Proteomics-based screening of the target proteins associated with antidepressant-like effect and mechanism of nimesulide

Nimesulide is an inhibitor of COX-2 with antioxidant and anti-inflammatory effects. However, few studies have explored the antidepressant mechanism of nimesulide. Here, we evaluated the therapeutic effects of nimesulide on CUMS rats. iTRAQ technology was used to identify the differentially expressed protein in the hippocampus between CUMS and nimesulide-treated rats to identify the possible molecular mechanism of its effects. We found that nimesulide had positive effects on depressive-like behaviors and inflammatory factors in depressed rats. Using proteomics technologies, we screened 16 differentially expressed proteins in CUMS-exposed rats after nimesulide treatment, 5 of which were related to inflammation. Overall, these results show that nimesulide might mediate its antidepressant effect on depressed rats through the inhibition of oxidative stress inflammatory response.

Depression is reported to be associated with many health problems like low mood, cognitive impairment, and even suicidal behaviours 1 . With the increase in competitive pressure in modern society, the number of people suffering from depression is growing day by day 2 . Furthermore, depression may induce substance abuse 3,4 and suicide 5 . Brain imaging studies showed a reduced hippocampus volume of depressed patients 6 . However, a disadvantage of conventional antidepressants is a slow onset of antidepressant action. Therefore, the pathogenesis of depression needs further studies to search for new therapeutic targets.
Substantive evidence shows inflammation being a driver of neuropsychiatric symptoms by negatively impacting on neuronal proliferation, survival, and differentian [7][8][9] . A population-based, prospective cohort study in Denmark followed a total of 3.56 million people for 24 years and found that those who were hospitalized for an infection or visited a hospital for treatment of an autoimmune disease had a significantly higher risk for developing a depressive disorder 10 . The cyclooxygenase (COX) −2 catalyzes the synthesis of prostaglandins (PGs) and induces inflammation responses 11 . The COX pathway potentiates the inflammatory process and may exacerbate brain inflammation and injury. Clinical studies found that enhanced COX-2 expression in patients with neuropsychiatric diseases such as depression and Alzheimer's disease [12][13][14] . Consistent with these results, an animal model of depression in rats demonstrated that the upregulation of COX-2 may induce hyperactivity in inflammatory responses and lead to depression-like behavior 15 . Interestingly, the COX-2 inhibitor can relieve depressive-like behaviors by inhibiting inflammatory factors 16 . In people with major depression, a randomized, double-blind study found compared reboxetine plus celecoxib with reboxetine plus placebo a significant therapeutic effect of celecoxib 17 . These results point out that a strong correlation between COX-2 and depression.
Fluoxetine is the first selective serotonin reuptake inhibitor antidepressant with the longest half-life and high bioavailability. Moreover, fluoxetine is one of the most widely prescribed antidepressants in clinical settings and has been experimentally widely used for studying depression 18 . As one of only two antidepressants, fluoxetine was approved for depression in youth by the United States Food and Drug Administration (FDA) 19 . Besides, fluoxetine was used as the control in a large number of animal experiments 20,21 .
Isobaric tags for relative and absolute quantification (iTRAQ) is an advanced technique used for identifying biomarkers and examining pathophysiological processes of various diseases 22 . In our study, we used iTRAQ to screen target proteins regulated by nimesulide in the hippocampus of CUMS rats, to provide insight into the pathogenesis of depression and the role of nimesulide in depression.

