Introduction
Major depressive disorder (MDD) is one of the most common diseases leading to disability and loss of quality of life worldwide [13]. A previous study found a relationship between proinflammatory cytokines and MDD, and an increase in the production of interleukin 6 (IL-6), tumour necrosis factor a (TNF-a), interleukin 1b (IL-1b), and C-reactive protein (CRP) was observed in patients with MDD [17,24]. A previous study showed that this effect occurs in a dose-dependent manner, and that higher expression of these factors predicts the subsequent development of depressive symptoms [42]. Oleuropein is a natural product extracted from olive leaves and has drawn much attention from researchers for its biological effects, including antiproliferative, antidiabetic and anti-inflammatory activities [7]. Oleuropein has many biological functions, including the ability to reduce blood pressure through up-regulation of blood nitric oxide synthase inhibitors (L-NAME) [29], and the ability to decrease apoptosis in a mouse model of splanchnic arterial occlusion (SAO) shock [30]. Moreover, oleuropein could decrease the production of interleukin 1 (IL-1) by 80% in cultured cells [31], and therefore, may be useful in the treatment of MDD. The neurotrophic hypothesis of depression demonstrated that depression is initially caused by a reduction in brain-derived neurotrophic factor (BDNF) and a decrease in neurogenesis, leading to the impairment of synaptic transmission [25]. BDNF belongs to the neurotrophin family, which contains nerve growth factor (NGF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4). And a previous study found that BDNF regulates the development, maintenance, and plasticity of the nervous system, and also regulates the apoptosis of neuronal cells [23]. Moreover, BDNF regulates central nervous system (CNS) development in the immature CNS and regulates synaptic homeostasis in the mature CNS [10]. It is well known that mood disorders are closely related to pathophysiological changes in inflammation, particularly depression. In addition, inflammatory-related factors, including CRP, IL-6, IL-1, and TNF-a, are elevated in depression models and are regarded as markers of depression [22]. Thus, we speculated that BDNF overexpression might contribute to the therapeutic effect of oleuropein and performed the following experiments to test this hypothesis. Here, using behavioural tests, we found that oleuropein treatment could help to improve the brain function of MDD mice, as well as to reduce the expression and concentration of proinflammatory factors using in vivo experiments. Moreover, the combination of oleuropein and BDNF overexpression increased the viability of cells by inhibiting the production of pro-inflammation cytokines using in vitro experiments, and further experiments found that this effect might be mediated by the RhoA/ROCK signalling pathway using in vitro experiments.
Material and methods
Animal model of depression
Forty C57/BL mice aged 6-8 weeks were purchased from the Medical Laboratory Animal Centre. Mice were housed at 22-24°C with 50% humidity, a 12/12-h light-dark cycle, and freely available water and food. Mice were divided into four groups as follows: the control group (CG), the lipopolysaccharide (LPS) model group (LG), the oleuropein treatment group (OG) and the LPS model combined with the oleuropein treatment group (LO). In the LPS model group, mice were treated with 5 mg/kg LPS (No. tlrl-smlps, InvivoGen) 24 h before performing the following experiments through intraperitoneal injection according to a previous study [8], and in oleuropein treatment, mice were treated with 10 mg/kg oleuropein (No. 12247, Sigma) 60 min before performing the following experiments through intraperitoneal injection according to a previous study [36] (Fig. 1).
Behaviour tests
Sucrose preference test
The sucrose preference test (SPT) was performed to evaluate the anhedonic state of mice [46]. Low-grade stress is a factor contributing to the aetiology of depression. According to this study, stress leads to increased production of corticosteroids, resulting in an increase in blood sugar levels. High blood sugar in animals leads to a low intake of sucrose, which explains the reduced sucrose preference in mice with MDD. The mice were grouped and treated as described in the manuscript, then, mice were given two bottles of 1% sucrose on the first day. On the following day, one of the bottles was changed to pure water, and, the mice were fasted for the next 24 h. Subsequently, the mice were given two pre-weighed bottles of drinks, one was a 1% sucrose solution and the other was drinking water. The mice were given freely available water and food for 1 h, following which, the two bottles were weighed. Sucrose preference (%) was calculated as follows: sucrose consumption/(sucrose consumption + water consumption) × 100%.
