IGF-1 defends against chronic-stress induced depression in rat models of chronic unpredictable mild stress through the PI3K/Akt/FoxO3a pathway.

Introduction

Depression is a common mental disturbance that results in impaired role functioning and poor quality of life [1] . Major symptoms of depression include loss of pleasure, sustained low mood, loss of appetite and sleep disorders [2] . Depression has been regarded as a major factor in the global burden of diseases and is the 4th leading cause of disability worldwide according to the World Health Organization (WHO) [1] . It has been reported that depression is mainly caused by the interactions between environmental factors and genetic predispositions, yet its etiology and mechanisms are still unrevealed [3] . Although many patients respond to psychotherapy and first‐line medications, approximately 20–40% of patients fail to benefit from these treatments [4] . Therefore, it is crucial to develop a suitable and effective antidepressant therapy. Chronic stress is known as a major risk factor for depression [5] . Exposure to environmental stimulation can trigger chemical, physical and mental responses at the molecular and cellular levels, resulting in impairment of antioxidant defenses and oxidative damage [2] . Chronic mild stress has been broadly used to model depression in animal experimental studies, since it can induce a hedonic‐like state in animals that resembles major symptoms of endogenous depression in humans [6] .

Accumulating evidence shows that the pathology of depression is associated with neuronal inflammation and can be alleviated by neurogenesis [7] . Akt is known as a serine/threonine protein kinase and a target of phosphatidylinositol 3‐kinase (PI3K), which functions as a signaling molecule for many downstream target genes and a precise regulator of the cell cycle [8] . A recent study has demonstrated that PI3K and Akt can activate immune cells by regulating the key inflammatory cytokines, so changes in the PI3K/Akt pathway may produce specific therapeutic effects for depression [8] . In addition, Akt phosphorylation inhibits FoxO3a, which is a FoxO subclass of the Forkhead transcription factors [9] . One way in which Akt promotes cell proliferation and survival is via inhibiting FoxO3a, thus resulting in its sequestration in the cytoplasm away from death‐inducing genes [10] . Bim, a member of the BH3‐only group of the Bcl‐2 protein family, is a mediator of apoptosis as well as a sensor of cell stress, and FoxO3a has been demonstrated to be crucial for the initiation of Bim‐dependent apoptosis [11] . Insulin‐like growth factor 1 (IGF‐1) is regarded as a regulator of the somatotropic axis and can pass through the blood‐brain‐barrier [12] . Additionally, IGF‐1 is produced by the brain itself [13] and is deemed to influence mood regulation [14] . It has been reported that IGF‐1 regulates anti‐depressive effects in the brain [15] . Therefore, we aimed to study the effect of IGF‐1 on depression in a rat model of chronic unpredictable mild stress (CUMS) via regulating the PI3K/Akt/FoxO3a pathway, hoping to develop a new potential pharmacological treatment for depression.

Materials and methods

Experiment animals

The experimental protocols were approved by the ethics committee for animal experiment and conformed to National Institutes of Health guidelines for the care and use of laboratory animals. Forty‐eight healthy and clean Sprague–Dawley (SD) male rats (275 ± 20 g) were supplied by Beijing Vital River Laboratory Animal Technology Co., Ltd. (Animal license number: SCXK (Beijing) 2012‐0001). All rats were housed under standard conditions of light (12/12 h, light/dark circle), temperature (20–25 °C) and humidity (55% ± 2%) with free access to water and food.

Rat modeling and grouping

CUMS is described as a chronic unpredictable mild stress‐induced depression model, which is a typical modeling approach for depression. The procedures of CUMS modeling was designed according to the classical method set up by Katz et al., with some modifications by Willner et al. [16] . The experimental procedures were as follows: wet litter, turning night into day, fasting, water deprivation, swimming in cold water, oscillation, heat, noise, foot shock, restraint and other procedures. One of the procedures was selected randomly every day and performed to avoid the prediction of stimulation from the animals. The CUMS model was close to the mechanism of human depression and easily used, as it mainly simulates the lack of pleasure in the core symptoms of human depression. After 1 week of acclimation, the 48 rats were randomly divided into the control, CUMS and CUMS + IGF‐1 groups. Rats in the control group did not receive stimulation and were housed under standard conditions. Rats in the CUMS group were subjected to CUMS for 3 weeks. Various stimulations were adopted in the procedure (total of 21 days), including wet litter, food deprivation and water deprivation for 24 h, nighttime illumination for 12 h, oscillation for 30 min, tail pinch for 3 min and restraint for 2 h. Each kind of stimulation was performed three times on average [17] . Rats in the CUMS + IGF‐1 group were injected with 5 μl of IGF‐1 (5 μg/μl) every day before exposure to the same stimulation as the CUMS group.

