Brain-derived Neurotropic factor (BDNF) mediates the protective effect of Cucurbita pepo L. on salivary glands of rats exposed to chronic stress evident by structural, biochemical and molecular study

Abstract Acute and chronic stresses affect the salivary glands, representing the source of plasma BDNF during stressful conditions. Pumpkin is a medicinal plant with an evident antioxidant, anti-inflammatory and potential antidepressant effects. Objective To assess the structural and biochemical effects induced by exposure to chronic unpredictable mild stress (CUMS) on salivary glands of albino rats, and to evaluate the role of pumpkin extract (Pump) in ameliorating this effect. Methodology Four groups (n=10 each) of male albino rats were included in this study: the control, CUMS, Fluoxetine-treated and Pump-treated. The corticosterone, the pro-inflammatory cytokines, tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), and the oxidant/antioxidant profile were all assessed in the serum. The level of BDNF mRNA was measured in the salivary glands using qRT-PCR. Histopathological changes of the salivary glands were also assessed. Results The depressive-like status was confirmed behaviorally and biochemically. Exposure to CUMS significantly up-regulated (p<0.001) the level of serum corticosterone. CUMS induced degenerative changes in the secretory and ductal elements of the salivary glands evident by increased apoptosis. Both Fluoxetine and Pumpkin significantly up-regulated (p<0.001) BDNF expression in the salivary glands and ameliorated the CUMS-induced histopathological and biochemical alterations in the salivary glands. Pumpkin significantly (p<0.001) increased the serum levels of antioxidant enzymes SOD, GPX and CAT, and reduced the serum levels of the pro-inflammatory cytokines TNF-α, IL-6. Conclusion Pumpkin ameliorates the depressive-like status induced in rats following exposure to chronic stress through exerting a promising anti-inflammatory, antioxidant and anti-depressant-like effects. The pumpkin, subsequently, improved stress-induced structural changes in the salivary glands that might be due to up-regulation of BDNF expression in the glands.


Introduction
Stress is a critical risk factor for numerous neuropsychiatric disorders, including depression and schizophrenia. Moreover, exposure to chronic stress results in neurochemical alterations and depressive behavior. 1 The World Health Organization (WHO) expected that, in 2020, depression can be the second important disability cause in the world and, in 2030, the major depressive disorders will represent the highest among of healthcare spending. 2 However, the precise mechanism underlying depression is not fully elucidated and the results obtained from the animal models are not consistent with those of clinical trials. 3 The model of chronic unpredictable mild stress (CUMS) in rodents has been thought to be the suitable model for discovering the pathophysiology of depression due to its high face, structural, and predictive validity. 4 Salivary glands are sensitive to stress, and their secretion include brain-derived neurotropic factor (BDNF), which is produced by the salivary glands and amygdala in rats in response to acute stress. 1 On the other hand, chronic stress reduces BDNF expression in amygdala, hippocampus, and cingulate cortex. This is the reason why several studies on stressrelated disorders have focused on it as a biomarker of stress. 1,5 High level of BDNF is a crucial neuroprotective response under acute immobilization-associated stressful conditions. Furthermore, BDNF crosses the blood brain barrier and plays a vital role in homeostasis under stressful conditions. 6 However, the relationship between chronic unpredictable stress and salivary compounds remains unclear.
Inflammation was described to be an underlining mechanism in the development of depression induced by chronic stress. 7 Moreover, previous studies postulated that depression is an oxidative and inflammatory disorder. 8 Therefore, we assess the inflammatory cytokines tumor necrosis factoralpha (TNF-α) and interleuin-6 (IL-6) in this study.

Fluoxetine (FLU), a classic antidepressant belonging
to the selective serotonin reuptake blockers, was used for pharmacological validation of new therapies and drugs. 9 It also induces its effects on rats exposed to CUMS through reduction of neuroinflammation in prefrontal cortex. 10 Herbal medicine is used individually or in combination with pharmacological drugs in treatment of different medical disorders. Pumpkin (Cucurbita pepo L.; Cucurbitaceae) is a popular plant with many medical properties such as anti-diabetic, antioxidants, anti-inflammatory, anti-carcinogens and phytochemical, among others. 11 Forced-swimming and tail-suspension tests were used to study the antidepressant-like effect of pumpkin seed extract compared to imipramine. Although the extracts showed a significant anti-depressive-like effect, the mechanism of this effect was not clear. 12 Furthermore, pumpkin seeds are defined among antidepressant food with an score of 47%. 13 Pumpkin (Cucurbita moschata) was has a promising anti-fatigue and an ergogenic activity as it increased the maximal swimming time of in mice a dose-dependent manner. 14 In a recent study, methanolic extract of Cucurbita pepo seeds could inhibit haloperidol-induced motor dysfunction and anxiety and recover locomotor activity. 15 This study was conducted to assess the structural and biochemical effect induced by exposure to CUMS on the of albino rats' salivary glands, and to evaluate the role of pumpkin extract in ameliorating this effect. Thus, as the primary outcome of this study, we hypothesized that pumpkin extract ameliorates the chronic stress-induced structural changes in the salivary glands of rats.