Forced swimming test
The forced swimming test (FST) was performed to evaluate the giving-up-like behaviour of mice [1]. Briefly, mice were forced to swim for 10 min adaptively 1 day before the test. The mice were then placed on a transparent cylinder, and the immobility time in 5 min was recorded using a Super Maze Behaviour Analysis System (Shanghai Xinruan Information Technology Co., Ltd., Shanghai, China).
Open field test
The open field test (OFT) was performed to measure spontaneous locomotor activity and exploratory behaviour in a novel environment [2]. The experiment was performed in a round open-field apparatus, with a 60 W bulb positioned above the centre of the apparatus [39]. The mice were placed in the centre of the apparatus to observe their behaviour over a 3-min period, and the number of crossed squares and rearing time were recorded using a Super Maze Behaviour Analysis System (Shanghai Xinruan Information Technology Co., Ltd., Shanghai, China).
Vector construction
The BDNF knockdown vector was constructed according to a previous study [38]. Briefly, oligos were acquired using the following primers: forward: 5˘-CACCGTCTACCCACACGCTTCTGTA-3˘, reverse: 5˘-AACTACAGAAGCGTGTGGGTAGAC-3˘. The primers were incubated at 95°C for 4 min and 70°C for 10 min. Oligos and the CRISPR vector were digested with BsmBI (No. R0580S, NEB). The BDNF knockdown vector was constructed after annealing with T4 PNK enzyme (No. M0201S, NEB). The lentivirus vector was constructed after transfection into 293T cells using Lipofectamine 2000 (No. 11668030, Thermo) transfection reagent, and stable expressing cells were screened using 2 µg/ml puromycin (No. P8230, Solarbio).
The cDNA of BDNF was obtained using PCR with the following primers: forward: 5˘-TCTGACGACGGACATCGGC-3˘; reverse: 5˘-CCAGCAGAAAGAGTAGAGGAGGC-3˘.Primers and the pcDNA3.1 vector were digested with XbaI (No. R0145S, NEB) and XhoI (No. R0146S, NEB), annealed with T4 enzyme, and transfected into SH-SY5Y cells using Lipofectamine 2000 transfection reagent. Stably expressed cells were screened using 800 µg/ml G418 (No. G8161, Solarbio).
Cell model of depression
SH-SY5Y cells (No. CRL-2266) were purchased from ATCC. The cells were cultured in H-DMEM supplemented with 10% foetal bovine serum (FBS) (No. 10100, Gibco) at 37°C with 5% CO2. The cells were treated with 1 µg/ml LPS for 4 h to construct a major depression model in SH-SY5Y cells [8]. Cells were divided into four groups as follows: the LPS treatment group (L), the LPS treatment combined with oleuropein treatment group (G), the LPS and oleuropein treatment combined with BDNF overexpression group (O) and the oleuropein treatment combined with BDNF inhibition group (I). In the oleuropein treatment group, cells were treated with 10 µM oleuropein for 1 h [26] before performing the following experiments.
MTT assay
Cells were first seeded into a 96-well plate at a concentration of 1 × 104 cells per well, and then the cells were cultured and grouped as previously described. After treatment, the cells were incubated with MTT for 4 h, and the OD value was detected using SpectraMax iD3 at 570 nm. Viability rate = (ODExperiment – ODBlank)/(ODControl – ODBlank). Each experiment was repeated three times independently.
RNA extraction
RNA extraction was performed using a Total RNA Extraction Kit. Briefly, cells were cultured and grouped as previously described, lysed with lysis buffer and incubated with chloroform. After centrifugation at 12,000 rpm for 10 min, the water phase of the sample was transferred to an absorption tube. The samples were then washed with washing buffer and eluted with elution buffer. The RNA concentration was measured using a Nanodrop 2000. Samples were stored at –80°C until further use.
