which contains neuropeptide Y (NPY)/agouti-related peptide neurons and pro-opiomelanocortin neurons, could regulate the external stress and injury (5). NPY is a 36-amino acid neuropeptide that is secreted by NPY neurons and can regulate multiple physiological processes, including appetite, body temperature and energy metabolism (6, 7). Recent studies have indicated that NPY plays an important role in the regulation of pain (8, 9). In the CIBP model, the intrathecal administration of NPY could efficiently alleviate pain, and selective inhibition of the Y1 receptor (Y1R) or Y2 receptor (Y2R) reversed the effect of NPY on pain (10). In addition, electrical stimulation inhibited was reported to inhibit chronic pain after exposure to alcohol by increasing the expression of hypothalamic NPY (9).

Ghrelin, as a growth hormone-releasing peptide, is mainly secreted by the stomach and transmits hunger signals to the hypothalamus to enhance appetite and maintain energy balance (11). Patients with advanced breast cancer often exhibit anorexia, cachexia, and depression. Serum ghrelin remains at low levels in patients with cachexia (12). Ghrelin could penetrate the blood-brain barrier and bind to growth hormone secretagogue receptor 1α (GHS-R1α) in the hypothalamus, which promotes the release of intracellular Ca2+ in arcuate nucleus neurons to activate the CaMKKβ pathway and phosphorylate AMPK (13). Ghrelin can also induce the production of hypothalamic NPY through the AMPK-mTOR pathway for the treatment of obesity (5). Thus, the administration of ghrelin to induce the production of hypothalamic NPY might be a new treatment for CIBP. In this regard, we investigated whether ghrelin could induce the production of hypothalamic NPY through the AMPK-mTOR pathway to alleviate CIBP.

Materials and Methods

Animals. Six-week-old Sprague-Dawley rats obtained from Soochow University (Suzhou, Jiangsu, PR China) were used in this study. All rats were maintained under specific pathogen-free conditions in which the temperature was 21°C-25°C and the humidity was 55%-65%. The experimental procedure was in accordance with the regulations of the Medical Ethics Committee for Animal Experimentation of Soochow University.

Cell culture and treatment. Rat breast cancer cells (SHZ-88 cells) were purchased from Procell Life Science & Technology Co., Ltd (Wuhan, PR China). The cells were cultured in medium consisting of RPMI-1640 (PM150110), 10% FBS (164210-50) and 1% P/S (PB180120) for 4-5 passages and used for modelling. The specific methods were as following: The medium in the Petri dishes overgrown with cells was aspirated and discarded. Then, 1 ml of Hank’s balanced salt solution (HBSS, Gibco14175-053, Shanghai, PR China) was added to wash out the remaining medium. Then, the cells were trypsinized and resuspended. After the cells were resuspended, 0.5 ml of medium was added to inactivate trypsin. The supernatant was then removed, the cells mixed with 1 ml HBSS and were counted using a cell counter. Finally, the cells were diluted to the target concentration and placed on ice until use.

Intracerebroventricular catheterization. Rats were anaesthetized with 2% pentobarbital sodium and fixed on a stereotactic frame. The anterior and posterior fontanels were sufficiently exposed and drilled through the skull with an electric drill at the coordinates (1.5,1.1, 0). A special 26-g stainless steel tube was placed in the above target position with a stereotactic instrument, and a small amount of cerebrospinal fluid could be seen after reaching the ventricle. The fresh premixed denture support resin was dripped into the rat skull to fix the tube. After the denture support resin was dried and formed, the skin was sutured. Rats then received an intragluteal injection of 40,000 UI of penicillin.

CIBP model. Rats were anaesthetized with 2% pentobarbital sodium, and the subcutaneous tissue and fascia were separated to expose the upper 1/3 of the right tibia. A hole was made in the upper 1/3 of the tibia with an electric drill, and 10 μl (3×103 cells) SHZ-88 cells were slowly injected into the bone marrow cavity of the tibia with a microsyringe. The needle of the microsyringe was left in the bone marrow cavity for 5 min to prevent the leakage of cells. Then, the hole was sealed with bone wax, and the incision was stitched. After surgery, rats were subcutaneously injected with 40,000 UI of penicillin.

Study protocol. Firstly, rats were divided into sham and CIBP groups (n=6 per group). The body weight, food intake, mechanical allodynia, mechanical hyperalgesia, limb use score and weight bearing ratio were measured on the day before the operation and on days 3, 6, 9, 12, 15, and 18 after the operation. An X-ray of the right tibia was taken on day 18 after the operation. The rats were then euthanized, and the hypothalamic arcuate nuclei and the tibia were extracted.

