MATERIALS AND METHODS

Study area: The study was carried out at the Research Center, College of Medicine, King Khalid University, Abha, Saudi Arabia from September-December, 2019.

Animals: All rat studies were overseen and approved by the Medical Research Ethical Committee at King Khalid University, Abha, Saudi Arabia. Ref no: (Rec. No. 2014-06-09). Male Wistar rats (total 24 rats) at 8 weeks and weighing 150-200 g were used for these studies. They had free access to food and water and were housed under a constant temperature of 23±1°C with a 12 hrs light/dark cycle.

Experimental design: After a one-week adaptation period, rats were randomly assigned into 4 groups (n = 6 each) as follows: 1. Control group: Received normal saline. 2. Quercetin treated group (QUR): Rats received QUR (50 mg kg^–1)²⁰. 3. Chronic unpredictable stress (CUS) group: A model group and were exposed to CUS protocol, as detailed below and received normal saline. 4. CUS+QUR treated group: were exposed to CUS with a concomitant daily dose of QUR (50 mg kg^–1). All treatments were administered as 1 mL, i.p. for three consecutive weeks daily.

Chronic unpredictable stress (CUS) protocol: CUS protocol was induced as previously described²¹ with modification. Briefly, a set of chronic unpredictable stressors were used to induce brain injury in rats that lasted for 3 weeks.

Assessment of depressive-like behaviours; forced swimming and sucrose preference tests: The experiment of assessing depressive-like behaviour in the form of a forced swimming test was carried out as previously described²² to induce brain injury in rats for three weeks. All scoring was done by a single trained person, blind to experimental conditions. The sucrose preference test procedure was performed as previously described²³. The sucrose preference was calculated using the following formula:

Preparation of brain tissues for analysis: At the end of the experimental period, blood samples were collected by cardiac puncture under anaesthesia (sodium thiopental at 40 mg kg^–1 b.wt.) and rats were then culled using cervical dislocation. Brain tissues were harvested and washed with iced Phosphate-Buffered Saline (PBS). The brains were cut into longitudinal sections. Parts of the brains were homogenized in a cold phosphate buffer, pH 7.4, containing EDTA. The supernatant obtained was distributed in separate tubes and stored at -70°C for biochemical analysis. Other parts of the brains were stored at -70°C for RNA extraction.

Detection of p53 and Bax messenger RNAs by reverse transcriptase-polymerase chain reaction: As previously described²⁴, total RNAs were isolated from freshly dissected rats’ brains using the RNeasy Mini Kit (Qiagen Pty, Victoria, Australia). The RNA was reverse-transcribed for a single strand cDNA synthesis (Invitrogen) and amplified by PCR for the tumour suppressor p53, apoptosis regulator Bax and β-actin. The PCR products were separated by 2% agarose gel electrophoresis and visualised by ethidium bromide. Gel images were scanned and quantified by densitometry using the NIH image software.

Determination of tissue levels of nitrosative and oxidative stress and apoptosis and survival biomarkers: ELISA kits were used according to the manufacturers’ instructions to determine the tissue levels of malondialdehyde (MDA) for lipid peroxidation (Cat No. NWK-MDA01, NWLSS, USA), Superoxide Dismutase (SOD) (Cat. No. 706002, Cayman Chemical, Ann Arbor, MI, USA),Glutathione Peroxidase (GPx) (Cat. No. 703102, Cayman Chemical, Ann Arbor, MI, USA), caspase 3 (Cat. No. R5814), B-cell lymphoma 2 (Bcl-2) (Cat. No. R6813) and inducible Nitric Oxide Synthase (iNOS) (Cat. No R6663) were purchased from STZ ELISA (USA).

