Antinflammatory, antioxidant, and behavioral effects induced by administration of growth hormone-releasing hormone analogs in mice

Growth hormone-releasing hormone (GHRH) antagonist MIA-690 and GHRH agonist MR-409, previously synthesized and developed by us have demonstrated potent antitumor effects. However, little is known about the effects of these analogs on brain functions. We investigated the potential antinflammatory and antioxidant effects of GHRH antagonist MIA-690 and GHRH agonist MR-409, on isolated mouse prefrontal cortex specimens treated with lipopolysaccharide (LPS). Additionally, we studied their effects on emotional behavior after chronic in vivo treatment. Ex vivo, MIA-690 and MR-409 inhibited LPS-induced inflammatory and pro-oxidative markers. In vivo, both MIA-690 and MR-409 induced anxiolytic and antidepressant-like effects, increased norepinephrine and serotonin levels and decreased nuclear factor-kB, tumor necrosis factor-α and interleukin-6 gene expression in prefrontal cortex. Increased nuclear factor erythroid 2–related factor 2 expression was also found in mice treated with MIA-690 and MR-409. MIA-690 showed higher efficacy in inhibiting all tested inflammatory and oxidative markers. In addition, MR-409 induced a down regulation of the gene and protein expression of pituitary-type GHRH-receptor in prefrontal cortex of mice after 4 weeks of treatment at 5 µg/day. In conclusion, our results demonstrate anxiolytic and antidepressant-like effects of GHRH analogs that could involve modulatory effects on monoaminergic signaling, inflammatory and oxidative status.


Discussion
GHRH and mRNAs for GHRH-R have been found in rat cortex and brain stem 22,23 , and various reports suggested that GHRH might play a key role in cognitive and mood disorders 20,24-28 . GHRH-antagonists compounds can exert powerful antitumor effects, possibly related in part to their antinflammatory and antioxidant properties 17,[29][30][31][32][33] . In the present study we show that MIA-690, a GHRH antagonist, and MR-409, a GHRH analog, exhibit antinflammatory and antioxidant effects on prefrontal cortex specimens, ex vivo (Figs. 1, 2 and 3). Accordingly, various studies showed that GHRH and GHRH antagonists can influence the inflammatory and reduction/oxidation (redox) status in cancer and other tissues 29,33 .  www.nature.com/scientificreports www.nature.com/scientificreports/ In particular, MIA 690 decreased inflammation by reducing the infiltration of macrophages and leucocytes, the production of TNF-α, IL-1β, and monocyte chemotactic protein-1 (MCP-1) in tissue after insult with lipopolisaccaride (LPS) and the production of the pro-inflammatory markers in carrageenan-induced chronic prostatis 31,32 . In addition, MIA-690 showed antioxidant and neuroprotective properties 28 . In a model of Alzheimer's disease as well as in cancer and other tissues, MR-409 has been described to exert antinflammatory and antioxidant effects, as well as in early experimental diabetic retinopathy 31 . In this context, the authors suggested that the protective effects of the MR-409 could be mediated by its direct and/or GH-mediated action.