Secondly, all rats received i.c.v. catheterization and SHZ-88 cells to establish the CIBP model. Rats were randomly divided into CIBP, CIBP+NPY, CIBP+NPY+BIBO3304, CIBP+NPY+BIIE0246 and CIBP+NPY+BIBO3304+BIIE0246 groups (n=6 per group) on day 18 after the operation. The doses of reagents were 30 μg of NPY (Aladdin, Shanghai, PR China), 3 μg of BIBO3304 (MedChemExpress, Shanghai, PR China) and 3 μg of BIIE0246 (MedChemExpress). The reagents were dissolved in 10 μl dimethyl sulfoxide (DMSO) and injected into the lateral ventricle through the ventricle tube. Rats in the CIBP group were injected with 10 μl DMSO into the lateral ventricle. Mechanical allodynia, mechanical hyperalgesia, limb use score and weight bearing ratio were measured every half hour. The behavioral indicators were compared to investigate whether NPY exerted its antinociceptive effect through binding to Y1R or Y2R.

Thirdly, all rats received i.c.v. catheterization and were divided into sham, CIBP, CIBP+ghrelin and CIBP+ghrelin+[D-Lys3]-GHRP-6 (D-Lys) groups (n=6 per group). Rats in the CIBP+ghrelin and CIBP+ghrelin+D-Lys groups were intraperitoneally injected with 0.33 mg/kg/d ghrelin (BOC Science, New York, NY, USA). Rats in the CIBP+ghrelin+D-Lys group were injected with 3 μg/10 μl/d D-Lys (GLPBIO) into the lateral ventricle through the ventricle tube. Behavioral indicators were measured every three days until day 18 after the operation. The rats were then euthanized, and the hypothalamic arcuate nuclei and the tibia were extracted.

Mechanical allodynia. Mechanical allodynia was measured using von Frey filaments. Rats were acclimated to the environment by placing them in a plexiglass cage with a metal sieve plate at the bottom. The plantar surface of the right hind paw was stimulated vertically using a Von Frey filament so that the filaments were bent to the point where rats had a paw withdrawal response. Each intensity was stimulated five times and held for approximately 6-8 s. When more than three paw withdrawal responses occurred in five trials, the minimum intensity of the stimulation was recorded as the paw withdrawal threshold (PWT). The strongest intensity was 26 g, and the PWT was recorded as 26 g if the strongest intensity still did not cause the response.

Mechanical hyperalgesia. Mechanical hyperalgesia was measured using a hot plate experiment. Rats were placed in a hot-plate apparatus, and the temperature of the hot plate was then increased to 52.5°C. The paw withdrawal latency (PWL) was the time from the onset of heat radiation to the occurrence of nociceptive responses. The assay was repeated three times, and the PWL was the average of three replicates.

Limb use score. Rats walked freely in clear plastic cages and were observed separately for 3 min. The score of gait was measured as following: 3=normal use; 2=mild or insignificant lameness with normal weight distribution; 1=significant lameness with a shift in weight distribution to the healthy limb. 0=partial or no use (14).

Weight bearing ratio. The weight bearing ratio was measured using a Dual Channel Weight Averager machine. Rats were placed in a plexiglass chamber, and hind paws were placed on transducer pads. The two numbers that appeared represented the distribution of the rat’s body weight on the left and right paws. The results were expressed as the ratio of the bearing weight of the right paw to both paws.

General observation. In addition to the behavioral tests described above, changes in body weight and food intake were also recorded.

Western blotting. The hypothalamus homogenate was centrifuged at 4°C and 12,000 rpm for 15 min, and the supernatant was collected and stored at −20°C. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) was performed for protein electrophoresis. The wet transfer method was used to transfer proteins onto PVDF membranes after electrophoresis. After sealing in serum at room temperature for 2 h, membranes were incubated with primary antibodies against ghrelin (#PA1-1070, Thermo Fisher Scientific, Waltham, MA, USA), NPY (sc-133080, Santa, Shanghai, PR China), mTOR (#2983, CST, Boston, USA), p-mTOR (#5536, CST), AMPK (#2603, CST), p-AMPK (#2537, CST), CaMKKβ (#16810, CST), and β-Actin (#4967, CST) at 4°C overnight. The secondary antibody (R-21459, Thermo Fisher Scientific) was then added after washing with TBST.

Hematoxylin-eosin staining. The tibia was fixed with 4% polyformaldehyde overnight and then placed in a decalcifying agent. The tibia was then embedded in paraffin and cut into slices. The slices were deparaffinized and dehydrated with xylene and gradient ethanol, respectively. A hematoxylin-eosin (HE) staining kit (Saint-bio, Shanghai, PR China) was used to stain the slices. Changes in the tibia were observed under a light microscope.

Immunofluorescence. Brain sections were incubated with anti-ghrelin (#PA1-1070, Thermo Fisher Scientific) and NPY (sc-133080, Santa) overnight, followed by incubation with Alexa Fluor 555- and 488- conjugated anti-goat antibody (A30677 and A-11094, Thermo Fisher Scientific) at room temperature for 1 hour. Finally, the sections were observed using a fluorescence microscope.