Open‐field test

An open cube box (80 cm × 80 cm × 40 cm) was prepared. The peripheral wall and bottom of the box were black and opaque, and the bottom was divided into 25 pieces with equal size. Then, the rats were placed in the center of the box, and their movements within 5 min were recorded. The horizontal (number of horizontal crossing) and vertical (rearing times) activity of the rats were evaluated. The number of squares crossed referred to the bottom pieces crossed by the rats, and the number of rearings referred to the times that their forelegs lifted off the ground. The open‐field test was carried out one day before modeling and on the 21st day. Forty‐eight hours before modeling, a sucrose intake test was performed. The procedures were as follows: the rats were given 5% sucrose, followed by 12 h of water deprivation. Then, sucrose consumption (during 2 p.m.–3 p.m.) was measured. The sucrose consumption test [18] was carried out on the 21st day. Body weights of all rats were measured one day before and after the test, and the comparisons of rats were analyzed.

Reverse transcription quantitative polymerase chain reaction (RT‐qPCR)

Total RNA in the hippocampus was extracted using Trizol reagent (Takana, Israel). The OD 260/280 value in each RNA sample was measured by ultraviolet spectrophotometer, and the RNA concentration was calculated. Then, the samples were stored at −80 °C. According to gene sequences published by GenBank database, primers were designed by Primer 5.0 software ( Table 1 ) and synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). Reverse transcription was conducted using Reverse Transcription System A3500 (Promega, Madison, WI, USA), as described by the manufacturer. cDNA was obtained to undergo RT‐qPCR using reverse transcription. The reaction conditions were pre‐denaturalization at 94 °C for 5 min, followed by 40 cycles of denaturalization at 94 °C for 1 min, annealing at 58 °C for 1 min and extension at 72 °C for 1 min, with a final extension step at 72 °C for 10 min. The reaction system for RT‐qPCR consisted of 12.5 μl of Premix Ex Taq or SYBR Green Mix, 1 μl of forward primer, 1 μl of reverse primer, 1–4 μl of DNA template and double distilled H 2 O (ddH 2 O) to reach a total volume of 25 μl. Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) was taken as the internal reference, and a melting curve was adopted to evaluate the reliability of the PCR products. The relative expression level was calculated according to the 2 −△△CT method.

Western blotting

The extracted hippocampus of each group was boiled in a loading buffer (30 μg/well) at 95 °C for 5 min. The protein samples were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) with a 10% polyacrylamide gel (Boster biological technology Co. Ltd., Wuhan, China) from 90 V through a concentrated gel to 120 V through a separating gel and then transferred to a PVDF membrane (wet transfer) at 100 V for 90 min. Subsequently, the membrane was stained with ponceau S and incubated with 5% bovine serum album (BSA) (diluted by 1 × tris‐buffered saline Tween‐20, TBST) for 1 h at room temperature. The membrane was then incubated with rabbit anti‐p‐Akt/Akt primary antibody (1: 10000, Cell Signaling Technology, Danvers, MA, USA), goat anti‐p‐FoxO3a/FoxO3a (1: 10000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and Bim (1:1000) at 4 °C in a refrigerator overnight. After washing with TBST three times (10 min per time), the membrane was incubated with horseradish peroxidase (HRP)‐labeled secondary antibody (rabbit anti‐rat IgG, 1: 2000, Sigma, St. Louis, MO, USA) at room temperature for 1 h, followed by washing with TBST three times (10 min per time). The membrane was then developed with electrochemiluminescence reagent (Amersham Biosciences, Piscataway, NJ, USA), and GAPDH was taken as the internal reference. The gel was imaged using a Bio‐Rad Gel Doc™ EZ Imager (Bio‐Rad, Hercules, CA, USA), and the gray scale of each band was analyzed by ImageJ software.