Quantitative real-time polymerase chain reaction
The UltraSYBR One-step RT-PCR kit (No. CW0659, CWBio) was used to perform qPCR with 500 pg RNA sample on a StepOnePlus™ Real-Time PCR System (No. 4376600, Applied Biosystems, Thermo). Briefly, the reaction mixture was prepared as recommended to a total volume of 25 µl, and the reaction was performed with the following primers: IL-1b: forward: 5˘-AGGTGGTGTCGGTCATCGT-3˘, reverse: 5˘-GCTCTCTGTCCTGGAGTTTGC-3˘; IL-6: forward: 5˘-ATGCTTCCAATCTGGGTTC-3˘, reverse: 5˘-TGAGGATAATCTTTGCGTTC-3˘; TNF-a: forward: 5˘-ACGGGCTTTACCTCATCTACTC-3˘, reverse: 5˘-GCTCTTGATGGCAGACAGG-3˘. Reverse transcription was performed at 50°C for 20 min, and pre-denaturation was performed at 95°C for 2 min.
The following steps were repeated for 45 cycles: denaturation at 95°C for 15 s, annealing at 55°C for 25 s, and extension at 72°C for 50 s. The expression of each target gene was measured using the 2-DDCq method [4]. Fold change of each target gene = expression of target mRNA/expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA. GAPDH was used as an internal control. Each experiment was repeated three times independently.
Protein extraction and western blotting analysis
The cells were cultured and grouped as described previously. Subsequently, the cells were lysed with RIPA buffer (No. R0010, Solarbio), the protein samples in the supernatant were collected after centrifugation at 12,000 rpm for 10 min, and the concentration of protein was detected using a BCA assay (No. PC0020, Solarbio). SDS-PAGE electrophoresis was performed with 60 µg of protein, followed which proteins were transferred onto a PVDF membrane. Membranes were then blocked with 5% skim milk and incubated with the following primary antibodies (1 : 1000) (BDNF (ab108319), GAPDH (ab8245), p-TrkB (ab229908), TrkB (ab134155), p-PKC (ab109539), PKC (ab181558), Ca-MKII (ab134041), AC-3 (ab125093), RhoA (ab187027), ROCK (ab45171), EGF (ab184265), NT-3 (ab263864) and NT-4 (ab150437)) overnight at 4°C. Following incubation, the membranes were incubated with secondary antibody at room temperature for 4 h. Protein expression was detected using a chemiluminescent immunoassay (SW2020, Solarbio), and the grey values of proteins were analysed using Image-Pro Plus (version 6.0). Fold change of each target protein = grey value of target protein/grey value of GAPDH.
Enzyme-linked immunosorbent assay
Enzyme-linked immunosorbent assay (ELISA) was performed according to the protocols of the ELISA kits (mouse IL-1b (ab197742), IL-6 (ab222503), TNF-a (ab208348) and calcium (ab102505) kits). Blood samples were first collected into a 1.5 ml tube, and after clot formation, samples were centrifuged at 2000 g for 10 min. The culture medium of cells, brain tissue lysates and serum samples of mice were collected and added into each well of a 96-well plate and incubated at room temperature for 1 h. After washing with washing buffer, the samples were incubated with TMB solution for 10 min at room temperature. Stop solution was added and the OD value was detected at 450 nm using SpectraMax iD3.
Ethics statement
Animal studies were performed in accordance with the guidelines of the Declaration of Helsinki and according to the principles and procedures outlined in the National Institutes of Health Guide for the Care and Use of Animals. Animal experiments were performed in the Third Hospital of Heilongjiang Province and were approved by the Health Animal Care and Use Committee of the Third Hospital of Heilongjiang Province (SCXK 2020-0016 of July 2020).