X-ray. Rats were placed in a supine position after anesthesia, and the right tibia was scanned by X-ray (Senographe DS, GE, San Francisco, CA, USA) to record bone destruction after CIBP.

Statistical analysis. Data analysis was performed using GraphPad Prism 5 (San Diego, CA, USA). Data are presented as the mean±standard error of the mean (SEM). Independent sample t-tests were used for comparisons between two groups, and one-way analysis of variance followed by Tukey’s test was used for comparisons among multiple groups. A p-value of less than 0.05 was considered statistically significant.

Results

In the first study, body weight, food intake and behavioral indicators were measured before the operation and on days 0, 3, 6, 9, 12, 15 and 18 after the operation. We found that body weight and food intake showed no differences between the sham and CIBP groups (Figure 1B, C). However, compared with the sham group, a significant decrease in PWT, PWL and weight bearing ratio was observed in the CIBP group from day 9 after the operation (Figure 1D-G). The limb use score in the CIBP group was significantly decreased from day 12 after the operation (Figure 1F). Meanwhile, X-ray showed bone destruction in the right tibia of cancer-bearing rats, and H&E staining showed that tumor cells occupied the entire field of vision accompanied by the absence of normal bone cells on day 18 after the operation (Figure 1H, I). The expression of hypothalamic ghrelin and NPY was significantly decreased in the CIBP group compared with the sham group (Figure 2A-C). We also examined the expression of the AMPK-mTOR pathway and found that the expression of CaMKKβ and p-AMPK was significantly decreased and that the expression of p-mTOR was increased in the CIBP group (Figure 2D, E).

Figure 1.
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.
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 p-mTOR/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.

Then, we investigated whether hypothalamic NPY exerted an antinociceptive effect through binding to NPY receptors. We found that i.c.v. administration of NPY significantly increased PWT at 90 min and increased PWL, limb use score and weight bearing ratio at 120 min, compared with the CIBP group. However, the antinociceptive effect of NPY was reversed with the i.c.v. administration of the Y1R antagonist BIBO3304 and the Y2R antagonist BIIE0246 (Figure 3).

Figure 3.
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.

Finally, we investigated whether exogenous ghrelin could alleviate CIBP by inducing the production of hypothalamic NPY through the AMPK-mTOR pathway. The body weight and food intake still had no significant differences with the administration of ghrelin (Figure 4B, C). We found that the administration of ghrelin could sharply improve PWT and PWL from day 9 after the operation (Figure 4D, E), limb use score from day 12 after the operation (Figure 4F) and weight bearing ratio from day 6 after the operation (Figure 4G) compared with the CIBP group. However, the antinociceptive effect of ghrelin was reversed with the administration of the GHS-R1α antagonist D-Lys (Figure 4). The administration of ghrelin increased the expression of hypothalamic NPY. Meanwhile, the administration of ghrelin increased the expression of CaMKKβ and p-AMPK and decreased the expression of p-mTOR. However, the expression of CaMKKβ, p-AMPK and NPY was decreased, and the expression of p-mTOR was increased with the administration of D-Lys (Figure 5).

Figure 4.
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.
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 p-mTOR/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.

Discussion

CIBP is a persistent and unbearable pain that is usually derived from breast, prostate, and lung cancers (2). Up to 65% of bone metastases come from breast cancer and lead to persistent and unbearable pain, hypercalcemia, pathological fracture, spinal cord or other nerve compression and increased mortality (15). Moreover, CIBP commonly presents around bone lesions and radiates to other sites, such as the spine, pelvis, and ribs (16-17). Nonopioids as well as weak and strong opioids are often used to alleviate varying degrees of pain (18). However, side effects and addiction restrict the volume of drugs, which finally causes insufficient efficacy (1, 18). In the current study, we used breast cancer cells to establish a CIBP model and found that the administration of ghrelin could induce the production of hypothalamic NPY through the AMPK-mTOR pathway to alleviate CIBP in cancer-bearing rats.

