Introduction
Sleep problems (insomnia) are a major public health problem that affecting approximately 10~30% of the population [3]. The common insomnia drugs targets the γ-aminobutyric acid (GABA) receptor, melatonin receptor, histamine, and serotonin receptor [19]. Melatonin is a hormone that is secreted by the pineal gland and then released into the bloodstream, and it plays an important role in sleep patterns [5].
Stress can also cause depression and sleep problems [15] Corticosterone (CORT) is increased by stress-induced dysfunction of the hypothalamic-pituitary-adrenal (HPA) axis [16]. CORT is the biological glucocorticoid in animals, and that is high CORT levels have been observed are seen in stress-related disorders. Previous studies have shown that stress can cause endoplasmic reticulum (ER) stress and lead to neuronal damage, and which involves in the mitogen-acti- vated protein kinase (MAPK) pathways (MEK/ERK) [18]. The cAMP-response element binding protein (CREB) is a neuronal survival factor and lead to neuroprotective effects via the promotion of phosphorylated CREB [2].
Ptecticus tenebrifer (Walker) belongs to the Diptera Stratiomyidae family and is commonly used as a raw material for feed material. It is known that the nutritional value and economic feasibility are superior to those of other insects [1]. Previous studies have reported that GABA can be by effectively synthesized by the bio-transformation of monosodium glutamate (MSG) to GABA via microorganisms, including lactic acid bacteria, and that GABA-enhanced materials can be use as effective feed additives [6, 7].
Thus, in the present study, we investigated the anti-stress of a GABA-enhanced PT extract through modulation of ERK/CREB signaling pathways in SH-SY5Y cell death, and to further explored the associated serum CORT in chronic restraint stress (CRS)-exposed mice. Moreover, we evaluated sleep-enhancing of the GABA-enhanced PT extract on pento- barbital-induced sleeping behavior in mice via the regulation of melatonin levels.
Materials and Methods
Materials
Minimum Essential Medium (MEM), phosphate buffered saline (PBS), and fetal bovine serum (FBS) were purchased from Invitrogen Inc. (Grand Island, NY, United States). Bovine serum albumin (BSA), anti-β-actin, CORT, dimethyl sulfoxide (DMSO), GABA, and 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Antibodies against p44/42 MAPK (ERK1/2), phosphorylated-p44/42 MAPK (p-ERK1/2), phosphorylated-cyclic AMP-responsive element-binding (CREB), anti-CREB, and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG secondary antibodies were purchased from Cell signaling Technology (Beverly, MA, USA). Bicinchoninic acid assay (BCA) protein assay were purchased from Thermo Scientific Inc. (Waltham, MA, USA).
Preparation of the extract
P. tenebrifer (PT) used in this study were provided in GreeNest Company (Jeollanamdo, Republic of Korea). The larvae (instar 4) of PT were mixed with MSG (150 g) and water, and then grown for 10 days. After 10 days, it was dried with hot air drier in 80℃ for 24 hr. PT was heated to 60℃ and press to remove oil, except for non-PT (before feeding MSG). The 50 g with PT was extracted using water or 70% ethanol at 30℃ for 24 hr. The extracted solution was collected, centrifuged at 13,000 rpm for 3 min to remove impurities, and then freeze-dried. Finally, dried P. tenebrifer water extract (PTW) or P. tenebrifer 70% ethanol extract (PTE) was stored at 4℃ until it was used in the analysis.
IdentificationofGABAinPtecticustenebrifer ex- tract
To carry out the analysis of GABA in PT, we was performed by using the high-performance liquid chromatography (HPLC) system (Waters series e2695, MA, USA) comprised of a of a photodiode array detector (2998) and a evaporative light scattering detector (2424). The column was Triart-C18 (250 mm×4.6 mm, 5 μm, YMC, Japan) and the detection wavelength was set at 254 and 280 nm. The column thermostat was maintained at 35℃. The evaporative light scattering detector (ELSD) system temperature was maintained at 65℃ and the nitrogen gas flow was injected 4 bar. Mobile phase A was Acetonitrile and mobile phase B was 0.1% Formic acid in water with the elution profile as follows: Initial 100% of B; 0-8 min, 100-99% of B; 8-15 min, 99-97% of B; 15-20 min, 97-85% of B; 20-25 min, 85-0% of B; 25-30 min, 0% of B; 30-35 min, 0-100% of B; 35-45 min, 100% of B; 50min. The flow rate was 0.5 ml/min, and the injection volume was 10 μl.