In the present study, MIA-690 and MR-409 have been also able to modulate emotional behaviors, in mice. We observed that both peptides induced anxiolytic and antidepressant-like effects following chronic treatment, without affecting locomotor activity (Figs. 4-7). The role of GH in regulation of mood is somewhat contradictory and little is known about the action of GHRH on brain functions. Human studies indicate that in adults with childhood onset GH deficiency, long term treatment with GH improves mood and memory 34 . On the other hand, somatostatin, which inhibits the release of several hormones, including GH, reduces anxiety-like behavior 35,36 . Mood disorders might be related to GH deficiency 37,38 , however the anxiolytic-antidepressant effects of a GHRH antagonist, MZ-4-71, suggests that GHRH itself may be involved in control of behavior 20,[25][26][27] . Similarly, our research group described anxiolytic and antidepressant-like behavior in both young and old mice with generalized ablation of the GHRH gene 21,39,40 . Thus, we can hypothesize that the beneficial behavioral effects of MIA-690 and MR-409 could be at least in part related to their antinflammatory and antioxidant effects (Figs. 8, 9), also described in different reports 14  of chronic diseases, including cancer, cardiovascular diseases, aging, neurodegenerative, and psychiatric disorders, such as anxiety and depression [40][41][42][43] . The activation of the inflammatory and oxidative stress response leads to the release of inflammatory cytokines and mobilization of immune cells that can get access to brain 42,43 . In particular, some studies have demonstrated an increase in pro-inflammatory markers, such as NF-kB, IL-1 and IL-6, in anxiety-and depression-related conditions [44][45][46] . In addition, major depression and posttraumatic stress disorder are characterized by an increased activity of pro-oxidants over antioxidants 47,48 . Cytokines and their signaling pathways have significant effect on the metabolism of multiple neurotransmitters such as 5-HT and DA through impact on their synthesis, release and reuptake. Through their effects on neurotransmitter systems, cytokines lead to significant changes in motor activity and motivation as well as anxiety, arousal and alarm 49 . In this context, we also evaluated monoamine levels and NF-kB, TNF-α and IL-6 gene expression in prefrontal cortex of mice, after chronic administration with MIA-690 and MR-409. We observed an increase of NE and 5-HT levels, paralleled by a decrease of inflammatory markers in both MIA-690 and MR-409 treated mice (Table 1; Fig. 8, panel A, B and C).
In agreement, the anxiolytic-antidepressant activity of MZ-4-71 was previously suggested to be mediated by α1/ α2-adrenergic and 5-HT1/5-HT2 serotonergic receptors 25,26 . Because several studies established a link between oxidative stress, anxiety and depression, we investigated the expression of Nrf2 in prefrontal cortex by immunohistochemistry. Nrf2 is a key transcription factor controlling various homoeostatic processes, at cellular level, in response to oxidative stress and toxic stimuli 50 and regulating oxidative/xenobiotic stress response, also repressing inflammation 51 . Activation of Nrf2 results in up-regulation of cytoprotective and antioxidant enzymes-proteins www.nature.com/scientificreports www.nature.com/scientificreports/ in brain, by protecting against oxidative stress, in the brain 50,51 . Less Nfr2 signaling could reflect a "normalization" of oxidative parameters also evidenced from the molecular markers.
We found a significant increase of positive area percentage for Nrf2 in MIA-690 or MR-409 treated mice, suggesting an evident Nrf2 activation (Fig. 9). Surprisingly, both the agonist and the antagonist peptides induced similar effects in all experimental paradigms. However, we observed a higher efficacy of MIA-690 compared to MR-409. An accurate behavior analysis showed that MIA-690 was able to modulate emotional behavior beginning from week 2 of treatment, while MR-409 induced significant behavioral change only at week 4 of treatment. At the end of treatment, we found a reduction of P GHRH-R gene and protein expression in MR-409 treated mice (Figs. 10, 11) (Supplementary Figs. S1 and S2). As previously observed by Schally et al. 16 , our results confirmed that chronic administration of MR-409 results in a down-regulation of P GHRH-R, which could justify the effects of MR-409 on emotional behavior. On the other hand, we can not rule out the possible involvement of other mechanisms in emotional behavior induced by the peptide.