Methodology
Preparation of pumpkin extract (PE) Fresh pumpkin (Cucurbita pepo L.) was purchased from the local market at Jeddah, Saudi Arabia and was identified by a specialist at the Botany department, Faculty of Science, King Abdulaziz University (KAU).
Pumpkin extract was obtained according to the method previously described. 14 The fruits had their seeds and skin removed, and then were cut with the aid of a slicer. Next, the fruits were dried by using the lyophilize machine freeze-drier (FD5508; ILShinBase Co., Ltd., Korea). Finally, they were crushed by grinding with electrical machine. The powder was passed through a 40-mesh sieve to get the fine powder, which was stored in an airtight container.
The dried powder (50g) of pumpkin was used to prepare the ethanolic extract as was previously described. 16 The yield of extraction was 41%. 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. 14 J Appl Oral Sci. and water ad libitum. At the time of starting the experiment they were weighted 150 to 200 g and aged from 2 to 3 months. The rats were randomly distributed, using simple random technique, into control and experimental groups. The control group included 10 rats that were not exposed to stress.
The experimental group included 30 rats that were subjected to CUMS procedure through exposure to diverse types of stressors at different times during the day for four weeks, to prevent habituation to the stressors. The latter included tilting the cage at 30 degrees, placing rats in an empty cage with water at the bottom, with wet sawdust or with in soiled cages of other mice, as well as restrain stress and reversal of the light/dark cycle. The rats were exposed for each stressor for 4 hours at different points during the day to prevent habituation. The CUMS procedure was fully described in previous works. 17 These 30 rats were then divided into three groups (n=10): untreated (CUMS), FLU-treated group (CUMS+FLU) and Pumptreated (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. 18 After two weeks the behavioral tests were performed and the sampling process was started.

Assessment of behavioral changes
To confirm the effect of CUMS, the forced swim test (FST) was conducted in all rats after 4 weeks, as previously described. 19 Preswim exposure was performed 24 h before the test session. During the latter, the total time in seconds spent by the rat without mobility during the 6 minutes was determined. The immobility was considered as "the cessation of limb movement, except for the minor movement necessary to keep the rat afloat". at 13,000 g. The supernatant was discarded, the pellet was washed twice with 1 mL 100% ethanol centrifuged and 1 mL Trizol was added to the pellet. 22 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. 23 The cDNAs obtained were amplified 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 The assay was performed according to a previous study. 22 The PCR reactions were kept track of by 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 a histopathologist blinded to the experimental groups.
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).

Data normality was tested using Kolmogorov-Smirnov
test. For the parametric data, the different groups were compared using one-way ANOVA (F test), followed by Bonferroni post-hoc test. For nonparametric data (e.g., immunohistochemistry and gene expression variables), one-way analysis of variance (ANOVA) (Kruskal-Wallis test) followed by a Dunn's post-hoc test was done to avoid a multiple-comparison effect.
The sample size was determined using power analysis.
The experimental unit of the study was the rat. No inclusion or exclusion criteria were adopted regarding the animals. No experimental units were excluded during the analysis. Significance was considered at a p<0.05.

Analysis of the pumpkin extract
The constituents of PE were determined using GC-MS and were found to include mainly Oleic acid (about 56%), Hexadecanoic acid (about 9%), Decenoic acid (5%) and other components, whereas the Linoleic acid represent only 1% (Table 1).
Effect of exposure to CUMS on corticosterone After 4 weeks of exposure to CUMS, a significant (p<0.001) increase in the serum corticosterone level in CUMS-exposed rats was observed. Both FLU (p=0.002) and Pump (p<0.001) administration significantly reduced serum corticosterone level ( Figure 1A, Table   1).

Effect of exposure to CUMS on behavior
Exposure to CUMS leads to development of depressive-like behavior that was evident in the rat by the significant increase (p<0.001) in the mean immobility time compared to the control.
Administration of FLU and Pump along with the CUMS reduced the depressive-like behavior, as evidenced by the significant decrease (p=0.02, p<0.001) in immobility time compared to CUMS, respectively (Table   1 and Figure 1B). decreased it compared to the CUMS group (Table 1 and Figure 1C, 1D).