SH-SY5Y cell cultures and MTT assay
The SH-SY5Y human neuroblastoma cell line was purchased from the American Type Culture Collection (ATCC, CRL-2266; Manassas, VA, USA) and cultured in MEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin, at 37°C, in a humidified atmosphere containing 5% CO. For cell viability assay, undifferentiated 2 SH-SY5Y cells were seeded at a density of 1.0×104 cells/well in a 96-well plate for 24 hr, and exposed to 1, 3, 10, and 30 μg/ml concentrations of PTW or PTE in either the presence or absence of CORT for 24 hr. At the end of the treat- ment, MTT solution (5 mg/ml; 20 μl/well) was added to each well and incubated for 4 hr at 37℃ in 5% CO. After in- 2 cubation, the supernatants were removed and the formazan crystals were solubilized in 150 μl DMSO.
Immunoblot analysis
To evaluate ERK and CREB protein levels, undifferentiated SH-SY5Y cells were seeded at a density of 2×105 cells/well in a 60 mm cell culture dishes and pretreated with 10 and 30 µg/ml of PTW or PTE for 24 hr and then exposed to 1 mM CORT for 20 min (ERK) or 12 hr (CREB). The cells were washed with cold PBS and lysed with NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, Inc., Waltham, MA, USA) according to the manu- facturer’s instructions. Nuclear and cytosolic lysates were either used immediately or stored at -80℃ until assayed. Protein concentration was determined by the BCA protein assay reagent using BSA as a standard. Protein lysates were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis using the Power Pac Basic electrophoresis apparatus (Bio-Rad, Hercules, CA, USA). The protein samples were transferred to a polyvinylidene difluoride (PVDF) membrane (0.45 µm pore size, Merck Millipore, Darmstadt, Germany). The membranes were blocked with 1× TBS/0.2% Tween-20 supplemented with 5% skim milk for 1 hr at 21± 2℃. Immunoblotting was performed overnight at 4°C with p-ERK1/2, ERK1/2, p-CREB, CREB, and β-actin antibodies. The membranes were incubated with diluted HRP-conjugated anti-rabbit IgG secondary antibodies for 1 hr at 21±2℃. Detection was performed by using a chemiluminescent western blot detection kit (Thermo Scientific, Inc.) in accordance with the manufacturer’s instructions.
Animals
Male C57BL/6J mice (Seven-week-old, weight 23-26 g) were purchased from Samtako Bio Korea (Osan, Republic of Korea). The animals were maintained at a constant room temperature of 22±3℃, with a humidity of 50±15%, and were kept at a 12/12 hr light/dark cycle. Mice were given free access to water and food. The all mice were acclimatized for 7 days prior to the experiments. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at Jeollanamdo Institute of Natural Resources Research (approval no. JINR-2201-2022).
Drug administration and chronic restraint stress (CRS) procedure
The mice were randomly assigned to five groups was as follows: Group I, (control group), treated with saline; Group II, (vehicle group), was exposed to CRS and treated with saline; Group III, (EO 10 group), was exposed to CRS and treated with escitalopram oxalate (EO, 10 mg/kg/day, positive control); Group IV, (PTW 100 group), was exposed to CRS and treated with PTW (100 mg/kg/day); and Group V, (PTW 200 group), was exposed to CRS and treated with PTW (200 mg/kg/day). All drugs were administered orally for three consecutive weeks. Thirty minutes after drug administration, CRS was induced for a period of 6 hr by clear plastic tubes, under a 60 W light for three consecutive weeks (11:00 and 5:00 p.m.).
