PDF
Citation
Print
INTRODUCTION
Breast cancer, the fifth most common malignant tumor overall, has a high tendency to metastasize to the bone, which causes bone pain, hypercalcemia, pathological fractures, compression of the spinal cord or other nerves and reduced mobility [1,2]. Cancer-induced bone pain (CIBP) includes both inflammatory and neuropathic pain, which is common in advanced cancers and can significantly impact patient quality of life [3,4]. A variety of drugs, including nonsteroidal anti-inflammatory drugs (NSAIDs), opioids, antidepressants, anticonvulsants, bisphosphonates and denosumab, have been used for the treatment of CIBP [5]. However, patients with advanced cancer usually suffer from anorexia and cachexia. The oral administration of NSAIDs for CIBP could further stress the burden on the gastric mucosa and lead to ulcerative bleeding and indigestion, which further aggravate the physical condition of patients [6]. Moreover, the addictive nature of opioids constrains the dosage of drugs, thereby reducing their therapeutic efficacy [7]. Thus, alleviating CIBP is still a serious challenge.
Ghrelin, which is produced primarily by the stomach, can stimulate appetite and regulate energy balance and glucose metabolism [8,9]. Ghrelin can inhibit the inflammatory response through binding to the growth hormone secretagogue receptor (GHS-R1α) in the hypothalamus, thereby alleviating inflammatory pain [10,11]. Ghrelin can also alleviate acute pain by upregulating the expression of the endogenous δ-opioid peptide proenkephalin [12]. NPY is found mainly in the mammalian central and peripheral nervous systems and is involved in regulating appetite regulation and energy balance [13–15]. In addition, NPY can hyperpolarize Y1 receptor-expressing excitatory interneurons to inhibit peripheral neuropathic pain, and exogenous administration of NPY can alleviate nociceptive behaviors in rats, which can be reversed by selectively inhibiting NPY Y1 or Y2 receptors [16,17]. Electrical stimulation significantly alleviated alcohol-induced chronic pain via the activation of hypothalamic NPY [18].
Octanoic acid (OA), a member of the medium-chain fatty acids (MCFAs), plays a role in controlling energy balance by regulating hypothalamic pro-opiomelanocortin neuronal activity [19]. A recent study indicated that a lower level of OA is associated with a greater risk of breast cancer [20]. OA has the potential to treat glioblastoma through the stimulation of apoptosis [21]. Moreover, OA also has anticancer effects on human colorectal, skin and breast cancer [22]. A clinical study demonstrated that OA-rich enteral nutrition for cachectic patients for two weeks could significantly increase serum levels of acyl-ghrelin, thus improving patient nutrition and mental condition [23]. However, whether an OA-rich diet can alleviate CIBP via the acyl-ghrelin/NPY pathway remains to be elucidated.
Therefore, the authors initially examined the effect of diets with different ratios of OA on CIBP. Next, they validated whether an OA-rich diet could induce the expression of hypothalamic NPY through the upregulation of acyl-ghrelin to alleviate CIBP in cancer-bearing rats.
MATERIALS AND METHODS
1. Animals
This study employed a total of sixty Sprague–Dawley rats aged six weeks at the time of the experiment and obtained from the laboratory at Soochow University (Suzhou, Jiangsu, PR China). All the rats were housed under specific pathogen-free conditions and a standard 12 hr day/night cycle with an ambient temperature of 21°C –25°C and a humidity of 55%–65%. Food and water were provided ad libitum. Subsequently, the authors generated a randomization sequence and numbered the rats one by one, then grouped the rats according to the randomization sequence. The procedures were conducted in accordance with the ethical standards of the Soochow University Medical Ethics Committee for Animal Experiments (JC-2024-01-0013).
2. Breast cancer cell culture
The rat breast cancer cell line (SHZ-88 cells) was purchased from Procell Life Science & Technology Co., Ltd. The SHZ-88 cells were cultured in DME media supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution in a humidified atmosphere containing 5% CO2 at 37°C.
