Introduction

Depression is the fourth leading cause of disability worldwide, leading to cognitive, psychomotor, and physiological abnormalities (Murray et al. 1996; Kessler and Bromet 2013) and affects roughly 17% of the population during their lifetime (Kessler et al. 2003). Depression is associated with central nervous system atrophy, especially in the hippocampus, which shrinks by 20% during major depression. Depression-induced atrophy persists for decades and does not reverse by remission (Sapolsky 2000; Salehpour et al. 2019). It is thought that stressors that play a role in the pathophysiology of depression contribute to the inhibition of neurogenesis in the hippocampus and its subsequent volumetric changes (Gould et al. 1998). Besides suicidal ideation and impaired mood that is frequently experienced in patients with major depression, most patients with depression show signs of cognitive impairments (Wingenfeld and Wolf 2014; Mahmoudi et al. 2015). Exposure to stressful stimuli for a long time and not being able to cope with it cause gradual behavioral changes known as learned helplessness (LH). LH affects emotion, cognition, motivation, and subjective relationships and imposes social and economic burden (Wang et al. 2003). In rodent, LH is induced by painful stressors including, foot-shock and is widely used for modeling behavioral changes observed in depression and development of anti-depressants (Anisman and Merali 2001). Interestingly, the vulnerability of the male mice to the induction of the stress due to their territory makes them a suitable candidate for stress assessing studies (Bartolomucci et al. 2004). At the molecular level, LH changes the level of BDNF (brain-derived neurotrophic factor) (Su et al. 2016), synaptic proteins including synaptophysin, synapsin-1, and post synaptic density-95 (PSD-95) (Iwata et al. 2006; Seese et al. 2013) and promotes neuronal death via expression of Bcl-2, Bax, caspase-3, and caspase-9 molecules (Hunsberger et al. 2011; Kubera et al. 2011). Sericin, as a globular and water-soluble protein, makes up a natural silk polymer in association with fibroin. Its anti-tumor, antibacterial, and neuroprotective effects make it an ideal molecule for biomedical researches (Zhang 2002; Aramwit et al. 2012; Yellamma 2014; Kunz et al. 2016). Findings show that sericin reduces behavioral abnormalities in the mouse model of restraint stress by protecting neuronal cells in the hippocampus (Mohammadi et al. 2019). Also, sericin reduces neurodegeneration caused by diabetes mellitus in rats by activating the signal transduction pathway and its anti-apoptotic and neuroprotective properties (Chen et al. 2012).

Chronic stress extensively stimulates the adrenal–pituitary-hypothalamus axis resulting in increased corticosterone levels in rodents and leading to the development and persistence of depressive symptoms (Morris et al. 2012). Findings show that administration of sericin in stressed mice reduces depression and anxiety signs and lowers corticosterone levels (Mohammadi et al. 2019). Also, sericin increases the levels of antioxidant enzymes in the brain of demented rats and improves cognition (Peera and Yellamma 2015).

Given the neuroprotective properties of sericin in different animal models, this study aimed to study the effects of the sericin on the cognitive deficit, depression, and anxiety caused by LH in male mice.

Materials and Methods

Sixty male Balb/c mice, 3 months old and weighing 25–30 g were obtained from Homa teb Tabriz, Co. (Tabriz, Iran) and kept under standard ambulatory conditions (24 ± 2 °C with a 12 h light/12 h dark cycle) throughout the experiment with ad libitum access to water and standard food. All experimental processes were confirmed by the Animal Ethics Committee of Baqiyatallah University of Medical Sciences, Tehran, Iran (IR.BMSU.REC.1399.357) and conducted under the guidelines of the National Institutes of Health (NIH; Publication No. 85–23, revised 1985).

Study Design

Animals were divided into five groups (n = 12 in each) of control, normal saline (NS), Ser100, Ser200, and Ser300. Mice in the control group received normal saline and were not subjected to LH. Mice in the Ser100, Ser200, and Ser300 groups were pretreated with normal saline, 100, 200, and 300 mg/kg of sericin (Xi’an Lyphar Biotech Co., Ltd, China), respectively and then subjected to LH (Fig. 1). The dose of Ser was chosen based on previous reports (Mohammadi et al. 2019; Mahmoudi et al. 2021). All solutions were freshly prepared before administration and given orally (gastric gavage) at the constant volume of 10 ml/kg for 21 consecutive days. To avoid the effect of circadian rhythms on the SIT, all animals were tested at the same time frame between 10 and 13 A.M.

Fig. 1
figure 1

The time scale of the study

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Learned Helplessness Paradigm

Apparatus

We used an automated shuttle-box apparatus (20 cm × 40 cm × 20 cm) divided into two equal compartments by a retractable automated gate to perform LH. The apparatus was equipped with a grid floor of stainless-steel bars (2 mm in diameter and spaced 0.4 cm apart). The floor was connected to the electric scrambled shocker with an adjustable voltage and duration.