Statistical and morphometric analysis: Statistical analysis was performed by using the Graph pad Prism statistical software package (version 6). The data were expressed as mean±standard deviation (SD).One-way analysis of variance (ANOVA) was performed followed by Tukey’s t-test. Using the "Leica Qwin 500 C" image analyzer (Cambridge, UK), the degree of cerebral cortex damage was obtained in 10 non-overlapping high-power fields/ rat of H and E-stained sections. Quantitative data were tabulated as a means and Standard Deviations (SD) and compared using analysis of variance (ANOVA) followed by post hoc analysis (Tukey test). A significant difference was considered when p-value 0.05. Calculations were made on SPSS software (version 19).

RESULTS

Quercetin inhibits CUS-induced brain injury biomarkers and depression: To test the hypothesis that quercetin in this modified model of CUS can protect against brain injury and depression caused by CUS, we measured iNOS, MDA as a by-product of lipid peroxidation and the antioxidants SOD and Gpx using the ELISA method in brain tissue homogenates of all groups of rats. Compared with the control untreated group, CUS significantly (p<0.0001) augmented iNOS (Fig. 1a) and MDA (Fig. 1b) and ameliorated SOD (Fig. 1c) and Gpx (Fig. 1d) tissue levels, which were significantly (p<0.0001; CUS vs CUS+QUR) protected by quercetin. However, the levels of MDA in the treated group (CUS+QUR) were still significant (p<0.046) to the two control groups (Fig. 1b) which mean, partial inhibition of MDA was achieved by quercetin. We further assessed depression-like behaviour levels in these animals in the presence and absence of quercetin. As shown in Fig. 2, CUS significantly (p<0.0001) inhibited mobility (Fig. 2a; increased immobility) and climbing ability (Fig. 2b) that were significantly (p<0.0001; CUS vs. CUS+QUR) protected by quercetin. However, exposing rats to CUS for 3 weeks did not affect their swimming ability (Fig. 2c). Sucrose consumption (Fig. 2d) was inhibited by CUS and protected with quercetin. The degree of protection exerted by quercetin was partial in (A).

Quercetin protects CUS-modulated apoptosis and survival biomarkers in brain tissues: We then tested the apoptotic p53-Bax-caspase-3 axis cell signalling in brain tissue CUS strongly (i) augmented p53 and Bax cDNA message (Fig. 3a-c); (ii) augmented caspase-3 protein expression (Fig. 3d) and (iii) inhibited the survival protein Bcl-2 (Fig. 3e), which were effectively (p<0.0001; CUS vs. CUS+QUR) protected by quercetin. However, complete protection was only seen in (E).

Correlation between p53 or Bcl-2 and biomarkers of depression and nitrosative and oxidative stress: The correlation between either p53 or Bcl-2 scorning and tissue levels of nitrosative and oxidative stress and depression biomarkers were determined to further support the link between p53 and Bcl-2 and brain injury and to further confirm and characterize the role of QUR as being stable and an appropriate agent in brain injury rats. A significant (p<0.0001) correlation was observed between p53 and iNOS (r = 0.849) (Fig. 4a), p53 and MDA (r = 0.918) (Fig. 4b), p53 and climbing ability (r = -0.898) (Fig. 4c), p53 and sucrose consumption (r = - 0.915) (Fig. 4d), Bcl-2 and iNOS (r = -0.936) (Fig. 4e), Bcl-2 and MDA (r = - 0.946) (Fig. 4f), Bcl-2 and climbing ability (r = 0.921) (Fig. 4g) and Bcl-2 and sucrose consumption (r = 0.955) (Fig. 4h).

Fig. 1(a-d):

Quercetin protects the modulation of nitrosative and oxidative stress and anti-oxidants biomarkers caused by CUS Brain tissue levels of (a) iNOS, (b) MDA, (c) SOD and (d) GPx were measured at the end of the experiment in all groups of rats; control, QUR, CUS and CUS+QUR groups. Results represent the mean (±SD); n = 6 for each group. All shown p-values are significant; ap<0.05 versus control, b p<0.05 versus QUR, cp<0.05 versus CUS. CUS: Chronic uncontrolled stress, QUR: Quercetin, iNOS: Inducible nitric oxide synthase, MDA: Malondialdehyde, SOD: Superoxide dismutase, GPx: Glutathione peroxidase