3. CIBP modeling
The rats were anesthetized via an intraperitoneal injection of pentobarbital sodium and placed on the operating table. Then, the right tibia was shaved and disinfected, and the subcutaneous tissue and periosteum were dissected carefully. A hole was subsequently drilled in the upper 1/3 of the tibia, and 3*103/10 μL of SHZ-88 cells was slowly injected into the marrow cavity via a microsyringe. In addition, the rats in the sham group received 10 μL of phosphate buffered saline instead. Finally, the microsyringe was withdrawn gently, the hole was closed with bone wax, and the skin was sutured. All the rats were injected subcutaneously with 40,000 UI penicillin to prevent infection after surgery. During the study, rats did not develop adverse events, such as skin or bone infections and death.
4. Study protocol
In the first part of the study, thirty-six Sprague–Dawley rats were randomly divided into the following groups: the sham, CIBP, CIBP + OA (20), CIBP + OA (40), CIBP + OA (60) and CIBP + OA (80) groups (n = 6 per group). The rats in the Sham and CIBP groups received a standard chow diet, and the rats in the CIBP + OA (20), CIBP + OA (40), CIBP + OA (60) and CIBP + OA (80) groups received an OA diet (20, 40, 60, and 80, respectively) for 18 days. The diet composition is detailed in Table 1. Body weight, food intake, and nociceptive behaviors, including mechanical allodynia, mechanical hyperalgesia, limb use score, and weight bearing ratio, were measured at baseline and 3, 6, 9, 12, 15, and 18 days after surgery. X-ray imaging was performed on day 18 after surgery. All the rats were then euthanized for collection of serum, right tibias and hypothalamic arcuate nuclei.
In the second part, twenty-four rats were randomly divided into the sham, CIBP, CIBP + OA, and CIBP + OA + Go-Coa-Tat groups (n = 6 per group). The rats in the sham and CIBP groups received a standard chow diet, while the rats in the CIBP + OA and CIBP + OA + Go-Coa-Tat groups received the OA diet (60). In addition, rats in the CIBP + OA + Go-Coa-Tat group were intraperitoneally injected with 96 µL/kg ghrelin O-acyltransferase (GOAT) inhibitor, Go-Coa-Tat (Phoenix Biotech) after surgery, and those in the other groups were injected with the same amount of saline instead. All the rats were subsequently euthanized, and the serum, right tibias, and hypothalamic arcuate nuclei were extracted.
Power analysis was calculated to assess the sample size using GPower 3.1.9.7 according to the author’s previous study’s nociceptive behaviors [24]. It was found that the effect size was 2.305, the significance level was 0.05, the actual power was 0.889 using the paw withdrawal threshold (PWT) as the effect parameter; the effect size was 5.182, the significance level was 0.05, the actual power was 0.995 using the paw withdrawal latency (PWL) as the effect parameter; the effect size was 3.223, the significance level was 0.05, the actual power was 0.834 using limb use score as the effect parameter; the effect size was 8.001, the significance level was 0.05, and the actual power was 0.998 using weight-bearing ratio as the effect parameter, which provided a valid basis for the chosen sample size.
5. Mechanical allodynia
Von Frey filaments were used to measure mechanical allodynia, which was recorded as the PWT. The procedure was as follows: first, the rats to be measured were placed in a transparent plexiglass cage on a metal sieve plate for a period of 5 minutes to acclimate them to the environment. Each von Frey filament was subsequently applied vertically to the plantar surface of the modeling side (right) and contralateral side (left) paws, and the intensity of the filament was increased sequentially until the rat withdrew its paw [25]. The trials were repeated five times for each filament, and a minimum of 2 minutes was allowed between each trial. Finally, the minimum intensity that caused the rat to withdraw its paw more than two times in five trials was defined as the PWT. If the maximum intensity (26 g) did not result in paw withdrawal, the value of 26 g was recorded as the PWT.