Statistical analysis

SPSS 17.0 (SPSS Inc., Chicago, IL, USA) was utilized for statistical analysis. The measurement data were expressed as the mean ± standard deviation. The t ‐test and paired t ‐test were used for comparisons between two groups. One‐way analysis of variance analysis followed by the least‐significant difference method was adopted for comparisons among multiple groups. P < 0.05 indicated statistical significance.

Results

IGF‐1 increases the body weight of CUMS rats

An open‐field test was employed to verify the body weights of rats. The body weight of rats among the control, CUMS and CUMS + IGF‐1 groups did not show significant differences one day before the test ( P > 0.05) ( Table 2 ). After the test, a lower body weight of rats was observed in the CUMS and CUMS + IGF‐1 groups compared to the control group ( P < 0.05), while the body weight of rats in the CUMS + IGF‐1 group was significantly heavier than those in the CUMS group ( P < 0.05). Taken together, IGF‐1 increases the body weights of CUMS rats.

IGF‐1 promotes horizontal and vertical activity in CUMS rats

An open‐field test was applied to confirm the horizontal and vertical activity of rats. There were no significant differences among rats in the control, CUMS and CUMS + IGF‐1 groups one day before the test ( P > 0.05). After the test, the horizontal crossing number and rearing time of rats in the CUMS and CUMS + IGF‐1 groups were significantly reduced compared with the control group ( P < 0.01), while the horizontal crossing number and rearing time of rats in the CUMS + IGF‐1 group were significantly increased compared with those in the CUMS group ( P < 0.01) ( Table 3 and Table 4 ). In conclusion, IGF‐1 promotes horizontal and vertical activity of CUMS rats.

IGF‐1 increases sucrose consumption in CUMS rats

Sucrose consumption test was performed to identify the sucrose consumption of rats. There was no significant difference in sucrose consumption among rats in the control, CUMS and CUMS + IGF‐1 groups one day before the test ( P > 0.05) ( Table 5 ). After the test, the sucrose consumption of rats in the CUMS and CUMS + IGF‐1 groups was significantly lower than that of rats in the control group ( P < 0.01), while the sucrose consumption of rats in the CUMS + IGF‐1 group was significantly increased compared with that in the CUMS group ( P < 0.01). Based on these findings, IGF‐1 is likely to increase sucrose consumption in CUMS rats.

PI3K, Akt and FoxO3a mRNA expression is higher and Bim mRNA expression is lower in the CUMS + IGF‐1 group compared to the CUMS group

RT‐qPCR was adopted to detect the mRNA expressions of PI3K, Akt, Bim and FoxO3a. As shown in Fig. 1 , the mRNA expression of PI3K, Akt and FoxO3a in the hippocampus of the CUMS + IGF‐1 and CUMS groups were significantly lower compared to the control group, while Bim mRNA expression in those two groups was significantly higher than that in the control group ( P < 0.05). Compared with the CUMS group, the mRNA expression of PI3K, Akt and FoxO3a was significantly increased in the CUMS + IGF‐1 group, while Bim mRNA expression was significantly reduced in the CUMS + IGF‐1 group ( P < 0.05). These results provide evidence that PI3K, Akt and FoxO3a mRNA expression is higher, while Bim mRNA expression is lower in the CUMS + IGF‐1 group compared to the CUMS group.

Protein phosphorylation of PI3K, Akt and FoxO3a is higher, while Bim protein expression is lower in the CUMS + IGF‐1 group than in the CUMS group

Western blotting was used to determine the protein expression of PI3K, Akt, Bim and FoxO3a. As seen in Fig. 2 , protein phosphorylation of PI3K, Akt and FoxO3a in the hippocampus of the CUMS and CUMS + IGF‐1 groups was significantly lower than that in the control group, while the protein expression of Bim was significantly higher than that in the control group ( P < 0.05). Compared with the CUMS group, protein phosphorylation of PI3K, Akt and FoxO3a in the CUMS + IGF‐1 group was elevated significantly, while the protein expression of Bim was down‐regulated remarkably ( P < 0.05).