Materials and methods
Animals. Male Sprague-Dawley rats (180-220 g) were purchased from the Laboratory Animal Center of Chongqing Medical University. They were housed in groups of 5 each cage at constant room temperature (25 ± 2 °C) with a 12 h light and dark cycle. Food and water were continually available for 1 week as rats adapted to the environment. All procedures were performed following Chongqing Science and Technology Commission guidelines for animal research and approved by the Chongqing Medical University Animal Care Committee.
Drugs and treatment groups. A total of 80 rats were randomly divided into a control group (CON, n = 20) and a CUMS-exposed group (n = 60). CUMS-exposed rats (n = 60) were further subdivided into the following 3 groups, with 20 rats each group: CUMS model group (CUMS), fluoxetine+ CUMS group (FLX), and nimesulide+ CUMS group (NIM). The dose of nimesulide for rats was derived from a human dose by following a conversion equation 23 . The rats in the NIM and FLX groups were treated with nimesulide (Hubei Jianyuan Chemical Co., Ltd. China) and fluoxetine (Wuhan Sheng Tianyu Biotechnology Co., Ltd. China) through oral gavage at a dose of 12 mg/kg/d and 3 mg/kg/d, respectively for 21 days. The rats in the CON and CUMS groups received an equal volume of 0.5% sodium carboxymethylcellulose (CMC-Na) (National Chemical Reagent, China) through oral gavage for 21 days. Behavioral tests were performed following drug intervention. CUMS procedure. The CUMS procedure was conducted according to the previous study 24 . All of these stressors were randomly arranged, including tail pinching for 1 min, 4 °C cold water swimming for 5 min, 45 °C hot water swimming for 5 min, fasted (food and water) for 24 h, cage tilting at 45 ° for 24 h, wet bedding for 24 h, inverted day/night cycle and odor stimulation. Rats were individually exposed to the stressors in random order once a day for 21 days. No single stressor was performed consecutively.
Open-field test (OFT). The OFT was performed with minor modifications as described previously 25 . Rats were individually placed in the center of a black box (100 cm × 100 cm × 50 cm) in a dark and quiet room with a 5-m visibility distance. Horizontal (crossing times) and vertical (rearing times) exploratory activity were measured for a 5-min session. The apparatus was cleaned with detergent before each test session to remove any olfactory cue.
Forced swimming test (FST). The FST was conducted as previously described with minor modifications 26 .
Rats were individually placed in a transparent cylindrical bucket (45 cm in height and 25 cm in diameter) filled with water (23 ± 2 °C) to a depth of 30 cm. Total immobility time in 6 min was calculated. The water was completely replaced after each test.

iTRAQ of CUMS hippocampus samples
Protein extraction and iTRAQ sample labeling. The iTRAQ procedure was provided by the Institute of Life Sciences of Chongqing Medical University. Removed hippocampus tissues of 3 rats/group were immediately snapped-frozen within liquid nitrogen and stored in a refrigerator (−80 °C) until isobaric labeling. The 5 μL phosphatase inhibitor, 1 μL protease inhibitor, and 10 μL phenylmethylsulfonyl fluoride (PMSF) were added to lysis buffer, and the mixture was homogenized. The lysis buffer and glass homogenizer were then on ice. The cryopreserved rat hippocampus tissue was washed with phosphate Buffered Saline (PBS), and lysis buffer was added to rat hippocampal tissue, which was manually ground to a homogenate using a glass homogenizer. Next, we added trichloroacetic acid (TCA)-ice acetone to the homogenate and centrifuged it at 3000 rpm in 30 min at 4 °C after precipitation at 20 °C for 2 h, ultimately taking the precipitate without the supernatant. The precipitate was then added to acetone, as described previously, precipitating for 30 min at −20 °C and centrifuging at 3000 rpm for 30 min at 4 °C. Then, the supernatant was removed and the precipitate was kept. The above operation was repeated until the precipitate became white and stored at −80 °C. Bioinformatics analysis. Proteins with a greater than 1.2-fold change or a fewer than 0.83-fold change and a P-value < 0.05 were considered differentially expressed. All significant proteins were uploaded to DAVID for Gene Ontology (GO) annotations of biological process, molecular function, and cellular component. Pathway enrichment analysis was performed based on the KEGG database.

Statistical analysis.
All the data were presented as mean ± standard error (SEM) and analyzed with GraphPad Prism 5.0. The data were analyzed in a One-way ANOVA followed by Dennett's t-test and P < 0.05 was considered significant.

