Main Research Findings

1) Treatment with GHRP-2 was found to produce antinociceptive effects at the supraspinal level by acting on opioid receptors indicating its potential analgesic effects.

2) Administration of GHRP-2 has the potential to elicit anti-inflammatory effects in arthritic rats in a manner directly mediated by ghrelin receptors that act on immune cells.

 

Selected Data

1) Researchers Zeng et al conducted this study to investigate the antinociceptive properties of growth hormone–releasing peptide-2 (GHRP-2) and the mechanisms underlying its effects in mice. All experiments were conducted using male Kunming strain mice weighing 18–22 grams. The animals were housed under controlled environmental conditions, including a constant temperature of 22 ± 2 °C, a 12-hour light/dark cycle, and 50–60% relative humidity. Mice were allowed a three-day acclimatization period before experimental procedures began, with food and water provided ad libitum. Efforts were made throughout to minimize both the number of animals used and any potential suffering [1].

The primary compounds investigated were GHRP-2 and the ghrelin receptor antagonist GHRP-6. GHRP-2 is a synthetic hexapeptide that activates the growth hormone secretagogue receptor (GHS-R1a), whileGHRP-6 serves as its competitive antagonist. In addition, morphine hydrochloride was used as a reference opioid analgesic. To explore the potential involvement of opioid receptors in the effects of GHRP-2, several opioid receptor antagonists were employed: naloxone hydrochloride, β-funaltrexamine (β-FNA), naltrindole hydrochloride (NTI), and nor-binaltorphimine dihydrochloride (nor-BNI). All drugs were prepared in normal saline and stored as frozen stock solutions, with aliquots thawed immediately prior to use.
Drug administration was carried out via intracerebroventricular injection following a standardized method described in previous literature. The injection coordinates relative to the bregma were 1.5 mm posterior, 1.0 mm lateral, and 3 mm deep from the skull surface. A microsyringe was used to deliver 3 μL of the drug solution at a constant infusion rate of 10 μL per minute. At the conclusion of behavioral testing, histological verification of injection sites was performed; only animals with dye properly distributed throughout the ventricles were included in data analysis [1].

To assess the analgesic effects of GHRP-2 and related treatments, the tail withdrawal test was employed. This test measures nociceptive thresholds by immersing the distal portion of a mouse’s tail in warm water with the latency to withdraw the tail served as an index of pain sensitivity. Experiments were conducted beginning at 10:00 a.m., and each animal was tested only once to avoid confounding factors. Baseline withdrawal latencies were first established for each mouse, with only those exhibiting baseline latencies between 3–5 seconds included for further study. A cutoff latency of 15 seconds was set to prevent tissue damage. Before drug administration, six baseline measurements were recorded at 10-minute intervals, and the average of the last four readings was used as the control value. Post-treatment latencies were subsequently recorded at 5, 10, 20, 30, 40, 50, and 60 minutes following drug administration [1].

To elucidate the mechanisms underlying GHRP-2-induced antinociception, several pharmacological manipulations were performed. Specifically, GHRP-2 was co-administered with the ghrelin receptor antagonist GHRP-6 to determine whether its analgesic effects were mediated via the GHS-R1a receptor. Additionally, naloxone was co-administered with GHRP-2 to assess the role of opioid receptors in mediating its antinociceptive actions. To further specify which opioid receptor subtypes were involved, selective antagonists for μ-, δ-, and κ-opioid receptors (β-FNA, NTI, and nor-BNI, respectively) were used in combination with GHRP-2.

After behavioral testing, data analysis focused on quantifying changes in tail withdrawal latency (TWL). The primary metric used was the percentage change from baseline latency, calculated as: [(post-drug latency – pre-drug latency) / pre-drug latency] × 100. To provide an integrated measure of drug effects over time, the area under the curve (AUC) for TWL from 0 to 60 minutes post-treatment was calculated for each animal. Results were expressed as mean ± standard error of the mean (SEM), with each treatment group consisting of 8–12 mice [1].

