Preparation of pumpkin extract (PE)
Fresh pumpkin (L.Cucurbita pepo) was purchased from X local market at, X and was identified by a specialist at the Botany department, Faculty of Science, X University. Extraction of pumpkin was done according to [12]. Removal of seeds, cutting the raw fruits and its skin with a slicer, then drying by using the lyophilize machine freeze-drier (FD5508; ILShinBase Co.,Ltd., Korea) and finally crushing by grinding with electrical machine. The powder was passed through a 40-mesh sieve to get the fine powder which is stored in an airtight container.
The dried powder (50g) of pumpkin was used to prepare the ethanolic extract of as was previously described [13]. PE was stored in a suitable container till use after being dissolved in distilled water at a dose of 100 mg/kg and administered by gavage once daily for two weeks [12].
Analysis of the PE
The components of PE were identified using Trace Gas Chromatography and Mass Spectrometer (GC–MS) (Thermo Scientific, Austin, TX, USA) with a direct capillary column TR–5MS (30 m 0.25 µm 9 0.25 µm film thickness.
Experiment design:
Forty male albino rats (30–40g), purchased from animal house at X Research Center, were used in this study. The male gender was selected to nullify the effect of gender as a confounder. No other confounders existed in this study. Rats were left for one week to acclimatize in the laboratory conditions under the standard laboratory condition. The rats were randomly, using simple random technique, into control and experimental groups. The control group included ten rats that left unexposed to stress. The experimental group included thirty rats were subjected to CUMS procedure by through exposing them to diverse types of stressors at different times during the day for 4 weeks in order to prevent habituation to the stressors. The CUMS procedure was fully described in previous works [14]. These thirty rats were then divided into three groups (n=10); untreated (CUMS), FLU-treated group (CUMS+FLU) and Pump-treated (CUMS+Pump) groups. Fluoxetine (Dar Al Dawa Pharmaceuticals Co., Ltd., Amman, Jordan), was dissolved in 0.03% sodium carboxymethyl cellulose (CMC-Na), administered by gavage (20 mg/kg) once daily for two weeks [15]. After two weeks the behavioral tests were performed and the sampling process was started.
Assessment of behavioral changes
In order to confirm the effect of CUMS, the forced swim test (FST) was conducted for all rats after 4 weeks, as was previously described by [16]. The total time, in seconds, spent by the rat without mobility during the 6 minutes was determined. Immobility was considered as "the cessation of limb movement, except for the minor movement necessary to keep the rat afloat".
Regarding the elevated plus maze (EPM) test; it was performed according to [17]. The number of closed arm entries during 6 min and time spent by each mouse inside the open and closed arms were recorded in seconds.
Assessment of serum corticosterone, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) levels
Blood samples were obtained for biochemical assessment from the intra-orbital sinus, centrifuged at 3000 rpm for 15 min at 4°C to obtain the serum then kept at −18°C.
Corticosterone level was measured using ELISA kits (ALPCO Diagnostics, Orangeburg, NY, USA) according to the manufacturers 'instructions. TNF-α, IL-6 (quantakin R&D system, USA Kit) were measured in the serum by ELISA according to the manufacturer instructions.
Assessment of malonaldehyde [18] in the serum:
Malonaldehyde (Biodiagnostic; Egypt) was measured for the estimation of damage by reactive oxygen species (H2O2) according to the method described by [19].
Assessment of Superoxide dismutase (SOD), Glutathione peroxidase (GPX), catalase (CAT) in the serum:
To assess the antioxidant effect of PE, the levels of SOD (Biodiagnostic; Egypt), GPX (Randox Labs, Crumlin, UK) and CAT (Biodiagnostic; Egypt) were measured in serum according to the previously described methods [19].
Quantitative real-time PCR (qRT-PCR):
Ribonucleic acid (RNA) was extracted from 100 mg of formalin-fixed paraffin-embedded (FFPE) section; 1 mL of xylene was used for deparaffinization, incubation at 56°C for 15 minutes, centrifugation for 10 minutes at 13 000 g. The supernatant was discarded and the pellet washed twice with 1 mL 100% ethanol centrifuged, the supernatant was discarded, and 1 mL Trizol was added to the pellet [20]. Extraction of total RNA using Trizol was done according to the supplier instruction (Invitrogen Life Technologies, Carlsbad, CA, USA). The details of the procedure was previously described [21]
The cDNAs obtained were amplified by using PCR Master Mix (Bioneer) with primers designed by Metabion International (Semmelweisser, Germany) as follows: BDNF (forward 5′-TATTTCATACTTCGGTTGC-3; reverse 5′-TGTCAGCCAGTGATGTCG-3′) and β-actin (forward 5′-TCTGGCACCACA CCTTCTA-3; reverse 5′-AGGCATACAGGGACAGCAC-3).
The assay was performed according to [20].The PCR reactions were kept track of by determining the strength of the fluorescence brought on by SYBR Green Dye intercalation to the double-stranded DNA (dsDNA), melting curve evaluation was done to verify the specificity of the products.
Histopathological assessment:
At the end of the experiment, rats were anesthetized with 4% Isoflurane (SEDICO Pharmaceuticals Company, Cairo, Egypt) in 100% oxygen then euthanized by cervical dislocation. Salivary glands were dissected out, fixed in 10% neutral buffered formalin then processed into paraffin blocks to be sectioned at 4-μm thickness and stained with Haematoxylin and Eosin (H&E).
A set of slides were immunohistochemically stained using the streptavidin–biotin–peroxidase technique. Anti-alpha Smooth muscle actin (ASMA) antibody (Biocare Medical, Pacheco, USA, at 1/100 dilution), a marker of myoepithelial cells (MECs) [22]. Anticaspase-3 (Dako Company, Cairo, Egypt, at 1/200 dilution), a marker of apoptosis was used. Anti-BDNF antibody (Santa Cruz Biotechnology, Texas, USA at 1/400 dilution) was also used.
Olympus BX-51 (Tokyo, Japan) microscope connected to digital camera was used to for photographing. Immunoexpression of the studied antibodies (expressed as percentage area) was assessed in 30 fields at ×200 magnification using Pro Plus image analysis software version 6.0. Histopathological assessment was performed by histopathologist blind to the groups of the experiment. The principal investigator was aware of the group allocation at the different stages of the experiment.
Statistical analysis
Statistical Package for the Social Sciences (SPSS, v.16) was utilized to analyze the data and the results were presented as mean and standard deviations (SD). One way ANOVA (F test), followed by a Bonferroni post hoc test was done to avoid a multiple-comparison effect. The sample size was determined a prior using power analysis. The experimental unit of the study was the rat. There were no inclusion or exclusion criteria of the animals. No experimental units were excluded during the analysis. Significance was considered at a p value ˂ 0.05.