Forced swim test (FST)
An FST was conducted in mice according to our previous reports [17]. The mice were individually placed in a plexiglass cylinder (diameter 15 cm) filled with 20 cm of water at 23±2℃. The mice were exposed to a 15 min of pre-test session. After 24 hr, the animals were forced to swim for a 6 min post-test session, and their immobility behavior was recorded and measured for the last 5 min using the SMART video tracking system (SMART v3.0, Panlab SL, Barcelona, Spain).
Open field test (OFT)
The general locomotor activity was evaluated using the OFT. Thirty minutes after the final drug administration, the mice were placed into a 60 cm×60 cm wooden box with 20 cm boundary walls, divided into 25 equal squares. Each mouse was gently placed in a corner of the apparatus and counted when the mice totally crossed from one square to the next for a 5 min session.
Pentobarbital-induced sleeping test and drug ad- ministration
To the evaluation of the PTW activity when administered in combination with pentobarbital sodium at a hypnotic dos- age, the mice were pretreated with PTW (100 and 200 mg/kg, oral) and diazepam (2 mg/kg, oral, positive control) for 45 min. And then, Pentobarbital sodium (45 mg/kg, intraperi- toneally; Sam Eung Ind. Co. Ltd, Seoul, Republic of Korea) was diluted in 0.9% saline and administered 45 min after the last drugs administration. After administration of pentobarbital sodium, the mice were observed for the sleep latency (loss of righting reflex), and duration of sleep (time between loss and regain of the righting reflex).
Serum sampling and measurement of CORT and melatonin levels
Mice were sacrificed immediately after each experiment. Blood samples were collected during the decapitation. The serum was separated by centrifuging the blood at 4,000 rpm for 20 min, and stored at -80℃ until further analysis.
Serum CORT levels were determined using ELISA according to the manufacturer’s instructions (Abnova Corp., Taipei City, Taiwan). Briefly, the serum was diluted to 1:100 with the diluted assay buffer that was provided. The 50 µl sample or standard was added to the pre-coated antibody plate that was provided with the kit, and then 25 µl of CORT conjugate and 25 µl of CORT antibody were immediately added to each well and incubated at 21±2℃ for 1 hr. After incubation, the plates were washed four times and, 100 µl of TMB substrate was added to each well for 30 min and stopped with 50 µl of stop solution.
Serum melatonin levels were determined using ELISA according to the manufacturer’s instructions (LSBio, Seattle, WA, USA). Briefly, the serum was diluted to 1:10 with the diluted assay buffer that was provided. The 50 µl sample or standard was added to the pre-coated antibody plate that was provided with the kit, and then 50 µl of biotinylated detection antibody was immediately added to each well and incubated at 37℃ for 45 min. After incubation, the plates were washed three times and 100 µl of HRP conjugate was added to each well for 30 min. After incubation, the plates were washed and TMB substrate were added to each well at 37℃ for 15 min and stopped with stop solution. Optical density values was determined at 450 nm using a spectrophotometer .
Statistical analysis
Data are presented as the mean ± standard error of the mean (SEM). Data were statistically evaluated by a Student’s t-test or one-way analysis of variance (ANOVA) using the GraphPad Prism version 5.00 for Windows (GraphPad soft- ware, San Diego, CA, USA) software program. The differences between the groups were assessed by using Duncan’s multiple range tests. A value of p<0.05 was considered statistically significant.
Results
Identification of PTW on GABA content
We determined the GABA content of the PTW extract using both HPLC-UV and HPLC-ELSD analysis. The results showed that GABA peak were detected in HPLC-ELSD analysis near 5.2 min, but were not detected in the HPLC-UV spectrum. As shown in Fig. 1C and Fig. 1D, GABA content was confirmed at 5.2 min in PTW by confirmed to HPLC- ELSD analysis, but not in non-PTW.
Fig. 1. HPLC-ELSD analysis. (A) Identify using HPLC chromatogram of standard GABA. (B) Analysis of standard GABA by HPLC with ELSD. Analysis of (C) non-PTW and (D) PTW by HPLC with ELSD. The retention time of GABA in PTW was detected approximately 5.2 min.