Statistical analysis
Experimental data are presented as the mean ± SD, and each experiment was repeated three times independently. One-way ANOVA was performed to detect the differences between groups followed by Tukey’s post hoc test [degree of freedom (DF) n = 3, DF d = 8). Significant difference was set at p < 0.05.
Results
Mouse behaviour tests
As shown in Figure 2A, the percentage of sucrose consumption in the CG, LG, OG and LO groups was 1.00 ±0.08, 0.62 ±0.03, 1.03 ±0.09, and 0.94 ±0.06, respectively. The percentage of sucrose consumption was significantly lower in the LG group than in the other three groups (p = 0.0003, DF = 22.5). The immobility time in these groups was 65.2 ±3.5, 113.6 ±5.2, 58.3 ±2.9, and 74.4 ±4.6 s, respectively. The immobility time in the LG group was significantly increased compared to the CG group, and was significantly decreased in the OG and LO groups (p = 0.0001, DF = 106.4) (Fig. 2B). The number of locomotor crossings in these groups was 92.3 ±7.3, 63.4 ±5.2, 97.1 ±7.6, and 86.6 ±6.9, respectively. The number of locomotor crossings was significantly decreased in the LG group compared to the CG group, and was significantly increased in the OG and LO groups compared to the LG group (p = 0.0014, DF = 14.4) (Fig. 2C).
Expression of pro-inflammation cytokines in the brain tissues of the mice model of depression
As shown in Figure 3, the expression of IL-1b in the CG, LG, OG and LO groups of MDD mice was 1.00 ±0.08, 1.32 ±0.12, 0.94 ±0.07, and 1.06 ±0.10, respectively. The expression of IL-1b was significantly increased in the LG group compared to the CG group, and was significantly decreased in the OG and LO groups compared to the LG group (p = 0.0053, DF = 9.412) (Fig. 3A). The expression of IL-6 in each group of MDD mice was 1.00 ±0.07, 1.26 ±0.11, 1.03 ±0.09, and 1.01 ±0.09, respectively. The expression of IL-6 was significantly increased in the LG group compared to the CG group, and was significantly decreased in the OG and LO groups compared to the LG group (p = 0.0234, DF = 5.554) (Fig. 3B). The expression of TNF-a in each group of MDD mice was 1.00 ±0.06, 1.42 ±0.15, 0.91 ±0.05, and 1.06 ±0.06, respectively. The expression of TNF-a was significantly increased in the LG group compared to the CG group, and was significantlydecreased in the OG and LO groups (p = 0.0006, DF = 18.64) compared to the LG group (Fig. 3C).
Detection of pro-inflammation cytokines in serum samples of the mice model of depression
As shown in Figure 4, the concentration of IL-1b in the serum samples of CG, LG, OG, and LO groups of MDD mice was 28.6 ±1.8, 56.1 ±3.5, 25.8 ±1.7, and 32.6 ±2.1 pg/ml, respectively. The concentration of IL-1b was significantly increased in the LG group compared to the CG group, and was significantly decreased in the OG and LO groups compared to the LG group (p = 0.0001, DF = 100.8) (Fig. 4A). The concentration of IL-6 in these groups was 102.1 ±4.2, 184.5 ±6.8, 95.3 ±3.7, and 126.4 ±5.6 pg/ml, respectively. The concentration of IL-6 was significantly increased in the LG and LO groups and was significantly decreased in the LG and LO groups compared to the CG group (p = 0.0001, DF = 181.1) (Fig. 4B). The concentration of TNF-a in these groups was 156.3 ±8.2, 246.1 ±11.3, 134.5 ±7.4, and 172.1 ±8.9 pg/ml, respectively. The concentration of TNF-a was significantly increased in the LG group compared to the CG group and was significantly decreased in the OG and LO groups compared to the LG group (p = 0.0001, DF = 85.54) (Fig. 4C). The concentration of calcium in the brain tissues from these groups was 8.6 ±1.3, 3.2 ±0.6, 9.1 ±1.5, and 7.4 ±1.1 mM, respectively. The concentration of calcium was significantly de creased in the LG group compared to the CG group, and the concentration of calcium was significantly increased in the OG and LO groups compared to the CG group (p = 0.001, DF = 15.64) (Fig. 4D). These results showed that the secretion of pro-inflammation cytokines was increased after LPS treatment and decreased after oleuropein stimulation, indicating that oleuropein had anti-inflammation effects. Oleuropein stimulation also increased the concentration of calcium in serum samples, which is another possible mechanism contributing to the improvement of MDD.