Effect of PE on the anti-inflammatory cytokines
The pro-inflammatory cytokines levels were measured to detect the anti-inflammatory effect of PE.
Serum TNF-α, IL-6 levels were significantly increased (p<0.001) in CUMS group compared to the control, Parameter (in the serum) The data were compared using one-way ANOVA test, followed by Bonferroni post-hoc test and one-way ANOVA (Kruskal-Wallis test) followed by a Dunn's post-hoc test, (n=10). Data are shown as mean ± SD. P represents p value versus control group. P# represents p value versus CUMS group. CUMS: chronic unpredictable mild stress; FLU: Fluoxetine; Pump: pumpkin; ASMA: alpha-smooth muscle actin; BDNF: brain-derived neurotropic factor; SMG: submandibular gland. whereas their levels significantly decreased (p<0.001) in both FLU and Pump-treated groups (Figure 2A, 2B and Table 1).

Effect of PE on the oxidant/antioxidant profile
Exposure to CUMS was associated with a significant increase (p<0.001) in serum MDA, whereas it significantly decreased (p=0.03) in Pump-treated group among the treated groups ( Figure 3C).
Exposure to CUMS was also associated with a Immunostaining with ASMA was performed to assess the integrity of the myoepithelial cells (MECs) present around the glandular acini and ducts. We observed that SMG of the control group had strongly positive immunoreactivity for ASMA, whereas exposure to CUMS resulted in a significant reduction (p<0.001) Figure 1-Confirmation of the depressive status in rats after exposure to CUMS was done through assessing serum corticosterone level (A), immobility time (B) during forced swimming test (FST) and elevated plus maze test (EPM) (C, D). CUMS: chronic unpredictable mild stress, FLU: fluoxetine, Pump: pumpkin. Data were compared using one way ANOVA test, followed by Bonferroni post-hoc test, (n=10). * indicates significant difference compared to the control group, # indicates significant difference compared to the CUMS group. Significance is considered at p˂0.05 J Appl Oral Sci. 2021;29:e20201080 7/14 in this reaction compared to the control. There was a significantly increase (p<0.001) of ASMA expression in both salivary acini and ducts in CUMS+Pump group compared to the CUMS group, whereas no such effect was detected in CUMS+FLU group ( Figure 3C, Figure   4I-L). BDNF was weakly expressed mainly in the duct system of SMG of the control group, whereas was significantly increased (p<0.001) after exposure to CUMS compared to the control rats. Further significant increase (p=0.03, p=0.01) of BDNF was observed in FLU-or Pump-treated groups when compared to the CUMS group ( Figure 3D, Figure 4M-P).

Parotid gland
The parotid gland of control rats was formed of closely packed, intact pure serous acini and striated ducts, besides the other ductal components. After exposure to CUMS, the serous acini appeared smaller in size and some of them were atrophic, whereas some of the acinar cells were vacuolated. These changes were also observed in the glands of rats treated with FLU or Pump, but to a lesser degree ( Figure   Immunoreactivity for BDNF was detected in striated ducts of the control parotid glands, which was significantly increased (p<0.001) following the exposure to CUMS compared to the control group, and further significantly increased (p<0.04, p=0.02) in FLU and Pump-treated groups compared to the CUMS group ( Figure 3D, Figure 5M-P).

Sublingual gland (SLG)
We observed that the SLG of the control group was formed of closely packed, intact pure mucous acini (MA), striated ducts and other ductal components.
After exposure to CUMS, many MA and ducts appeared smaller with disorganized architecture and vacuolated