Protective effects of PTW on cell death in SH- SY5Y cells by CORT
To determine the protective effects of PT (water and 70% ethanol) extract on CORT-induced cell death in SH-SY5Y cells, we first measured the various concentrations of 1, 3, 10, and 30 µg/ml using MTT assay (Fig. 2). As shown in Fig. 2A, non-PTW did not able to protect SH-SY5Y cells against CORT-induced cell death. As shown in Fig. 2B and C, when the concentrations of PTW and PTE were 1, 3, 10, and 30 μg/ml, the cell viability of SH-SY5Y cells was not altered (p>0.05). As shown in Fig. 2B, PTW showed significantly enhanced the cell viability to 74.4±3.9%, 73.8±2.2 % and 66.0±0.7% at 3, 10 and 30 μg/ml, respectively, in comparison with the viability of CORT-treated cell as 53.6± 1.4% (p<0.05 and p<0.01, respectively). As shown in Fig. 2C, PTE showed significantly enhanced the cell viability to 68.4±2.4% and 74.1±2.7% at 10 and 30 μg/ml, respectively, in comparison with the viability of CORT-treated cells (p< 0.05).
Fig. 2. Effects of PT extracts on cell viability in SH-SY5Y cells. (A) SH-SY5Y cells were treated with CORT (1 mM) for 24 hr in the absence or presence of non-PTW (before feeding MSG) extract at the indicated concentrations. (B) SH-SY5Y cells were treated with CORT (1 mM) for 24 hr in the absence or presence of PTW extract at the indicated concentrations. (C) SH-SY5Y cells were treated with CORT (1 mM) for 24 hr in the absence or presence of PTE extract at the indicated concentrations. Cell viability was measured by MTT assay. The values are expressed as the mean ± standard error of the mean (n=3). ##p<0.01 and ###p<0.001 compared with the control group; *p<0.05 and **p<0.01 compared with the CORT group. CORT, corticosterone; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; PTW, Ptecticus tenebrifer water extract; PTE, Ptecticus tenebrifer 70% ethanol extract.
To further confirm the mechanisms of protective effects, the actions of PTW and PTE on the ERK1/2 and CREB pathways were investigated in CORT-induced cell death in SH-SY5Y cells using western blotting. As shown depicted in Fig. 3A and C, CORT-treated cells showed down-regu- lation of phosphorylated ERK1/2, whereas, PTW and PTE attenuated the CORT-induced down-regulation of ERK1/2 phosphorylation (p<0.05). Moreover, as shown in Fig. 3B and Fig. 3D, the expression levels of CREB phosphorylation were down-regulated in CORT-treated cells as compared with the control, while PTW and PTE prevented the CORT-induced down-regulation of CREB phosphorylation (p<0.01 and p<0.001, respectively). These results suggest that PTW and PTE exerts neuroprotective effects by protecting against the CORT-induced down-regulation of phosphorylated ERK1/2 and CREB expression.
Fig. 3. Effects of PTW and PTE on ERK1/2 and CREB expression in SH-SY5Y cells induced by CORT. (A) SH-SY5Y cells were exposed to CORT (1 mM) for 20 min in the absence or presence of 10 and 30 µg/ml concentrations of PTW or PTE treatment 24 hr prior to CORT treatment. (B) SH-SY5Y cells were exposed to CORT (1 mM) for 12 hr in the absence or presence of 10 and 30 µg/ml concentrations of PTW or PTE treatment 24 hr prior to CORT treatment, and the cells were lysed nuclear fraction. (C, D) Quantitative analysis of protein levels showed the relative densities of the protein bands. The values are expressed as the mean ± standard error of the mean (n=3). ##p<0.01 and ###p<0.001 compared with the control group; *p<0.05, **p<0.01, and ***p<0.001 compared with the CORT group. CORT, corticosterone; PTW, Ptecticus tenebrifer water extract; PTE, Ptecticus tenebrifer 70% ethanol extract.