6. Mechanical hyperalgesia
A hot plate apparatus was used to measure mechanical hyperalgesia. The temperature of the apparatus was preset at 52.5°C. The rats were subsequently placed on the apparatus and the stopwatch or timer was started immediately. The rat was then observed until it exhibited a nociceptive response, such as licking or lifting its paw. The latency from placing the rat on the hot plate to the onset of paw withdrawal was recorded. Three trials were performed for the modeling side and contralateral side paws of each rat, and the average of the three trials was recorded as the PWL.
7. Limb use score
The rats were transferred to transparent plastic cages and were allowed to acclimate themselves to the environment for 5 minutes. The gait of the right limb was subsequently observed for 3 minutes. The gait scale from 3 to 0 was as follows: 3 = normal use of the right limb; 2 = slight lameness but normal body distribution in both limbs; and 1 = significant lameness and less body distribution to the right limb. Zero indicated little use of the right limb, defined as elevation of the right limb during locomotion and sitting.
8. Weight-bearing ratio
The weight-bearing ability of the rats was quantified via a dual-channel weight-averaging apparatus. Briefly, the rats were placed in a plexiglass chamber, and transducer pads were fixed to both hind paws. The rats were subsequently allowed to adapt to the new environment for 3–5 minutes. After the rats stabilized, the data derived from both pads were recorded. The weight bearing ratio was defined as the ratio of the weight bearing on the right paw to that on both paws.
9. X-ray
The rats were anesthetized and placed in the supine position on the operating plate on day 18 of modeling. The right paws of the rats were subsequently examined via X-ray (Senographe DS; GE), and the degree of bone destruction was recorded.
10. Hematoxylin-eosin (H&E) staining
After fixation in 4% paraformaldehyde for 12 hours, the tibias were immersed in decalcification agent. The decalcified tibias were subsequently dehydrated, embedded in paraffin and sliced. The slices were subsequently dewaxed with xylene and ethanol. Finally, the tibias were stained via a H&E staining kit (Saint-bio).
11. Measurements of ghrelin in the hypothalamus and serum
Total RNA was extracted from the hypothalami of the rats via TRIzol reagent. Following the isolation of total RNA, a reverse transcription reaction was performed to obtain cDNA. Real-time polymerase chain reaction (PCR) was performed on a PCR instrument via a PCR kit. A total of 40 cycles were set, with the respective primer sequences presented in Table 2.
The serum total ghrelin and acyl-ghrelin concentrations were quantified via a total ghrelin enzyme-linked immunosorbent assay (ELISA) kit and an acyl-ghrelin ELISA kit, respectively.
12. Measurements of inflammatory cytokines in the serum and bone marrow
The extracted tibias were cut into epiphyses and diaphysis and crushed into small fragments. Bone marrow was scraped from the fragments with a micro-scraper and resuspended in serum-free RPMI 1640 to obtain a suspension of bone marrow cells. The suspension was centrifuged at 1,500 rpm for 20 minutes to harvest supernatant. The levels of serum and bone marrow interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α were quantified via corresponding ELISA kits.
13. Western blotting
The extracted hypothalamus was mixed with RIPA lysis buffer, added to a centrifuge tube, and pulverized completely. After centrifugation at 12,000 rpm for 15 minutes at 4°C, the supernatant was collected, added to loading buffer and placed in a metal bath for 10 minutes before use. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used for protein electrophoresis. The proteins were subsequently transferred to a PVDF membrane and sealed with serum for 2 hours at room temperature. Primary antibodies were added, and the membranes were incubated overnight at 4°C. Then, the membranes were washed three times in TBST and incubated with a secondary antibody (R-21459, Thermo Fisher Scientific) for 1 hour. The following primary antibodies were used: ghrelin (#PA1-1070, Thermo Fisher Scientific), NPY (sc-133080, Santa), mTOR (#2983, CST), p-mTOR (#5536, CST), AMPK (#2603, CST), p-AMPK (#2537, CST), CaMKKβ (#16810, CST), GOAT (bs-13355R, Bioss), and β-Actin (#4967, CST).