Effects of nimesulide on hippocampal protein profiles in CUMS rats. Rats hippocampal samples
labeled with iTRAQ reagents were analyzed by LC-MS/MS. A total of 153 proteins were found to be significantly changed, with 88 changed in groups FLX vs. CUMS, 61 changed in groups NIM vs. CUMS, 75 changed in groups CON vs. CUMS. The 16 differentially expressed proteins shared among groups FLX vs. CUMS, groups NIM vs. CUMS, and groups CON vs. CUMS, with 11 downregulated (Fig. 3a, Table 1) and 5 upregulated (Fig. 3b, Table 2). Of the 16 differentially expressed proteins, 5 were related to inflammation.

GO functional annotations and KEGG pathway analyses. GO functional annotations and KEGG
pathway analyses were used to examine the differential proteins. The results show that, in terms of biological process, differential proteins were mainly involved in the cellular response to oxidative stress (8.33%) (Fig. 4a). Regarding molecular function, differential proteins were annotated as being associated with protein binding (38.46%), actin filament binding (23.08%), protein domain specific binding (23.08%), and structural constituent of the cytoskeleton (15.38%) (Fig. 4b). For cellular component, differential proteins are predicted to be in the cytoplasm (20.37%), extracellular exosome (14.81%), cytosol (12.96%), neuron projection (11.11%) and mitochondrion (11.11%) (Fig. 4c). Based on the KEGG database, the differentially expressed proteins were enriched in the following pathways: metabolic pathways, amyotrophic lateral sclerosis (ALS), Fc gamma R-mediated phagocytosis, carbon metabolism, biosynthesis of antibiotics, endocytosis and arginine and proline metabolism (Fig. 4d). www.nature.com/scientificreports www.nature.com/scientificreports/ Compared with the CUMS group, these proteins were downregulated by fluoxetine or nimesulide but upregulated in the CON group. (b) The red short arrow indicates 5 overlappings differentially expressed proteins. Compared with the CUMS group, these proteins were upregulated by fluoxetine or nimesulide but downregulated in the CON group.  www.nature.com/scientificreports www.nature.com/scientificreports/