Effects of PTW on behavioral changes and CORT levels in CRS-exposed mice
To investigate the effects of PTW on the duration of immobility behavior, we performed the FST. As shown in Fig. 4, CRS-exposed mice showed a significant increase in the immobility time compared with the control group (p<0.05). However, PTW 300 mg/kg (p<0.05) and EO (10 mg/kg, positive control) significantly led to a decreased in the immobility time compared to the CRS group. In contrast, as shown in Fig. 5, CRS-exposed mice showed a significant decrease in locomotor activity (p<0.01) compared with the control group. However, PTW 300 mg/kg significantly increased in the locomotor activity (p<0.05) compared to the CRS group.
Fig. 4. Effects of PTW in the FST in CRS-exposed mice. The effects of PTW (100 and 300 mg/kg/day) on the dura- tion of immobility behavior in CRS-induced mice. The values are expressed as the mean ± standard error of the mean (n=5). #p<0.05 compared with the control group; *p<0.05 compared with the CRS group. CRS, chronic restraint stress; EO 10, escitalopram oxalate 10 mg/kg; PTW 100, Ptecticus tenebrifer water extract 100 mg/kg; PTW 300, Ptecticus tenebrifer water ex- tract 300 mg/kg.
Fig. 5. Effects of PTW on locomotor activity in CRS-exposed mice. The values are expressed as the mean±standard error of the mean (n=5). ##p<0.01 compared with the control group; *p<0.05 compared with the CRS group. CRS, chronic restraint stress; EO 10, escitalopram ox- alate 10 mg/kg; PTW 100, Ptecticus tenebrifer water extract 100 mg/kg; PTW 300, Ptecticus tenebrifer wa- ter extract 300 mg/kg.
We investigated the effects of PTW on serum CORT levels in the CRS-exposed mice. As shown in Fig. 6, the levels of serum CORT were significantly increased in CRS-exposed mice (p<0.01) compared to those in the control group. However, PTW 300 mg/kg and positive control EO 10 mg/kg decreased the levels of CORT (p<0.05, respectively) levels compared to the CRS group. Our results suggest that PTW modulates anti-stress against CRS-exposed mice by decreasing CORT levels and inducing to behavioral changes, such as decreased immobility and increased movement.
Fig. 6. Effects of PTW on serum CORT levels in CRS-exposed mice. The levels of CORT were determined by ELISA kit (Abnova Corp.). The results were expressed as pg/ ml of CORT levels. The values are expressed as the mean±standard error of the mean (n=5). ##p<0.01 compared with the control group; *p<0.05 compared with the CRS group. CORT, corticosterone; CRS, chron- ic restraint stress; EO 10, escitalopram oxalate 10 mg/ kg; PTW 100, Ptecticus tenebrifer water extract 100 mg/kg; PTW 300, Ptecticus tenebrifer water extract 300 mg/kg.
Effects of PTW on pentobarbital sodium-induced sleep in mice
As shown in Fig. 7A, the PTW (100 and 300 mg/kg) led to significantly lower sleep latency compared to the control group (control, 214.4±5.2s; PTW 100, 166.6±1.7s; PTW 300, 138.0±2.5s, p<0.05 and p<0.01, respectively). As shown in Fig. 7B, PTW 300 mg/kg (13,089.8±462.6s, p<0.05) led to a significantly higher total sleep duration compared to the control group (8,999.4±264.5s). Similarly, the Diazepam 2 mg/kg also showed a decrease in the sleep latency (154.6± 5.9s, p<0.01) and an increase in the total sleep duration (14,741.9±509.6s, p<0.01) compared to the control group.
Fig. 7. Effects of PTW on the onset and duration of sleep in pentobarbital-treated mice. The sleep latency (A) and total sleeping time (B) were measured. The values are expressed as the mean ± standard error of the mean (n=10). *p<0.05, **p<0.01, and ***p<0.001 compared with the control group. DZP, diazepam 2 mg/kg; PTW 100, Ptecticus tenebrifer water extract 100 mg/kg; PTW 300, Ptecticus tenebrifer water extract 300 mg/kg.