In conclusion, both MIA-690 and MR-409 exhibit antinflammatory and antioxidant effects in ex vivo and in vivo experimental models. Surprisingly, both agonist and antagonist peptides induce anxiolytic and antidepressant-like behavior, which could be related to increased cortical NE and 5-HT levels, along with modulatory effects on the inflammatory and oxidative status. Further investigations are needed to confirm a role for GHRH analogs in mood disorders.   www.nature.com/scientificreports www.nature.com/scientificreports/ Animals. Adult C57/BL6 male mice (3 month-old, weight 20-25 g, n = 48) were housed in plexiglas cages (2-4 animals per cage; 55 × 33 × 19 cm) and maintained under standard laboratory (21 ± 2 °C; 55 ± 5% humidity) on a 14/10 h light/dark cycle, with ad libitum access to water and food. Only male mice were used to avoid any possible involvement of hormonal changes in adult female mice. Mice were fed with a standard rodent chow (Prolab RMH2500, PMI Nutrition International, Brentwood, MO). Housing conditions and experimentation procedures were strictly in agreement with the European Community ethical regulations (EU Directive n. 26/2014) on the care of animals for scientific research. In agreement with the recognized principles of "Replacement, Refinement and Reduction of Animals in Research", prefrontal cortex specimens were obtained as residual material from vehicle-treated mice randomized in our previous experiments approved by Local Ethical Committee ('G. d' Annunzio' University, Chieti-Pescara) and Italian Health Ministry (Project n. 885/2018-PR).

Methods
Ex vivo studies. Mice were sacrificed by CO 2 inhalation (100%CO 2 at a flow rate of 20% of the chamber volume per min), then brains were rapidly removed. The brains were cut into blocks containing the entire prefrontal cortex, frozen on dry ice, and stored at −80 °C before serial cryosectioning at a section thickness of 100 μm. A stereotaxic atlas of the mouse brain (Paxinos and Watson) was used during the cryosectioning procedure 52 . Tissue slices were maintained in a humidified incubator with 5% CO 2 at 37 °C for 4 h (incubation period), in RPMI buffer with added bacterial LPS (10 μg/mL), as previously described 53 . During the incubation period, the tissues were treated with MR-409 or MIA-690 (1-5 μM). Tissue supernatants were collected and PGE 2 and 8-iso-PGF 2α levels (pg/mg wet tissue) were measured by RIA, as previously reported 54 . Briefly, specific anti-PGE 2 and anti-8-iso-PGF 2α were developed in the rabbit; the cross-reactivity against other prostanoids was <0.3%. One hundred microliters of prostaglandin standard or sample was incubated overnight at 4 °C with the 3 H-prostaglandin (3000 cpm/tube; NEN) and antibody (final dilution: 1:120 000; kindly provided by the late prof. G. Ciabattoni), in a volume of 1.5 mL of 0.025 M phosphate buffer. Free and antibody-bound prostaglandins were separated by the addition of 100 μL 5% bovine serum albumin and 100 μL 3% charcoal suspension, followed by centrifugation for 10 min at 4000 g at 5 °C and decanting off the supernatants into scintillation fluid (UltimaGold ™ , Perkin Elmer) for β emission counting. The detection limit of the assay method was 0.6 pg/mL. Tissue supernatants were also assayed for nitrite determination by Griess assay, as previously described 55 . Briefly, nitrite production was determined by mixing 50 μL of the assay buffer with 50 μL of Griess reagent (1.5% sulfanilamide in 1 M HCl plus 0.15% N-(1-naphthyl) ethylenediamine dihydrochloride in distilled water, v/v). After incubation for 10 min, at room temperature, the absorbance at 540 nm was determined and nitrite concentrations were calculated from a standard curve for sodium nitrite.