Detection of the cellular viability rate and model constructs
As shown in Figure 5A, the viability rate of cells in the L, G, O and I groups of the cell model was 100.0 ±7.1%, 123.3 ±8.0%, 147.6 ±12.4%, and 103.2 ±7.5%, respectively. The viability rate was significantly increased in the G and O groups compared to the L group and was significantly increased in the O group and significantly decreased in the I group compared to the G group (p = 0.0007, DF = 17.91) (Fig. 5A). The expression of BDNF in the control, BDNF overexpression, and BDNF inhibition groups was 1.01 ±0.08, 1.33 ±0.11, and 0.16 ±0.01, respectively (Fig. 5B). The expression of BDNF was significantly increased in the BDNF overexpression group and was significantly decreased in the BDNF inhibition group (p = 0.0001, DF = 176.9).
Expression of pro-inflammation cytokines in each cell model of depression
As shown in Figure 6, the expression of IL-1b in the L, G, O and I groups was 1.00 ±0.07, 0.82 ±0.06, 0.51 ±0.04, and 1.06 ±0.09, respectively. The expression of IL-1b was significantly decreased in the G and O groups compared to the L group, and was significantly decreased in the O group and significantly increased in the I group compared to the L group (p = 0.0001, DF = 40.24) (Fig. 6A). The expression of IL-6 in these groups was 1.00 ±0.08, 0.84 ±0.06, 0.56 ±0.04, and 1.04 ±0.08, respectively. The expression of IL-6 was significantly decreased in the O group compared to the L group, was significantly decreased in the O group and was significantly increased in the I group compared to the G group (p = 0.0001, DF = 31.64) (Fig. 6B). The expression of TNF-a in these groups was 1.00 ±0.11, 0.76 ±0.07, 0.42 ±0.05 and 0.94 ±0.09, respectively. The expression of TNF-a was significantly decreased in the G and O groups compared to the L group, and was significantly decreased in the O group compared to the G group (p = 0.0001, DF = 29.57) (Fig. 6C).
Concentration of pro-inflammation cytokines in the culture medium of the cell model of depression
As shown in Figure 7, the concentration of IL-1b in the culture medium of the L, G, O, and I groups of the cell model was 75.1 ±10.3, 52.3 ±8.5, 30.4 ±5.6 and 68.6 ±9.6 pg/ml, respectively. The concentration of IL-1b was significantly decreased in the G and O groups compared to the C group and was significantly decreased in the O group compared to the G group (p = 0.001, DF = 15.78) (Fig. 7A). The concentration of IL-6 in these groups was 365.2 ±18.7, 308.6 ±16.4, 236.4 ±13.6, and 352.8 ±18.0 pg/ml, respectively. The concentration of IL-6 was significantly decreased in the G and O groups compared to the L group, and was significantly decreased in the O group and increased in the I group compared to the G group (p = 0.0001, DF = 36.06) (Fig. 7B). The concentration of TNF-a in these groups was 532.3 ±32.1, 463.7 ±27.4, 382.4 ±21.3, and 520.6 ±31.6 pg/ml, respectively. The concentration of TNF-a was significantly decreased in the O group compared to the C and G groups (p = 0.0007, DF = 17.4) (Fig. 7C). The concentration of calcium in these groups of cell lysates was 3.6 ±0.2, 7.1 ±0.8, 12.8 ±1.1, and 4.2 ±0.4 mM, respectively. The concentration of calcium was significantly increased in the G and O groups compared to the L group, and was significantly increased in the O group and significantly decreased in the I group compared to the G group (p = 0.0001, DF = 103.5) (Fig. 7D).