Fig. 2(a-d):

Quercetin inhibits CUS-induced depression-like behaviour levels in rats Markers of depression, (a) Immobility, (b) Climbing ability, (c) Swimming ability and (d) Sucrose consumption were assessed at the end of the experiment in the animal groups used in this study; Control, QUR, CUS and CUS+QUR groups. Results represent the mean (±SD), n = 6 for each group. ap<0.05 versus control,p<0.05 versus QUR, cp<0.05 versus CUS. CUS: Chronic uncontrolled stress, QUR: Quercetin

Fig. 3(a-e):

Quercetin protects the modulation of apoptotic and survival biomarkers caused by CUS (a-c) Brain tissue levels of p53 and Bax gene expression were measured using RT-PCR analysis, (d) Caspase-3 and (e) Bcl-2 protein expression measured by ELISA at the end of the experiment in all groups of rats; control, QUR, CUS and CUS+QUR groups. For (A): Lane 1: Control, Lane 2: QUR, Lane 3: CUS and Lane 4: CUS+QUR groups. Results represent the mean (±SD); n = 6 for each group. ap<0.05 versus control, bp<0.05 versus QUR, cp<0.05 versus CUS. CUS: Chronic uncontrolled stress, QUR: Quercetin, p53: Tumour suppressor p53, Bax: Apoptosis regulator Bax, Bcl-2: B-cell lymphoma 2

Quercetin partially protects brain tissue against CUS-induced injury: Given the results described above that showed substantial protection by quercetin to CUS-induced depression, apoptosis and nitrosative and oxidative stress, we assessed the degree of potential protection to brain tissue injury provided by quercetin in response to CUS. Compared to normal tissue structure in the control group (Fig. 5a), CUS caused profound tissue damage as demonstrated by substantial neuronal (N) damage with shrunken cells, disrupted atrophic nuclei (n), perineuronal vacuolation (arrows) and swollen glial cells (G) are seen (Fig. 5b). Whereas, tissue prepared from the cerebral cortex of the brain of the treatment group (CUS+QUR) showed partially preserved brain architecture (Fig. 5c-d), as shown by mixed populations of cells; intact and damaged.

We further determined the correlation between the score of the cerebral cortex brain injury and the tissue levels of p53 and Bcl-2 to support the link between brain injury and apoptosis. As displayed in Fig. 5(e-f), a positive correlation was scored between brain tissue injury and p53 (r = 0.919; p<0.0001) (Fig. 5e) and a negative correlation between brain tissue injury and Bcl-2 (r = -0.879; p<0.0001) (Fig. 5f).

Fig. 4(a-h):

Correlation between p53 or Bcl-2 and markers of depression and iNOS and MDA (a-d) P53 and Bcl-2 brain homogenate levels were measured in all groups of rats and the correlation between p53 and iNOS, p53 and MDA, p53 and climbing ability and p53 and sucrose consumption, respectively and (e-h) Correlation between Bcl-2 and iNOS, MDA, Bcl-2 and climbing ability and Bcl-2 and sucrose consumption, respectively

DISCUSSION

One of the main findings of our study was that exposure of rats to CUS for 3 weeks led to the activation of a p53-Bax-caspase-3 axis of apoptosis in brain tissue, which was inhibited by quercetin. We were required to generate this animal model of CUS-induced depression to investigate the apoptotic pathways induced by CUS in the presence and absence of quercetin. Also, our protective regimen of quercetin was used to assess brain tissue levels of the survival (anti-apoptotic) protein Bcl-2 and biomarkers of nitrosative and oxidative stress, which demonstrated beneficial effects of quercetin. Furthermore, our data show (i) a positive correlation between p53 score and the brain tissue levels of iNOS and MDA; (ii) a negative correlation between p53 score and climbing ability and sucrose consumption and (iii) a significant correlation between cerebral cortex injury score and p53 and Bcl-2 support our conclusion mentioned above. Our results were thus consistent with our working hypothesis that CUS can substantially induce the activity of brain tissue p53-Bax-caspase-3 axis, which is an apoptotic pathway located downstream of the oxidative and nitrosative stress in the cell signalling pathways^25,26. This axis was blocked by quercetin in a rat model of CUS-induced depression. Therefore, the findings of this study confirm those previously published that showed both types of stress, acute and chronic, induced several pathological manifestations of brain injuries in animal models^27,28, such as behaviour and oxidative status of restraint-stressed mice²⁹, psychological stress-induced enhancement of brain lipid peroxidation via nitric oxide³⁰ and quercetin pre-treatment protects against stress-induced anxiety and depression-like behaviour and improves memory in male mice³¹.