Overall, this study utilized a controlled experimental design to examine the pain-modulating effects of GHRP-2 administered directly into the brain and to determine the involvement of ghrelin and opioid receptor systems. The combination of precise drug administration techniques, validated behavioral pain testing, and careful statistical analysis provided a robust framework for exploring the neuropharmacological mechanisms of GHRP-2. The findings from these experiments contribute to the growing understanding of ghrelin analogs’ roles in nociception and may offer new avenues for analgesic drug development [1].

2) This study performed by Granado et al investigated the effects of GHRP-2 on arthritic and healthy rats, focusing on inflammatory and metabolic parameters. 6 week old male Wistar rats weighing 150–175 g were obtained from Charles River Laboratories. Arthritis was induced by a single intradermal injection of 1 mg heat-inactivated Mycobacterium butyricum emulsified in incomplete Freund’s adjuvant into the right hind paw. Control rats received an injection of mineral oil. The rats were housed three to four per cage under controlled lighting and temperature conditions, with free access to food and water throughout the study [2].

To monitor the severity of arthritis, each paw was scored on a scale of 0–4 based on the degree of erythema and swelling: 0 indicated no signs, while 4 represented complete ankylosis and inability to bend the ankle. The sum of the four limb scores produced an arthritis index with a maximum possible score of 16. On day 15 after arthritis induction, animals with an arthritis score below 9 were excluded from the study. The remaining 24 arthritic and 20 control rats were randomly assigned to treatment or placebo groups. Beginning on day 15 and continuing through day 22, one group in each condition received daily subcutaneous injections of 100 µg/kg GHRP-2, while the other group received 250 µl saline injections. All rats were weighed daily, and their arthritis scores were recorded. Food consumption was measured per cage by subtracting leftover pellets from the initial amount and expressed as grams per rat per 100 g of body weight [2].

On day 22, corresponding to eight days of GHRP-2 or saline treatment and 2.5 hours after the last injection, the rats were euthanized by decapitation. Trunk blood was collected into chilled tubes, allowed to clot, and centrifuged to obtain serum. Samples were stored at –20°C for nitrite and leptin assays and at –80°C for ghrelin and interleukin-6 (IL-6) analyses. Additionally, the left hind paw was amputated, and its volume was measured using a water displacement method to assess inflammation.

Parallel in vitro experiments were performed using peritoneal macrophages harvested from a separate set of eight-week-old male Wistar rats weighing approximately 250 g. Following euthanasia by decapitation, cells were collected by peritoneal lavage using 10 ml sterile saline, massaged, and re-extracted into sterile tubes kept on ice. The cell suspension was washed and centrifuged twice at 1,000 rpm at 4°C and resuspended in DMEM enriched with 2 mM glutamine, 50 µg/µl gentamicin sulfate, 10% fetal bovine serum, and 1 mM mercaptoethanol. A density of 5 × 10^5 cells per well in 500 µl medium was plated in 24-well culture plates and incubated at 37°C with 5% CO₂. After a two-hour preincubation to allow adhesion, wells were washed to remove non-adherent cells. Adherent macrophages were then exposed for 24 hours to supplemented DMEM containing various stimuli: lipopolysaccharide at 1 or 100 ng/ml , GHRP-2 at 10^-7 M, and rat ghrelin at 10^-7 M. This concentration of GHRP-2 and ghrelin was previously shown to stimulate pituitary GH secretion in vitro. After incubation, the culture medium was collected and stored at –20°C for nitrite/nitrate analysis and at –80°C for IL-6 determination. Each experiment was performed twice to ensure reproducibility [2].