14. Statistical analysis
The data are presented as the means ± standard errors of the means (SEMs). The box plot was used to find outliers. The lower and upper boundaries of the box are lower quartile - 1.5 × interquartile range (IQR) and upper quartile + 1.5 × IQR, respectively, and data outside of this range are usually considered outliers. Analysis of variance (ANOVA) was used for comparisons among multiple groups, while Tukey’s test was used for comparisons between groups. Statistical analyses were performed via GraphPad Prism 5 (GraphPad Software), and a value of P < 0.05 was considered statistically significant.
RESULTS
In the first part of the study, the authors investigated the effects of different ratios of OA-rich diets on nociceptive behaviors, protein expression and inflammatory cytokines in cancer-bearing rats. X-ray revealed that the rats subjected to CIBP modeling surgery exhibited breast cancer cell invasion, and H&E staining revealed that the bone tissue was destroyed by the breast cancer cells on day 18 after surgery (Fig. 1A, B). There were no differences in daily food intake between the groups (Fig. 1D). Compared with those in the sham group, the body weight, PWT, PWL, limb use score and weight-bearing ratio of the rats in the CIBP group were lower (Fig. 1C, E, G, I, J). The authors also measured the nociceptive behaviors on the contralateral side and found no significant differences in PWT and PWL between the groups (Fig. 1F, H). However, the OA-rich diet significantly increased the body weight (Fig. 1C) on day 18 after surgery and improved the PWT and PWL after the 9th day after surgery (Fig. 1E, G), the limb use score after the 12th day after surgery (Fig. 1I), and the weight-bearing ratio after the 6th day after surgery (Fig. 1J). In addition, inflammatory cytokines, including IL-1β, IL-6, TNF-α, were significantly elevated in the serum and bone marrow of the cancer-bearing rats (Fig. 1K–P). A significant reduction in the levels of serum IL-1β, IL-6, and TNF-α was observed in rats after receiving the OA-rich diet (60) and OA-rich diet (80) (Fig. 1K–M). However, the OA-rich diet had no significant effect on bone formation or repair processes and the levels of bone marrow IL-1β, IL-6, and TNF-α (Fig. 1A, B, N–P). The expression of the hypothalamic ghrelin-NPY pathway as well as the serum and hypothalamic levels of ghrelin and acyl-ghrelin in rats are shown in Fig. 2. Compared with those in the sham group, the expression of ghrelin, CaMKKβ, p-AMPK, and NPY were significantly lower and the expression of p-mTOR was increased in the CIBP group. Conversely, the administration of the OA-rich diet increased the expression of ghrelin, CaMKKβ, p-AMPK and NPY and decreased the expression of p-mTOR (Fig. 2A, B). And the original western blotting image is provided in Supplementary Fig. 1. The serum and hypothalamic levels of total ghrelin and acyl-ghrelin were significantly lower in the CIBP group than those in the sham group, whereas the OA-rich diet significantly increased the serum and hypothalamic levels of total ghrelin and acyl-ghrelin in a dose-dependent manner (Fig. 2C–F).
Then, the authors explored whether OA-rich diet induced the expression of hypothalamic NPY through the upregulation of acyl-ghrelin to alleviate CIBP. To inhibit the acylation of ghrelin, the expression of GOAT was downregulated with the administration of exogenous Go-Coa-Tat. The levels of acyl-ghrelin in the serum and hypothalamus were significantly decreased with the administration of exogenous Go-Coa-Tat (Fig. 3). Moreover, the expression of CaMKKβ, p-AMPK and NPY were increased, and the expression of p-mTOR was decreased with the administration of exogenous Go-Coa-Tat (Fig. 4). And the original western blotting image is provided in Supplementary Fig. 1. In addition, improvement in the body weight, PWT, PWL, limb use score, and weight bearing ratio by the OA-rich diet were aggravated with the administration of exogenous Go-Coa-Tat (Fig. 5A, C, E, G, H). And reduction in the levels of serum IL-1β, IL-6, and TNF-α by the OA-rich diet were aggravated with the administration of exogenous Go-Coa-Tat (Fig. 5I–K). However, both of OA-rich diet and OA-rich diet + Go-Coa-Tat have no significant effect on the levels of bone marrow IL-1β, IL-6 and TNF-α (Fig. 5L–N).