Hypothalamic NPY is involved in the regulation of energy homeostasis, food intake, adiposity, blood pressure, neuronal excitability, and memory (6, 19-20). Recently, the antinociception of NPY was investigated broadly. The effect of NPY on antinociception was achieved through inhibiting the release of substance P and other pain neurotransmitters in the spinal dorsal horn (21). Inflammatory pain could be alleviated by inducing calcium conduction and transmitter release to activate NPY neurons (22). The administration of exogeneous NPY alleviated cold and mechanical allodynia, and conditional knockout of NPY restored chronic pain (23). Meanwhile, the activation of NPY-interneurons could also reduce the behavior of acute pain (24). In the current study, the expression of hypothalamic NPY was significantly decreased in cancer-bearing rats, which was accompanied by CIBP. The i.c.v. administration of NPY could significantly alleviate CIBP in the short term. However, the relationship between NPY and CIBP needs to be further elucidated. A recent study indicated that NPY exerts biological and pathophysiological functions by activating several G protein-coupled receptors, which mainly include Y1R, Y2R, Y4R, Y5R and Y6R (20). The antinociceptive effect of NPY involves the inhibition of the transmission of nociceptive signals via Y1R and the inhibition of the release of excitatory neurotransmitters from the primary afferents via Y2R (10). The intrathecal injection of a Y1R selective agonist alleviated chronic pain via the hyperpolarization of Y1R-expressing excitatory interneurons (25). In a model of glyceryl trinitrate-induced migraine, NPY alleviated pain by binding to Y1R (26). Ablation of the Y1R or the Y1R antagonist showed an enhanced response to pain sensitization in chronic pain (21, 27-28). Y2R is closely associated with acute and neuropathic pain (29). Pharmacological inhibition of Y2R caused mechanical hyperalgesia, which revealed a unique role of Y2R in modulating mechanical pain (30). Therefore, we hypothesized that the i.c.v. administration of NPY might exert an antinociceptive effect through binding to Y1R and Y2R in the hypothalamus. Our study showed that the antinociceptive effect of NPY was significantly reversed with the i.c.v. administration of the Y1R antagonist BIBO3304 and the Y2R antagonist BIIE0246. The results strongly suggested that hypothalamic NPY exerted an antinociceptive effect through binding to Y1R and Y2R.

Ghrelin is a growth hormone-releasing peptide that is mainly produced in the stomach and plays a pivotal role in the regulation of appetite, body energy balance, lipid metabolism and glucose metabolism (31-33). The administration of exogenous ghrelin could also cause a positive effect on pain relief. In the formalin-induced pain model, ghrelin alleviated inflammatory pain by upregulating the levels of IL-10 and TGF-β (34). Ghrelin also alleviated neuropathic pain through the p38 MAPK/NF-κB pathway and acute pain through the activation of the opioid peptide PENK and opioid receptor OPRD (35-37). In the current study, the expression of hypothalamic ghrelin and NPY was simultaneously decreased in the CIBP group. In our previous study, ghrelin crossed the blood-brain barrier and bound to its receptor GHS-R1α in the hypothalamus, causing arcuate nucleus neurons to release intracellular Ca2+, activate CaMKKβ and promote the phosphorylation of AMPK (13, 38). mTOR activity was associated with pain, and inhibiting hypothalamic mTOR activity by using glucocorticoids upregulated the expression of NPY (29, 39-40). The phosphorylation of AMPK could downregulate mTOR activity and subsequently increase the expression of hypothalamic NPY (5, 41). In addition, inhibition of ghrelin o-acyltransferase can induce autophagy via the AMPK– mTOR pathway (42). Our current study found that the expression of p-AMPK was decreased and that of p-mTOR was increased in the CIBP group. Therefore, we hypothesized that cancer enhanced bone pain by inhibiting the expression of ghrelin to downregulate the expression of hypothalamic NPY through the AMPK-mTOR pathway. We found that the administration of ghrelin activated the AMPK-mTOR pathway and induced hypothalamic NPY production to alleviate CIBP in cancer-bearing rats. Moreover, this effect of ghrelin on NPY and antinociception was reversed with the administration of the GHS-R1α antagonist D-Lys.

Conclusion

CIBP has been a prevalent complication in patients with bone metastases from cancer for which there is a lack of effective treatments. Our study demonstrated that NPY could alleviate CIBP through binding to Y1R and Y2R in the hypothalamus. Meanwhile, the administration of ghrelin could induce the production of hypothalamic NPY through the AMPK-mTOR pathway to alleviate CIBP in cancer-bearing rats, which provides a novel therapeutic mechanism for CIBP.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (82202369) and the Suzhou Municipal Science and Technology Bureau (SKJY2021020 and SKYD2023073).

Footnotes

  • Authors’ Contributions

    Chun Cao and Xiaohua Li designed the study and analysed the data. Longjie Xu and Lili Hou performed the experiments and analysed the data. Longjie Xu and Xiaohua Li wrote the manuscript. All Authors revised the manuscript.

  • Conflicts of Interest

    The Authors report no potential conflicts of interest.

  • Received September 14, 2023.
  • Revision received November 10, 2023.
  • Accepted November 15, 2023.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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Ghrelin Induces the Production of Hypothalamic NPY Through the AMPK-mTOR Pathway to Alleviate Cancer-induced Bone Pain
LONGJIE XU, LILI HOU, CHUN CAO, XIAOHUA LI
In Vivo May 2024, 38 (3) 1133-1142; DOI: 10.21873/invivo.13548
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