cells. Most of the acini and ducts of SLG in FLU or
Pump-treated appeared intact (Figure 6 A-D). Saliva is essential to preserve oral health, since alterations in the salivary gland function could affect CUMS: chronic unpredictable mild stress, FLU: fluoxetine, Pump: pumpkin. ASMA: alpha-smooth muscle actin, BDNF: brain-derived neurotropic factor, SMG: submandibular gland Data were compared using one way ANOVA (Kruskal-Wallis test) followed by a Dunn's post-hoc test, (n=10). Data are shown as mean ± SD. * indicates significant difference compared to the control group, # indicates significant difference compared to the CUMS group. Significance is considered at p˂0.05 J Appl Oral Sci. 2021;29:e20201080 9/14 oral tissue integrity. 28 Function of the salivary glands is essentially controlled by the autonomic nervous system, so it is directly affected on exposure to stresses. 28 BDNF is among the stress markers in saliva that have been identified in previous studies.
We described BDNF measurement as a useful tool in evaluating the activation of the sympathetic adrenomedullary system. 29 This is the reason why our study focused on BDNF.
Our study aimed to investigate the effect of exposure to CUMS on the structure of rat salivary glands structure and the possible effect of PE in ameliorating this impact. We noticed that exposing rats to CUMS for 4 weeks resulted in the development of a depressive status and anxiety evidenced by the prolongation of the immobility time of the FST and by the EPM test, respectively. An increase in the amount of serum corticosterone was correlated with this depressive-like state. These results were in line with some previous studies. 9,30 The observed significant increase of serum corticosterone level, known as a stress marker, in the chronically stressed rats was due to the overactivity of hypothalamus-pituitary-adrenal (HPA) system mediating the reaction to stress. 31 The levels of salivary cortisol are strongly associated with the levels of blood Antioxidants act as "free radical scavengers" that prevent and recover the harmful effects of ROS. CAT is the main antioxidant enzyme implicated in hydrogen peroxide (H 2 O 2 ) degradation. 38 In our study, exposure to CUMS was associated with a significant reduction in serum SOD, GPX and CAT which indicates decreased total antioxidant capacity. Moreover, many studies showed that CUMS procedure markedly resulted in an oxidative damage in the prefrontal cortex, striatum, and hippocampus of rats, evident by up-regulation of MDA and down-regulation of SOD and CAT. 39 The previous data support our suggestions that oxidative stress, evident in our study, could be also behind the pathological alterations noted in CUMS-exposed salivary gland. Therefore, the structural observations were strengthened and explained by the biochemical results.
CUMS: chronic unpredictable mild stress, FLU: fluoxetine, Pump: pumpkin, ASMA: alpha smooth muscle actin, BDNF: brain-derived neurotropic factor. Since inflammation has been proposed to be a causative mechanism in the development of chronic stress-induced depression, 7 we assessed the inflammatory cytokines TNF-α and IL-6. The results showed a notable elevation of serum TNF-α and IL-6 of CUMS-exposed rats indicating the inflammatory process associated with chronic stress-induced depressive-like status. This is in agreement with previous studies that described depression as an oxidative and inflammatory disorder. 8 Furthermore, inflammatory cytokines, IL-1 β, IL-6 and TNF-α, reported to be elevated in rats that exhibited a depressive-like behavior. 42 These cytokines interfere with the metabolism of serotonin, an important neurotransmitter, and interfere with the synaptic plasticity by altering of BDNF and its receptor, tropomyosin receptor kinase B (TrkB) that plays a crucial role in the pathophysiology of depression in the prefrontal cortex and hippocampus. 43 In our study, FLU and Pump administration markedly reduced serum pro-inflammatory cytokines. These findings were consistent with those of Kim, et al. 34 (2016), who reported that pumpkin significantly reduced the protein levels of TNF-α and IL-6 in the serum of rats that exhibited a depressive-like behavior animals.
In our study, immunohistochemical detection of BDNF in the ductal cells confirmed that salivary glands produced BDNF protein. Immunoexpression of BDNF in SMG, SLG and parotid glands notably up-regulated on CUMS: chronic unpredictable mild stress, FLU: fluoxetine, Pump: pumpkin, ASMA: alpha smooth muscle actin, BDNF: brain-derived neurotropic factor. exposure to CUMS. This was confirmed by the increase in BDNF mRNA in the salivary glands. These findings also reported, by some researchers, an increase in salivary gland expression of BDNF after exposure to different types of stress. 9,28 This indicated that salivary gland is sensitive and responsive to stress as evidenced by increased BDNF expression.
In our study, treating rats from depressive-like status resulted from exposure to CUMS with fluoxetine or pumpkin induced a notable up-regulation of salivary BDNF expression compared to the untreated rats.
These findings are supported by previous studies that showed that FLU and Pump increased hippocampal BDNF level in CUMS-exposed mice. 6,34 Saruta, Sato and Tsukinoki 6 (2010) stated that "secretion of BDNF may represent a protective mechanism that plays important roles in homeostasis under stress conditions". Therefore, the structural, biochemical and molecular observations collected in this study, suggested the BDNF as a molecular mechanism through which Pumpkin could modulate the protective effect on the salivary glands during chronic stress.
In conclusion, our study showed that Pump ameliorates depressive-like status resulted from exposure to chronic stress by exerting a promising antioxidant, anti-inflammatory and antidepressant-like effects. Pump subsequently improves the structural changes induced by stress in the salivary glands which might be due to up-regulation of salivary BDNF expression. We also recommend future studies to test the efficacy of pumpkin in improving stress-related salivary alterations in humans.