A variety of hormonal and inflammatory markers were assessed. Serum leptin and active ghrelin concentrations were measured using commercial radioimmunoassay (RIA) kits, following the manufacturer’s instructions. Serum corticosterone levels were determined with a competitive protein-binding assay, while ACTH concentrations were quantified using RIA kits. IL-6 levels in serum and macrophage culture medium were measured using a rat-specific Biotrak ELISA system. Nitrite and nitrate concentrations in serum and culture medium were measured as indicators of nitric oxide production using a modified Griess assay. To prepare serum samples for this analysis, proteins were removed by ultrafiltration using a 30-kDa molecular mass filter by centrifugation at 15,000 rpm for one hour at 37°C for 300 µl samples. Filtered serum or diluted culture medium was mixed with vanadium chloride, followed by Griess reagents, incubated for 30 minutes at 37°C, and absorbance measured at 540 nm. Nitrite/nitrate concentrations were calculated against a sodium nitrite standard curve and expressed in micromoles per liter [2].

 

Discussion

1) The study conducted by Zeng et al examined the analgesic properties of GHRP-2 administered intracerebroventricularly in conscious mice using the warm-water tail immersion test. The results demonstrated that GHRP-2 produced a clear concentration- and time-dependent antinociceptive effect. Following intracerebroventricular administration of various doses of 0.1, 0.3, 1, 3, and 10 nmol/L, TWL significantly increased compared to saline controls. The analgesic effect began rapidly, peaked at approximately five minutes post-injection, and gradually declined, disappearing within 30–40 minutes. Interestingly, the effect followed a bell-shaped dose-response curve: the greatest analgesic activity occurred at 1 nmol/L, with both higher and lower doses being less effective. At the five-minute mark, percent changes in TWL were 23.2% for 0.1 nmol/L, 29.8% for 0.3 nmol/L, 100% for 1 nmol/L, 92.2% for 3 nmol/L, and 38.6% for 10 nmol/L. AUC analyses over 60 minutes supported these findings, with 1 nmol/L yielding the highest overall analgesic effect. These results suggested that GHRP-2’s analgesia is mediated by specific receptor mechanisms and not simply dose-proportional [1].

Figure 1: Dose dependent changes in tail withdrawal latency during 60 minutes post-injection period.

Given that GHRP-2 is a known ghrelin receptor agonist, the researchers explored whether activation of GHS-R1a was involved in this antinociceptive effect. To test this, they co-administered GHRP-2 with the GHS-R1a antagonist GHRP-6 at doses of 10 and 100 nmol/L. The antagonist alone had no significant impact on tail withdrawal latency. However, co-administration with GHRP-2 revealed a dose-dependent blockade, 10 nmol/L GHRP-6 partially inhibited GHRP-2-induced analgesia, while 100 nmol/L completely abolished it. These findings confirmed that GHRP-2’s analgesic action in this model is mediated through activation of ghrelin receptors in the central nervous system.

The study next assessed the role of opioid receptors in GHRP-2-induced analgesia. Naloxone, a non-selective opioid receptor antagonist, was administered intracerebroventricularly either alone or in combination with GHRP-2. Naloxone by itself did not alter TWL, but when co-administered with 1 nmol GHRP-2, it partially blocked analgesia at 10 nmol/L and completely blocked it at 100 nmol/L. This suggested that opioid receptor activation is also critical for GHRP-2’s effects. To further specify which opioid receptor subtypes were involved, the researchers administered selective antagonists: β-FNA, NTI, and nor-BNI, all at 100 nmol/L. None of these antagonists alone altered TWL. However, both NTI and nor-BNI significantly abolished GHRP-2-induced analgesia, while β-FNA had no significant effect. These results indicated that the delta- and kappa-opioid receptors, but not mu-opioid receptors, mediate GHRP-2’s analgesic effects. Thus, the analgesia observed from GHRP-2 involves a combined mechanism requiring activation of ghrelin receptors and specific opioid receptor subtypes [1].