Expression of the BDNF/TrkB/CaMKII signalling pathway in each cell model of depression
As shown in Figure 8, the expression of BDNF in the L, G, O and I groups of the cell model was 0.48 ±0.04, 1.01 ±0.08, 1.80 ±0.15, and 0.83 ±0.07, respectively. The expression of BDNF was significantly increased in all treatment groups compared to the L group, and the expression of BDNF was significantly increased in the O group and significantly decreased in the I group compared to the G group (p = 0.0001, DF = 105.7) (Fig. 8B). The ratio of p-TrkB/TrkB in these groups was 0.49 ±0.04, 0.72 ±0.06, 1.12 ±0.09, and 0.64 ±0.05, respectively, and the ratio of p-TrkB/TrkB was significantly increased in all treatment groups compared to the L group, and was significantly increased in the O group compared to the G group (p = 0.0001, DF = 55.01) (Fig. 8C). The ratio of p-PKC/PKC in these groups was 0.57 ±0.05, 0.81 ±0.07, 0.98 ±0.08, and 0.19 ±0.02, respectively. The ratio of p-PKC/PKC was significantly increased in the G and O groups, and significantly decreased in the I group compared to the L group, and was significantly increased in the O group and significantly decreased in the I group compared to the G group (p = 0.0001, DF = 99.12) (Fig. 8D). The expression of CaMKII in these groups was 1.05 ±0.09, 1.03 ±0.09, 1.41 ±0.12, and 1.08 ±0.09, respectively. The expression of CaMKII was significantly increased in the O group compared to the L and G group (p = 0.0044, DF = 9.992) (Fig. 8E). The expression of AC3 in these groups was 0.48 ±0.04, 0.92 ±0.08, 1.14 ±0.09, and 0.24 ±0.02, respectively. The expression of AC3 was significantly increased in the G and O group compared to L group, and significantly increased in the O group and decreased in the I group (p = 0.0001, DF = 121.7) compared to the G group (Fig. 8F).
Expression of the RhoA/ROCK signalling pathway in each cell model of depression
As shown in Figure 9, the expression of RhoA in the L, G, O and I groups of the cell model was 1.52 ±0.13, 1.31 ±0.11, 1.01 ±0.08, and 1.64 ±0.14, respectively. The expression of RhoA was significantly decreased in the O group compared to the L group, and significantly decreased in the O group and significantly increased in the I group compared to the G group (p = 0.0008, DF = 16.63) (Fig. 9B). The expression of ROCK in these groups was 0.62 ±0.05, 0.52 ±0.04, 0.35 ±0.03, and 0.83 ±0.07, respectively. The expression of ROCK was significantly decreased in the O group and significantly increased in the I group (p = 0.0001, DF = 48.73) compared to the L and G groups (Fig. 9C). The expression of epidermal growth factor (EGF) in these groups was 0.43 ±0.04, 1.03 ±0.09, 1.43 ±0.12, and 1.22 ±0.10, respectively. The expression of EGF was significantly increased in all treatment groups compared to the L group, and the expression of EGF was significantly increased in the O group compared to the G group (p = 0.0001, DF = 65.23) (Fig. 9D). The expression of NT-3 in these groups was 0.67 ±0.06, 0.92 ±0.08, 1.25 ±0.10, and 0.94 ±0.08, respectively. The expression of NT-3 was significantly increased in all treatment groups compared to the L group, and the expression of NT-3 was significantly increased in the O group compared to the G group (p = 0.0002, DF = 25.65) (Fig. 9E). The expression of NT-4 in these groups was 0.72 ±0.06, 1.17 ±0.10, 1.49 ±0.12, and 0.71 ±0.06, respectively. The expression of NT-4 was significantly increased in the G and O groups compared to the L group, and was significantly increased in the O group and significantly decreased in the I group compared to the G group (p = 0.0001, DF = 54.36) (Fig. 9F).