Tissue supernatants were also assayed for lactate dehydrogenase (LDH) activity 55 . LDH activity was measured by evaluating the consumption of NADH in 20 mM HEPES-K + (pH 7.2), 0.05% bovine serum albumin, 20 μM NADH and 2 mM pyruvate using a microplate reader (excitation 340 nm, emission 460 nm) according to manufacturer's protocol (Sigma-Aldrich, St. Louis, MO). LDH activity was measured by evaluating the consumption of NADH in 20 mM HEPES-K + (pH 7.2), 0.05% bovine serum albumin, 20 μM NADH and 2 mM pyruvate using a microplate reader (excitation 340 nm, emission 460 nm) according to manufacturer's protocol. Nitrite and LDH production data were expressed as relative variations compared to vehicle-treated specimens. Immediately after sacrifice, prefrontal cortex was rapidly removed, dissected and stored in RNAlater solution (Ambion, Austin, TX) at −20 °C until further processed. Total RNA was extracted from the prefrontal cortex using TRI Reagent (Sigma-Aldrich), according to the manufacturer's protocol. One microgram of total RNA extracted from each sample in a 20 μl reaction volume was reverse transcribed using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The samples were incubated in a 2720 Thermal Cycler (Applied Biosystems) initially at 25 °C for 10 min, then at 37 °C for 120 min, and finally at 85 °C for 5 s. Gene expression of COX-2, NF-kB and iNOS was determined by quantitative real-time PCR using TaqMan probe-based chemistry (Applied Biosystems), as previously described 56,57 . PCR primers and TaqMan probes were obtained from Applied Biosystems (Assays-on-Demand Gene Expression Products, Mm00478374_m1 for COX-2 gene, Mm00476361_ m1 for NF-kB gene, Mm00440502_m1 for iNOS gene, Mm00607939_s1 for β-actin gene. β-actin was used as the housekeeping gene. Gene expression data were calculated as previously reported 58 . In vivo studies. After 2-week acclimation, mice were randomized into three groups and treated daily for 4 weeks by subcutaneous administration of GHRH antagonist MIA 690 (5 μg), GHRH agonist MR 409 (5 μg) or vehicle solution 18 . All solutions were prepared freshly before use.The doses were selected based on previous studies including oncology. Injection volume was 0.1 ml for s.c. injection 16,18 . The animals were brought into the experimental room 30 min prior to the test in order to acclimate to the environment, and were kept in the testing chamber for 5 min prior to each test.
All treatments were administered at 09:00 am, and the experiments performed between 10:00 and 12:00 am. Each test session was recorded by a video camera connected to a computer; a single video frame was acquired with a highly accurate, programmable, monochrome frame grabber board (Data TranslationTM, type DT3153). The intelligent software Smart version 2.5 (Panlab, sl Bioresearch and Technology, Barcelona, Spain) was used for data processing. The apparatuses were purchased from 2 Biological Instruments (Besozzo VA, Italy) 21,39 . At the end of each test, the animals were returned to their home cages, and the apparatus was cleaned with 75% ethanol and dried before the next procedure. The behavioral parameters were recorded at 2 and then 4 weeks after the first treatment. Each test was conducted on the same group of animals (n = 18 animals for each group of treatment), after a 2 weeks rest period to avoid any interference on behavioral test performance, as previously reported 21 . Locomotor activity. Locomotor  Tail suspension test. This test is well characterized for assessing antidepressant-like activity. Mice were individually suspended by the tail to a horizontal bar (at the height of 30 cm from floor) using adhesive tape. Immobility time was recorded during a 6 min period. Mice were considered immobile only when they hung passively and completely motionless 21,39 . Prefrontal cortex monoamine extraction and high performance liquid chromatography (HPLC) determination. Immediately after sacrifice, brains were rapidly removed and prefrontal cortex were dissected and subjected to biogenic amine extractive procedures. Thereafter, samples were analyzed by HPLC coupled to electrochemical detection consisting of ESA Coulochem III detector equipped with ESA 5014 B analytical cell (selected potentials: electrode 1:−150 mV; electrode 2: +300 mV), as previously reported 40,59 . Monoamine levels were expressed as ng/mg wet tissue.

RNA extraction, reverse transcription and real-time reverse transcription polymerase chain reaction (PCR-RT).
Prefrontal cortex was rapidly removed, dissected and stored in RNAlater solution (Ambion, Austin, TX) at −20 °C until further processed as previously described. Gene expression of NF-kB, TNF-α, IL-6 and P GHRH-R was determined by quantitative real-time PCR using TaqMan probe-based chemistry (Applied Biosystems, Foster City, CA, USA). PCR primers and TaqMan probes were obtained from Applied Biosystems (Assays-on-Demand Gene Expression Products, Mm00476361_m1 for NF-kB gene, Mm00443258_m1 for TNF-α gene, Mm00446190_m1 for IL-6 gene, Mm01326479_m1 for P GHRH-R gene, Mm00607939_s1 for β-actin gene. β-actin was used as the housekeeping gene. Gene expression data were calculated as previously reported 58 .