Finally, the study investigated whether GHRP-2 could modulate morphine-induced analgesia. 1 nmol/L morphine alone produced a time-dependent antinociceptive effect, peaking around 30 minutes post-injection. When GHRP-2 at 1, 3, or 10 nmol/L was co-administered with morphine, there was a marked potentiation of morphine’s analgesic effect. At 10 minutes, the percent changes in TWL for morphine alone were approximately 89.6%, while co-administration with GHRP-2 increased these values to 122.4% with 1 nmol, 155.6% with 3 nmol, and 187.9% with 10 nmol. Notably, the analgesic response observed with 10 nmol/L GHRP-2 combined with morphine exceeded the additive effects of each drug alone, suggesting a synergistic interaction. The potentiation was not blocked by the ghrelin receptor antagonist GHRP-6, implying that the mechanism by which GHRP-2 augments morphine-induced analgesia may not depend on ghrelin receptor activation, even though GHRP-2’s own analgesic effects do [1].

In conclusion, intracerebroventricular administration of GHRP-2 induces a rapid but transient analgesic effect in mice that is maximal at an intermediate dose of 1 nmol/L. This effect is mediated through both ghrelin receptor activation and engagement of delta- and kappa-opioid receptors. Moreover, GHRP-2 can significantly enhance the analgesic efficacy of morphine, and this potentiation occurs via a mechanism that does not appear to require ghrelin receptor signaling. These findings highlight the interaction between GHRP-2 and central pain modulation systems that suggest potential therapeutic implications for pain management strategies involving ghrelin mimetics, especially in combination with opioids [1].

2) The study conducted by Granado et al examined the physiological and immunological effects of GHRP-2 in both arthritic and healthy rats. Arthritis significantly reduced cumulative body weight gain and food intake compared to controls. Specifically, arthritic rats displayed a 74% reduction in body weight gain relative to saline-treated controls, whereas food intake was reduced by about 10%. Administration of GHRP-2 over eight days increased body weight gain in both healthy and arthritic animals, however, the increase in food intake was observed only in control rats, not in those with arthritis. This suggests that while GHRP-2 can promote weight gain, the mechanisms underlying this effect may differ between healthy and arthritic states [2].

Figure 2: Changes in body weight and food intake in rats treated with saline and rats treated with GHRP-2.

The study also explored hormonal changes related to ghrelin and leptin. Arthritis produced an opposite pattern in these hormones: arthritic rats had higher serum ghrelin levels and lower serum leptin levels compared to healthy controls. Treatment with GHRP-2 significantly elevated serum leptin levels in both control and arthritic animals, indicating a potential role for the peptide in modulating energy balance and metabolism in inflammatory conditions. Conversely, GHRP-2 did not significantly alter ghrelin levels, suggesting that its effects on leptin are more pronounced [2].

Markers of hypothalamic-pituitary-adrenal (HPA) axis activity, such as corticosterone and ACTH, were also impacted by arthritis and GHRP-2 treatment. Arthritic rats exhibited significantly higher corticosterone and ACTH levels compared to controls, consistent with activation of the HPA axis during inflammation.GHRP-2 had different effects depending on the physiological state. In healthy rats, GHRP-2 increased ACTH levels significantly and corticosterone levels modestly. In contrast, arthritic rats treated with GHRP-2 exhibited significant reductions in both ACTH and corticosterone compared to saline-treated arthritic rats. These findings indicate that GHRP-2 modulates stress hormone production differently in healthy versus inflamed states, potentially contributing to its anti-inflammatory effects.

Consistent with these hormonal changes, GHRP-2 demonstrated significant anti-inflammatory effects in arthritic rats. Paw swelling, a measure of inflammation, was markedly increased in arthritic animals. GHRP-2 treatment reduced paw volume and improved arthritis scores, with significant improvements appearing after six days of treatment. These anti-inflammatory effects occurred without significant changes in serum nitrite/nitrate concentrations, which were elevated in arthritic rats. However, GHRP-2 significantly reduced serum IL-6 levels in both arthritic and control rats, indicating a role in suppressing pro-inflammatory cytokine production [2].

FIgure 3: Changes in paw volume and arthritic score in rats treated with saline and rats treated with GHRP-2.