Discussion
Brain-derived neurotrophic factor is a secreted protein belonging to the neurotrophin family. BDNF was first purified from the brain tissue of pigs and has been found to support the survival and differentiation of neurons [4]. In the central nervous system, BDNF protects neuronal cells from apoptosis, and increases the synaptic function and morphology of neuronal cells [34]. Loss of BDNF expression induces spinal atrophy via impairment of the GABA-
ergic system [37]. We first found that overexpression of BDNF promotes cell proliferation under LPS stimulation using an in vitro model. Moreover as the inflammatory response process is activated in the MDD model, the expression of pro-inflammatory cytokines including IL-1b, IL-6, and TNF-a, was significantly increased after LPS stimulation, and was significantly reduced after BDNF overexpression combined with oleuropein, this effect was also detected in a mouse model. Inflammation is critical for the development of psychiatric disorders in nervous system diseases, because the inflammation process affects the peripheral immune system [14]. The increased expression of pro-inflammatory cytokines in the CNS leads to the activation of microglia and astrocytes and further continuous production of these cytokines [40], resulting in the MDD and Alzheimer’s disease. In patients with MDD, the concentrations of IL-1b, IL-6, and TNF-a are elevated, which can lead to late-life depression [33], thus, a reduction in the concentration of IL-1b, IL-6, and TNF-a may have a therapeutic effect in MDD. Here, the combination of BDNF and oleuropein treatment significantly reduced the expression and secretion of IL-1b, IL-6, and TNF-a, suggesting that this treatment might be effective in the context of MDD.
BDNF is a major neurotrophic factor that regulates the proliferation and differentiation of neuronal cells. The biological function of BDNF depends on binding to TrkB, and, according to a previous study, administration of LPS or pro-inflammation cytokines led to a significant reduction in BDNF expression [48]. Moreover, a previous study suggested that these effects might be mediated by the BDNF/TrkB signalling pathway, and this interaction has been proven to promote synaptic efficiency and long-term potentiation [47]. Indeed, neuronal cell function is promoted when TrkB is overexpressed [32]. In addition, a previous study also found that the release of neurotransmitters is regulated by PKC, a downstream molecule of the TrkB signalling pathway. BDNF/TrkB regulates the activity of neuronal cells in an intracellular calcium-dependent manner at the release site of BDNF [6]. Here, we found that after oleuropein treatment, the expression of BDNF and TrkB was increased, and was further increased after overexpression of BDNF. BDNF/TrkB induces sustained calcium influx through canonical transient receptor potential channels, mediated by TrkB-PKC activation, which promotes the release of calcium from internal calcium stores through activation of CaMKII and other calcium-dependent signalling steps [27]. CaMKII has been reported to regulate the oxidative stress and anti-depressant response, and, a recent study reported that CaMKII is also associated with the development of MDD [28]. Thus, we considered that increased expression of BDNF/TrkB might induce the expression of CaMKII, which leads to the increased concentration of calcium in serum samples of MDD mice, and that this effect might contribute to alleviation of MDD symptoms. Adenylyl cyclase 3 (AC3) is considered a critical enzyme in the regulation of the cAMP signalling pathway in neuronal cells [15]. Membrane-associated ACs are divided into four groups, and AC3 belongs to class I,which is a calcium/calmodulin-sensitive form [44]. Using a genome-wide association method, researchers found that AC3 is one of the top-ranked genes for the development of MDD [45], and strong depression-like behaviour was found to be present in AC3 knockout mice [43]. Oleuropein treatment led to an increase in AC3 expression and the expression was enhanced after overexpression of BDNF, indicating that the combination of oleuropein treatment and BDNF overexpression might contribute to the therapeutic effect of MDD, potentially via activation of the BDNF/TrkB/PKC signalling pathway.