DISCUSSION
CIBP is a persistent and chronic pain that severely affects patients’ quality of life [26]. Over half of patients with progressive cancer suffer from CIBP [27]. The current treatment for CIBP is anticancer and symptomatic analgesic treatment [28]. However, the dosage of analgesic drugs is restricted due to their toxic effects, which ultimately prevents them from providing effective analgesic treatment. In the present study, the authors demonstrated that an OA-rich diet induced the expression of hypothalamic NPY through the upregulation of acyl-ghrelin to alleviate CIBP in cancer-bearing rats.
Patients with advanced breast cancers usually require adjuvant chemotherapy, which can cause gastrointestinal symptoms, such as nausea, vomiting and lack of appetite. These side effects of chemotherapeutic agents can further lead to malnutrition, which aggravates physical status [29,30]. Nutritional support can assist patients in maintaining their body weight, enhancing physical strength, reducing fatigue, augmenting the body's immunity, and reducing tolerance and response to treatments such as chemotherapy, radiotherapy, and surgery, which could prolong survival and improve quality of life [31]. MCFAs are saturated fatty acids that possess a backbone chain comprising 8–12 carbon atoms, which are important components of enteral nutrition. A review indicated that MCFAs could regulate glucose and lipid metabolism through the activation of the AMPK pathway and increase mitochondrial oxidative capacity through the activation of the PPARγ pathway [32]. OA is a member of the MCFAs, which could provide energy substrates and contribute to the acylation of ghrelin [22,33]. The acylation of ghrelin requires both acyl donors and GOAT to cross the blood-brain barrier to bind GHS-R1α. At present, most acyl donors provided by fatty acids (C2:0-C16:0) can be recognized by GOAT. However, the activating site of GOAT has a distinct preference for binding to OA (C8:0), with a sensitivity in vivo that is 50 times greater than that of C6:0 and C10:0 [19]. Thus, OA is the most effective of the MCFAs for promoting the acylation of ghrelin. A previous study demonstrated that enteral nutrition containing OA could be used to treat cachectic patients with chronic respiratory disease through increasing the level of acyl-ghrelin [23]. In the current study, an OA-rich diet was observed to increase the body weight of the cancer-bearing rats, which might slow down the progression of cachexia. In addition, the nociceptive behaviors of the cancer-bearing rats showed a gradient relief as the ratios of OA administered increased, which were significantly relieved in the CIBP + OA (60) or CIBP + OA (80) groups compared to the CIBP group. Meanwhile, it was also found that the OA-rich diet increased the level of acyl-ghrelin in a dose-dependent manner, which activated the AMPK-mTOR pathway and increased the expression of NPY. However, an OA-rich diet was unable to prevent breast cancer cells from invading and destroying bone tissue. In addition, an OA-rich diet had no significant effect on the levels of bone marrow inflammatory cytokines. Thus, an OA-rich diet might alleviate CIBP through the acyl-ghrelin/NPY pathway rather than directly affecting bone destruction or formation.