To further investigate the mechanisms underlying the IL-6 reduction, in vitro studies were conducted using peritoneal macrophages. When stimulated with lipopolysaccharide, macrophages showed significant increases in nitrite/nitrate and IL-6 production. Pre-incubation with GHRP-2 or ghrelin attenuated the LPS-induced increases. While neither GHRP-2 nor ghrelin altered basal mediator release, both completely prevented the LPS-induced IL-6 production and reduced nitrite/nitrate output compared to control cultures. These results suggest that GHRP-2 and its natural ligand ghrelin have direct immunomodulatory effects on macrophages.

Overall, the findings demonstrate that GHRP-2 exerts beneficial effects in arthritic rats by promoting weight gain, modulating HPA axis activity, and exerting anti-inflammatory effects, particularly through suppression of IL-6. The in vitro results support a direct action of GHRP-2 and ghrelin on immune cells, further highlighting their therapeutic potential for inflammatory diseases [2].

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] Zeng P, Li S, Zheng YH, et al. Ghrelin receptor agonist, GHRP-2, produces antinociceptive effects at the supraspinal level via the opioid receptor in mice. Peptides. 2014;55:103-109. doi:10.1016/j.peptides.2014.02.013

[2] Granado M, Priego T, Martín AI, Villanúa MA, López-Calderón A. Anti-inflammatory effect of the ghrelin agonist growth hormone-releasing peptide-2 (GHRP-2) in arthritic rats. Am J Physiol Endocrinol Metab. 2005;288(3):E486-E492. doi:10.1152/ajpendo.00196.2004

 

 

GHRP-2 is a growth hormone-releasing peptide that has been shown to mimic the effects of ghrelin in the way that it increases appetite and stimulates the release of growth hormone. A study conducted by Peroni et. Al, examined the effect that two different dosages of GHRP-2 had on growth hormone secretion and overall body weight of mice.

In the initial experiment conducted by the researchers, wild-type C57BL mice were injected with either 1 microgram of GHRP-2 or 10 micrograms of GHRP-2. Following the injection, the mice were euthanized and their blood was collected for up to 1 hour after injection at various different points in time to measure the amount of growth hormone in the body. Results of the first study showed that the C57BL mice injected with 10 micrograms elicited a far stronger secretion in GH than the mice injected with 1 microgram. Furthermore, the results of the study showed that GH secretion reached a peak of 163 ng/ml about 5-10 after the initial injection. It was shown that 20 minutes after the injection levels of GH returned to their baseline levels.

A separate, independent study was conducted by Peroni et. Al that treated lit/lit mice with 10 micrograms of GHRP-2 for two weeks, and rather than collecting the data over one hour, data was collected every 3 to 4 days in order to measure body weight, release of GH, IGF-1 and leptin levels. Body weight measurements were used to calculate an acceptable and measurable growth curve. The results of this second study found that when injected with 10 micrograms of GHRP-2, there was an expected overall increase in body weight. Additionally, results showed little change in GH or IGF-1 levels, but there was a noted increase in leptin levels on day 15 indicating reduced appetite (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3297037/).

 

GHRP-2 and Weight Gain

An additional study conducted by Thang Long et. Al studied the dose-dependent effects of GHRP-2 treatment in cross-bred castrated male swine. Over the course of treatment, the pigs were injected with doses of 2, 10, 30, and 100 mcgs/kg of body weight. The researchers were measuring peak GH secretion as well as the area under the GH response curve. Following the treatment period, it was found that both the peak concentration of GH and the area under the GH response curve were both greater than those in the control group injected with saline. The study also concluded that with long-term GHRP-2 treatment the average daily weight gain increased by 22.35% while feed efficiency increased by 20.64%, however, the actual amount of food ingested did not vary by notable amounts. Overall the results found that in castrated male swine, GHRP-2 increased GH secretion and overall growth (https://www.researchgate.net/publication/12524642_The_effects_of_growth_hormone-releasing_peptide-2_GHRP-2_on_the_release_of_growth_hormone_and_growth_performance_in_swine).

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