Moreover, we investigated the serum melatonin levels in PTW-treated mice. As shown in Fig. 8, PTW 100 and 300 mg/kg-treated mice displayed significantly increased serum melatonin levels compared with the control group (p<0.001). In contrast, positive control diazepam had no significant ef-fect on the serum melatonin levels. These results suggested that PTW improve sleep by increasing the melatonin levels.
Fig. 8. Effects of PTW on serum melatonin levels in mice. The values are expressed as the mean±standard error of the mean (n=10). ***p<0.001 compared with the control group. DZP, diazepam 2 mg/kg; PTW 100, Ptecticus tenebrifer water extract 100 mg/kg; PTW 300, Ptecti- cus tenebrifer water extract 300 mg/kg.
Discussion
GABA plays functions as the primary inhibitory neurotransmitter in the central nervous system (CNS), and is known to be involved in the modulation of physiology mechanisms in the CNS, which improvement of stress, depression, dementia, and insomnia by promoting brain cell metabolism [20]. Previous studies have reported that MSG is involved in GABA synthesis because it can produce glutamic acid. GABA is synthesized via glutamate catalyzed by glutamate decarboxylase (GAD), which is also involved in bio transformation of MSG to GABA by microorganisms, including bacteria strain [6, 9]. In the present study, we first investigated whether the GABA content was increased by feeding MSG to Ptecticus tenebrifer. We confirmed that GABA content was detected in the HPLC-ELSD analysis, but not in HPLC- UV spectrum. Moreover, our results confirmed that GABA content was confirmed in PTW, which confirmed the HPLC- ELSD analysis, but not in non-PTW. ELSD analysis does characterized not depend on the analyte's optical properties, and can detect almost all the components [14].
High CORT levels can lead to an induces ER stress [13]. ER stress is induced cell death and can also trigger the down-regulation of ERK1/2 and CREB, which mediate cell survival in oxidative stress [2]. In this study, we demonstrated that PTW and PTE extracts have neuroprotective effects via increased ERK1/2 and CREB phosphorylation compared with the CORT-treated cells. Furthermore, high levels of CORT are involved in the occurrence of insomnia and depression [11]. CRS models may induce behavioral changes characterized by increased immobility and on the other hand by decreased movement. It is also stimulates the release of CORT [12]. In this study, we demonstrated the anti-stress effect of PTW in CRS-exposed mice. The PTW decreased immobility time in FST test and increased locomotor activity in OFT test and significantly decreased serum CORT levels. These results suggested that the anti-stress effect of PTW may be related to the regulation of CORT and ERK/CREB signaling pathways.
Diazepam is one of known benzodiazepines, commonly used to sedative and hypnotic drugs [22]. It has been reported that diazepam, binds to the GABA receptor at the α subunits A (α3, α4 and α5), a β subunit (β2), and a γ subunit (γ3) [10]. The most common symptoms of insomnia patients is difficulty falling asleep or staying asleep, which is accompanied by related sleep problems as primary symptoms [8]. In the present study, PTW significantly decreased the sleep latency and increased the sleeping time in pentobarbital (45 mg/ kg)-treated mice. These results showed that PTW had a sleep improvement effects similar to those of diazepam, a positive control.
Additionally, melatonin is known to improve sleep quality. Melatonin is produced by the pineal gland, and studies exogenous melatonin supplements have shown no studies serious side effects to date [21]. Melatonin competes for benzodiazepine drug-binding sites (such as diazepam), which are shown to decrease shown chronic diazepam treatment decreased of melatonin levels in the pineal gland [4]. Our study suggests that PTW acts by enhancing melatonin levels. In contrast, diazepam, a positive control, had no significant effect on melatonin levels. These results suggest that PTW showed regulation of sleep latency and total sleep duration via regulation of melatonin.
In conclusion, the present study demonstrates that the anti stress and sleep-enhancing effects of PTW are mediated by increased ERK/CREB phosphorylation in CORT-induced cell death and are associated with inhibition of CORT levels in CRS-exposed mice.
Acknowledgement
This paper has been written with the support of Jeollan- nam-do (‘2021 R&D supporting program’ operated by Jeonnam Technopark).
The Conflict of Interest Statement
The authors declare that they have no conflicts of interest with the contents of this article.
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