Ghrelin, produced by the gastric oxyntic glands, is involved in the regulation of growth hormone secretion, food intake, energy metabolism, and chronic pain [11,34]. The binding of ghrelin to GHS-R1α results in the increased release of endogenous PENK, which in turn activates OPRD, thereby producing analgesia [12]. In the rat chronic constriction injury model, the administration of ghrelin could alleviate neuropathic pain via the binding to GHSR-1a, which inhibits the p38 MAPK/NF-κB pathway [35]. Ghrelin has been demonstrated to exert analgesic effects through increasing the levels of IL-10 and TGF-β in a rat of inflammatory pain [11]. Ghrelin exists in two forms in vivo: acyl-ghrelin and desacyl-ghrelin. The formation of acyl-ghrelin is mediated by GOAT, which specifically acylates serine 3 of ghrelin [36]. Only acyl-ghrelin has been found to be able to cross the blood-brain barrier and bind to hypothalamic GHS-R1α, thereby exerting its biological effects [33]. NPY, expressed in the major organs of the body, is involved in the regulation of a multitude of physiological and pathological mechanisms, including neuronal excitability, depression, anxiety, feeding, and energy balance [37]. NPY has been demonstrated to reduce the release of substance P and excitatory neurotransmitters from primary afferent nerves by binding to the Y1 receptor, thereby mediating antinociception [38]. Spinal NPY exerts a tonic and persistent, inhibitory control of nociception through acting on Y2 receptors and reducing the expression of Fos and the phosphorylated form of extracellular signal-related kinase [39]. Spinal NPY exerts analgesic effects by acting on Y2 receptors in a formalin-induced rat knee pain model [40]. The intrathecal administration of NPY can suppress injury signaling in the spinal cord by inhibiting Y1 receptors-positive excitatory interneurons and inhibit excitatory neurotransmitter release from primary afferents by acting on Y2 receptors, both of which lead to the alleviation of metastatic bone pain [41]. Moreover, the authors’ previous study also revealed that intracerebroventricular administration of NPY could alleviate CIBP by binding to the NPY Y1 and Y2 receptors, which was reversed with the intracerebroventricular administration of NPY Y1 or Y2 receptors antagonist [24]. Recent studies have indicated the existence of a correlation between ghrelin and NPY [42,43]. Ghrelin can activate feeding by regulating the expression of hypothalamic NPY [42]. NPY has been demonstrated to relieve neuropathic pain by acting on spinal Y1 receptors interneurons that co-express a ghrelin releasing peptide [43]. The authors’ previous study demonstrated that ghrelin could induce the production of hypothalamic NPY through upregulating the expression of CaMKKβ, promoting the phosphorylation of AMPK, and downregulating the expression of mTOR, thereby alleviating CIBP [24]. In the current study, it was found that the increase in acyl-ghrelin caused by an OA-rich diet upregulated the expression of CaMKKβ and p-AMPK, and downregulated the expression of p-mTOR, thereby increasing the expression of NPY, which contributed to the alleviation of CIBP. With the administration of GOAT inhibitor, the level of acyl-ghrelin significantly decreased and the acyl-ghrelin/NPY pathway was reversed. Moreover, the authors found that nociceptive behaviors were aggravated by the inhibition of GOAT. These results indicated that an OA-rich diet activated the acyl-ghrelin/NPY pathway which alleviated CIBP.
There are some limitations in this study. First, due to the fixed intensity and large gradient of von Frey filaments, two outliers were observed in the PWT at 12 and 18 days after surgery in the CIBP group. However, statistical analysis revealed no significant impact on the results whether these outliers were included or excluded, leading to their retention in the dataset. Second, an analgesic control group (e.g., NSAIDs, opioids, or NPY receptor agonists) was not included to evaluate the efficacy of the OA-rich diet in alleviating CIBP through the acyl-ghrelin/NPY pathway, which should be considered in future research. Third, while the study demonstrated that an OA-rich diet upregulated hypothalamic NPT levels in cancer-bearing rats, it did not show significant association with bone formation, repair processes, or bone marrow inflammatory cytokines, suggesting that its pain-alleviation effects may involve alternative mechanisms. Finally, the relationship between NPY and bone metabolism is unclear, underscoring the need for future studies to further investigate the dual role of NPY in both pain regulation and bone metabolism.
In conclusion, acyl-ghrelin played a pivotal role in the upregulation of NPY and the alleviation of CIBP through the activation of the AMPK-mTOR pathway. In addition, an OA-rich diet could promote the acylation of ghrelin and activate the acyl-ghrelin/NPY pathway, thereby exerting an analgesic effect. Therefore, in addition to improving nutritional status, an OA-rich diet might provide an effective treatment for CIBP in patients with bone metastasis.
XML Download