In cell signalling, nitrosative and oxidative stress are located upstream of the apoptotic biomarkers upon activation in many cases such as brain and vascular injury^25,32,33.

Fig. 5(a-f):

Quercetin partially protects brain architecture against injury induced by CUS (a) H and E stained images (400x) of harvested rat tissues obtained after 3 weeks from the brain cerebral cortex of the control group, (b) CUS group, (c) Protective group, CUS+QUR are visualized by light microscopy. Note that in (A), arrows point to the glial processes and in (B), point to the perineuronal vacuolation. N: Neurons, n: Nucleus, G: Glial cells. Histograms in (d) represent a quantitative analysis of the cerebral cortex brain injury in the three groups of rats. All shown p values are significant, ap<0.05 versus control, b p<0.05 versus QUR and (e-f) Correlation between the brain injury score versus (e) p53 and (f) Bcl-2. CUS: Chronic uncontrolled stress, QUR: Quercetin, p53: Tumour suppressor p53, Bcl-2: B-cell lymphoma 2

Oxidative and nitrosative stress and p53 were reported to be involved in Alzheimer disease²⁶. In addition, oxidative stress-activated a specific p53 transcriptional response, which regulates cellular senescence and ageing in mice and deletion of p53 and the redox that generates mitochondrial ROS by genetic means reduced apoptosis and increased longevity of transgenic mice²⁶. Furthermore, overexpression of iNOS caused induction of vascular endothelial cell apoptosis and caspase-3 and 9activities,which were reduced by SMT, a selective iNOS inhibitor³³. These reports are in agreement with our data that demonstrate the association between CUS-induced depression and apoptosis and oxidative and nitrosative stress (Fig. 1-4). Also, our data shown here are in agreement with our recently published report²⁴ that showed modulation of these parameters; p53, Bax, Bcl-2 and biomarkers of oxidative and nitrosative stress in acute liver injury. Finally, in vivo study of quercetin on humans is warranted to evaluate whether these animal findings can be translated into therapy. Also, a future study would use quercetin to augment the effects of an antidepressant drug in this model.

CONCLUSION

In summary, we have demonstrated that induction of CUS-induced depression in rats for three weeks causes the activation of brain tissue p53-Bax-caspase-3 axis of apoptosis associated with the induction of oxidative and nitrosative stress and inhibition of the survival protein Bcl-2 and quercetin can revert these parameters.

SIGNIFICANCE STATEMENT

This study represents a significant contribution in the study of brain injury and depression induced by chronic stress in rats since it investigates the brain tissue apoptotic and survival cell signalling molecules modulated by chronic stress and the potential protection of these molecules and inhibition of brain injury and depression by the antioxidant compound quercetin, which may offer therapeutic potential in humans.

ACKNOWLEDGMENTS

This work was supported by the Research Deanship of King Khalid University, Abha, Saudi Arabia; Grant number No. R.G.P.1/44/42. The author highly appreciates the support of Professors Bahjat Al-Ani, Mohamed A Haidara and Mohamed Samir Zaki from the College of Medicine, King Khalid University, Abha, Saudi Arabia. Thanks are due to Dr. Mariam Al-Ani for proofreading the manuscript.