Ras homolog gene family, member A (RhoA) is a member of the small GTPase protein family. Rho-associated protein kinase (ROCK) is a downstream effector of RhoA. RhoA/ROCK plays a critical role in cellular growth, transformation and cytoskeleton regulation [5]. Activation of the RhoA/ROCK signalling pathway is commonly observed in the regulation of CNS injury. A previous study found that the activation of RhoA started after 24 h, reached a peak after 3 days of injury and decreased until day 7 in a model of TBI [11]. A similar result was observed in a model of spinal cord injury (SCI), and researchers found that high expression of the RhoA/ROCK signalling pathway persisted for 3 months [12]. In addition, a recent study observed activation of the RhoA/ROCK signalling pathway in a seizure model induced by kainic acid, which is associated with axonal and synaptic retraction [11]. Thus, we hypothesized that the RhoA/ROCK signalling pathway plays an important role in the regulation of MDD.
Epidermal growth factor promotes the survival and proliferation of neural cells and activates growth factor signal transduction by binding to receptors. EGF knockout mice present behavioural abnormalities, including increased locomotor activity, damage to prepulse inhibition, and deficits in short-term memory. In addition, EGF knockout also reduces the activation of CaMKII and p21-activated kinase [35]. However, only a few studies focused on the function of EGF in patients with MDD, a previous study found that the concentration of EGF in plasma was significantly lower in patients with MDD [41], while we found that the expression of EGF was significantly increased after treatment with oleuropein combined with BDNF overexpression, which may contribute to reducing the inflammatory response after LPS stimulation. Thus, it is conceivable that a reduction in the inflammatory response might lead to a reduction in the symptoms of MDD. Neurotrophins (NTs) are one of the most studied factors in the brain, among which, NT-3 and NT-4 are most commonly observed [20]. NT-3 plays an important role in the development of excitatory synapses, and it has been previously shown that presynaptic differentiation of NT-3 occurs in a dose-independent manner [16]. In addition, using an in vitro model, researchers found that NT-3 promotes the survival of sympathetic neuroblasts, which further develop into sympathetic precursors [9]. Moreover, another NT member NT-4 can induce the differentiation of multiple neuronal cells, including cholinergic neurons, hippocampal neurons, and cerebellar granule cells [19], as well as improve the survival of trigeminal and jugular neurons during embryonic development [3]. The RhoA/ROCK signalling pathway has been shown to regulate the growth and differentiation of neuronal cells by regulating the expression of NGF, NT-3, and NT-4. Knockout of RhoA has been shown to enhance the outgrowth lengths of neurites compared to the control group, and inhibition of ROCK also promoted axon/neurite outgrowth by blocking the activation of the growth inhibitory pathway [18]. Here, we found that the expression of both NT-3 and NT-4 increased after treatment with oleuropein combined with BDNF overexpression, which may directly contribute to the protective effect in neuronal cells. Moreover, we also found that downregulation of RhoA/ROCK expression after treatment with oleuropein combined with BDNF overexpression increased the expression of NT-3 and NT-4, which further reduces the inflammatory response after LPS stimulation in a MDD model. Furthermore, oleuropein combined with BDNF overexpression enhanced the expression of neuron-protective factors via inhibition of the RhoA/ROCK signalling pathway in vivo, which might contribute to the therapeutic effect in MDD mice, and even contribute to the recovery of brain function.
With regards to the limitations of this study, this research was only performed in a mouse model, and the sample size was limited. Therefore, further experiments should be performed using more methods, including Co-IP and ChIP to explore the detailed underlying mechanisms. Moreover, further experiments on the effect of BDNF should be performed using primary neuron cells, as well as in clinical treatment. We intend to perform these experiments in the future to confirm the effect of BDNF in the treatment of MDD, which contributes to the clinical application of BDNF. However, according to a previous study, exogenous BDNF does not directly traverse the blood-brain barrier, which limits the therapeutic function of BDNF. In order to solve this problem, some researchers designed a liposome that can carry exogenous BDNF through the blood-brain barrier, this method will be verified in our further experiments.
Disclosure
The authors report no conflict of interest.
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