Discussion
Rodent animal models for CUMS has been widely used for investigating the pathophysiological mechanism of depression and evaluating the efficacy of antidepressants 27 . This stress-induced model of depression has good validity and reliability 28 . Recent research has identified that depression has been linked with inflammatory markers and be associated with regulating genes of the immune response [29][30][31] . COX-2 is a key mediator of the inflammation, being responsible for the production of PGE2 from arachidonic acid and mediates different physiological and pathophysiological reactions via its receptors 32 . In the present study, we observed the depressive-like behavior emerges and an increased level of the proinflammatory mediator in a rat model of CUMS-induced depression. In the hippocampus, we screened differentially expressed proteins between rats with and without CUMS exposure.
Nimesulide, already introduced as an anti-inflammatory agent, was recognized to be a COX-2 selective inhibitor 33 . Recently, the effects of COX-2 inhibitor have been examined in various clinical trials, suggesting that it reduces cerebral damage and neuronal death 34,35 . Reported data from clinical trials supported the protective effects of the COX-2 inhibitor in depression 36 . Similar findings have been documented in animal models of chronic stress 37 . Our results agreed with previous reports that nimesulide could relieve depressive-like behaviors and reverse the increase of inflammatory factors. The more interesting result was that the overlappings differentially expressed proteins regulated by nimesulide were screened in CUMS rats.
To clarify the functions and links among these proteins, GO annotation enrichment analysis and KEGG signaling pathway enrichment analyses were used. GO annotation shows that differential proteins were involved in the formation of cellular components, such as cytoplasm and extracellular exosome. For molecular function, they exert binding function and comprise the cytoskeleton. In biological processes, it is mainly involved in regulating the cellular response to oxidative stress and hippocampus and cortex development. KEGG pathway analyses suggest that differentially expressed proteins are mainly involved in metabolic and endocytosis pathways. As mentioned above, the pathogenesis of depression involves several systems and multiple proteins. Our findings showed that these differentially expressed proteins could regulate by nimesulide and may be potential drug targets for depression treatment. Remarkably, we found these proteins might be related to inflammation, such as Adaptor protein two complex subunit beta 1 (Ap2b1), Calcineurin B type 1 (Cnb1), 2-Oxoglutarate Dehydrogenase (Ogdh), Calretinin (Calb2) and NAD-dependent protein deacetylase sirtuin-2 (Sirt2).
Among the changes evoked by nimesulide in the hippocampus, we observed an increase in the level of Ap2b1. Ap2b1, a subunit of the AP-2 complex, is involved in clathrin-mediated endocytosis and migration 38,39 . The knockdown of AP2b1 lessened the number of dendrites in developing rat hippocampal neurons 40 . Recently research found that Ap2b1 might exert neuroprotection through inhibiting inflammatory reaction 41 .
Of the upregulated proteins after nimesulide treatment, the expression of Cnb1 was notable. Cnb1, a regulatory subunit of calcineurin, equally distributed between the cytoplasm and nucleus 42 . Studies reported that www.nature.com/scientificreports www.nature.com/scientificreports/ Cnb1 showed increased expression after venlafaxine treatment 43 . Notably, calcineurin interacts with the serotonin transporter modulating its plasma membrane expression and serotonin uptake 44 . Moreover, calcineurin has also direct antidepressant-like effects, while inhibition of calcineurin in the medial prefrontal cortex of rats induces depressive-like behaviour 45 . Variants in Cnb1 are associated with a more rapid functional decline in Alzheimer's disease 46 . Calcineurin-inhibitor agents also affect the activation of T cells, inhibit the expression of interleukins, and prevent the release of cytokines and inflammatory mediators from mast cells 47 .
In the present study, we found that nimesulide increased Ogdh level, known as a key enzyme in the tricarboxylic acid (TCA) cycles. Ogdh can catalyze the oxidative decarboxylation of αketoglutarate to form succinyl coenzyme A and release energy and it is known that energy deficiency would lead to dysfunction of organs and tissues, and then Alzheimer's disease, Parkinson's disease, and other diseases 48 . Besides, Ogdh is also closely related to chronic inflammation 49 . In short, Ogdh is speculated to be closely related to neuropsychiatric diseases and inflammation.
We noted the decrease in the expression of Calb2 after nimesulide administration. Calb2 is an important calcium-binding protein, which is mainly distributed in central neurons in the cerebral cortex and hippocampus 50 . Calb2 prevented calcium overload by binding excessive Ca 2+ , and thus reduce cell apoptosis 51 . Proteomics analysis in a global ischemic stroke also found that Calb2 associated with endoplasmic reticulum stress-induced neuronal cell apoptosis 52 . Treatment for nimesulide decreased the level of Sirt2 in our study. Sirt2 is an NAD + -dependent deacetylase in the brain. The pharmacologic inhibition of Sirt2 exerts neuroprotective effects in diverse models of neurodegenerative disease 53,54 . Studies have shown that microtubule acetylation is essential for normal neuronal development and function 55,56 and Sirt2 may function as the predominant microtubule deacetylase in mature neurons 57 . Sirt2 was found to be upregulated in response to oxidative stress, deacetylated FOXO3, and increased in the expression of genes targeted by FOXO3a, such as Bim 58 . Although Sirt2 is reported to a major inhibitor of microglia-mediated inflammation and neurotoxicity 59 , we cannot rule out the possibility that sirt2 also expression regulated by nimesulide through oxidative stress in the present research.
Summing up, our results suggest that nimesulide could improve depressive-like behaviors, suppress high levels of inflammatory factors, and regulate differentially expressed proteins in a rat model of CUMS-induced depression. Even though our findings highlight the role of nimesulide in depression and provide a promising therapeutic direction, the exact therapeutic mechanism behind the antidepressant effects of nimesulide has not been elucidated and future study is needed to explore the mechanism of action.