Light microscopy analysis and immunohistochemistry. Prefrontal cortex was fixed in 10%
phosphate-buffered formalin for 2.5 hours. Each tissue block was dehydrated in a series of alcohol solutions of 50%, 70%, 96% and 99% and then in Bioclear. Samples were then paraffin-embedded and cut into 7 μm-thick sections. Sections were de-waxed (Bioclear and alcohol in progressively lower concentrations), rehydrated and processed for haematoxylin-eosin and for anti-Nrf2 immunohistochemical analysis according to manufacturer protocol. Primary antibody anti-Nfr2 (rabbit polyclonal, sc-722, Santa Cruz Biotechnology, CA, USA) was applied for 2 hours at room temperature and diluted 1:200 in PBS. The immunohistochemical reactions was revealed with Rabbit specific HRP/DAB detection IHC kit (ab236469). Peroxidase reaction was developed using diaminobenzidine (DAB) chromogen and nuclei were counterstained with haematoxylin. Lastly, sections were dehydrated, cleared with xylene and mounted in Bio Mount (Bio Optica, Milano, Italy). Negative control was performed by omitting the primary antibody. Samples were then observe by means of LEICA DM 4000 light microscopy (Leica Cambridge Ltd., Cambridge, UK) equipped with a Leica DFC 320 camera (Leica Cambridge Ltd.) for computerized images 62,63 . Western blot analysis. Cortex samples obtained from mice treated or not with MIA-690 or MR-409 were homogenized in RIPA buffer (Sigma-Aldrich), sonicated and centrifuged at 14,000 rpm (4 °C for 15 min). Total protein lysates were quantified with Bicinchoninic Acid kit (BCA) from Sigma-Aldrich. Proteins (35 μg) were separated by 10% SDS-PAGE, transferred to a nitrocellulose membrane and incubated overnight at 4 °C with the specific P GHRH-R antibody (dilution 1:500, rabbit polyclonal P GHRH-R antibody, Abcam, ab76263). Blots were reprobed with actin (dilution 1:500, mouse monoclonal actin antibody, Santa Cruz Biotechnology, sc-376421) for protein normalization. Immunoreactive proteins were visualized using horseradish peroxidase-conjugated goat anti-mouse, goat anti-rabbit or mouse anti-goat (1:4000) secondary antibodies by enhanced chemiluminescence substrate (ECL) using ChemiDoc XRS (Bio-Rad), densitometric analysis was performed with Quantity One software (Bio-Rad) 18 .
Statistical analysis. Statistical analysis was performed using GraphPad Prism version 5.01 for Windows (GraphPad Software, San Diego, CA, USA). All data were collected from each of the animals used in the experimental procedure and means ± SEM were determined for each experimental group and analyzed by two way analysis of variance (ANOVA) followed by Bonferroni post-hoc test. F values are referring to repeated measure 2-way ANOVA. As for gene expression analysis, 1.00 (calibrator sample) was considered the theoretical mean for the comparison. Statistical significance was accepted at p < 0.05. As regards gene expression analysis, the comparative 2 −ΔΔCt method was used to quantify the relative abundance of mRNA and then to determine the relative changes in individual gene expression (relative quantification) 58 . Finally, as regards the animals randomized for each experimental group, the number was calculated on the basis of the 'Resource Equation' N = (E + T)/T (10 ≤ E ≤ 20) 64 according to the guidelines suggested by the 'National Centre for the Replacement, Refinement and Reduction of Animals in Research' (NC3RS) and reported on the following web site: https://www.nc3rs.org. uk/experimental-designstatistics.