Discussion

Depression is regarded as a multifactorial mental disturbance with many causes, including genetic, neurological, hormonal, immunological, gender and developmental factors [19] . As there is an increasing number of people suffering from depression, this study aimed to explore the role of IGF‐1 in depression through the PI3K/Akt/FoxO3a pathway in order to develop a potential effective treatment. By establishing a CUMS rat model, our findings demonstrated that IGF‐1 defends against chronic‐stress induced depression via activation of the PI3K/Akt/FoxO3a pathway.

First, in this study, the body weights of rats exhibited a lower level in the CUMS + IGF‐1 and CUMS groups in comparison to the control group, and the horizontal crossing number and rearing time, as well as sucrose consumption, were significantly decreased, indicating that the CUMS model was successfully established. In 1987, Willner et al. established the rodent CUMS model in order to induce anhedonia‐like behavioral change (including inability to experience pleasure), which was a major symptom of human depression [20] . Chronic stress is a major risk factor for depression [5] . Stress responses at the cellular and molecular levels can result in oxidative damage through the impairment of antioxidant defenses and can easily result in depression [21] . In addition, abundant evidence has shown that chronic stress not only altered the morphology of neurons, which led to changes in spine density, but also caused astrocyte morphological alterations and atrophy of process length, branching, and volume in the hippocampus or prefrontal cortex [22] . Consistently, a previous study indicated that the CUMS procedures showed a lower body weight of animals, sucrose preference, and locomotor activity in an open‐field test [23] .

Furthermore, we observed that IGF‐1 contributes to higher body weight, sucrose consumption, and horizontal and vertical activities in CUMS rats. There was a study investigating older adults in Britain that indicated that a moderate IGF‐1 level conferred a reduced risk of depression [24] . In addition, Kopczak A et al. demonstrated that up‐regulated IGF‐1 was significantly related to depression and impaired treatment response, indicating that IGF‐1 might exert an effect on the pathophysiology of depression and affect the response to antidepressant treatment [12] .

Moreover, compared with the CUMS group, mRNA expression and phosphorylation of PI3K, Akt and FoxO3a increased in the CUMS + IGF‐1 group, while Bim mRNA and protein expression were significantly reduced in the CUMS + IGF‐1 group, suggesting that IGF‐1 up‐regulated phosphorylation of PI3K/Akt/FoxO3a and down‐regulated Bim expression. The pathology of depression is reported to be associated with neuronal inflammation, while depression is believed to be alleviated by neurogenesis [7] . Akt is a target of PI3K, functioning as a signaling molecule for many downstream target genes; PI3K and Akt can activate immune cells by regulating key inflammatory cytokines; therefore, the PI3K/Akt pathway may have specific therapeutic effects for depression [8] . It was reported that IGF‐1 was a key signal molecule in the tumor micro‐environment that facilitated cell growth and survival, and is related to the activation of the PI3K/Akt/FoxO3a pathway [25] . Skarra DV et al. indicated that IGF‐1 is a major regulator of cell proliferation, metabolism and apoptosis in various cell types via regulation of the PI3K/Akt pathway and also reported that IGF‐1 in gonadotropes drove FOXO1 phosphorylation through the PI3K/Akt pathway in primary and immortalized cells, leading to the migration of FOXO1 from the nucleus to the cytoplasm [26] . It was reported that Bim could be significantly down‐regulated by IGF‐1 in multiple myeloma cells [27] . Furthermore, Bim, as a downstream target of FOXO3a, mediated a stress response when the PI3K/Akt levels were down‐regulated [28] . Based on the above remarks, it was inferred that depression could be controlled by IGF‐1 through activation of the PI3K/Akt/FoxO3a pathway and reduction of Bim protein expression.

In conclusion, activation of the PI3K/Akt/FoxO3a pathway by IGF‐1 defended against chronic‐stress induced depression in a rat model of CUMS. This may blaze a new path for developing novel effective pharmacological treatments for depression. In terms of the open‐field test, all rats were standardized to allow obvious comparisons to be made and achieve more objective results. Further efforts will be made in the future based on a larger sample size, and clinical experiments will be taken into consideration to validate the findings in the present study.

Acknowledgments

This study was supported by National Natural Science Foundation of China (Grants No.81621003) and Sichuan Science and Technology Plan Program (2015JY0072). We would like show sincere appreciation to the reviewers for critical comments on this article.

Conflicts of interest: All authors declare no conflicts of interest.

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