LH Training

Mice were exposed to inescapable, unpredictable, and uncontrolled electro foot shocks for two consecutive days to induce LH (Chourbaji et al. 2005). Each mouse was separately placed in the shock chamber with the closed gate position during the training session. After 3 min of habituation, mice received scrambled inescapable foot shock (0.150 mA for 1–3 s and interval-episodes of 1–15 s) for 60 min (Chourbaji et al. 2005).

Shuttle Escape Testing

Twenty-four hours after the training session, the mice entered the shuttle escape testing phase and were initially allowed to adapt for 3 min. In this phase, which was conducted to measure the avoidance-escape latency, the mice were located individually in the shuttle box and subjected to 30 trials with an interval of 30 s. Each trial started with a light stimulus of 3 s, and then foot shock was induced (0.150 mA for 10 s). In these trials, 3 s after the shock induction, the dividing gate was elevated for 30 s, and mice were allowed to escape from one side of the chamber to the other. Following induction of the shock, the time spent entering the shock-free chamber via the elevated gate was recorded (Smalheiser et al. 2014). As a non-shocked group, the control group was subjected to the same condition as the NS group, except that it did not receive any foot shock. After each trial, the apparatus was cleaned with 10% ethanol to remove any olfactory clues and residues. All behavioral instruments were purchased from the Arman Poshtiban Teb Co, Tabriz, Iran.

Lashley III Maze

The Plexiglas maze consisted of three separate parts, including the start box (8 cm × 9.5 cm × 7 cm), maze arms, and target box (19.5 cm long × 7 cm high × 5 cm wide). Lateral walls and floor were constructed of black Plexiglas and the ceiling was made of perforated transparent Plexiglas. The three longitudinal walls divided the maze into four equal corridors (each 45 cm long, 7 cm high, and 5 cm wide), which were connected by small doors. Briefly, on five consecutive days, each mouse was separately located in the maze and allowed to explore the smell of food. In order to complete the task appropriately, animals were food-deprived overnight and the palatable nutritional supplement (Chee toz, Iran) was placed in the target box.

For testing, each mouse was located in the start box for 15 min and then the Plexiglas gate was opened and mouse was allowed to enter to the corridors and explore the maze for 6 min. The latency to find nutritional supplements inside the target box and the number of errors (turn in an incorrect corridor) were recorded (Seyedaghamiri et al. 2021).

Social Interaction Test (SIT)

A transparent Plexiglas rectangular box (60 L × 45 W × 50 cm H), with three interrelated compartments, was used to test sociability in study groups. The box was divided by Plexiglas walls with sliding doors (6 × 6 cm), allowing the mouse to move and explore freely across the box. Each side compartment had a wire containment cup. This task was done in two sessions: habituation and sociability. In the habituation session, mouse was placed in the middle of the arena and allowed to move and explore chambers for 10 min. Before initiation of the sociability session, a mouse as a stranger was located in one of the wire containments of the side chamber. And then, the subject mouse was placed in the apparatus for 10 min. In the habituation session, total traveled distance of subject mouse was measured as the index of the locomotor activity. At the end of the test, the discrimination index was calculated based on the following formula (Denninger et al. 2018):

$$\frac{time\; spent\; for\; assessment\; stranger\; 2}{time \;spent \;for \;assessment \;stranger \;2\;+ \;time \;spent \;for \;assessment \;stranger \;1}$$

Preparation of Tissue Samples

Twenty-four hours after behavioral tests, mice were deeply anesthetized by intraperitoneal injection of ketamine and xylazine (200 mg/kg and 10 mg/kg, respectively) and then the brain was quickly removed. Then, hippocampus was isolated on the ice-cold surface and stored in −70 °C.

Western Blot Analysis

Hippocampal tissue samples were homogenized in Radio Immunoprecipitation assay (RIPA) lysis buffer ([150 mM sodium chloride, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris, pH 8.0] containing protease inhibitor cocktail). The prepared homogenized samples were then centrifuged at 13,000 g for 15 min at 4 °C. Then, the Bradford method was used to estimate total protein concentration in the supernatant. Equal amount of protein samples (20 μg) was loaded to 12.5% SDS–polyacrylamide gel electrophoresis and separated proteins were transferred onto a polyvinylidene difluoride membranes (PVDF; Roche, UK). Subsequently, the membranes were incubated with the primary antibodies overnight at 4 °C: anti PSD-95 (1:500, sc-32290), anti synapsin-1 (1:500, sc-17750), anti-synaptophysin (1:500, sc-17750), anti Bax (1:500, sc-493), anti Bcl-2 (1:500, sc-492), caspase-3 (1:500, sc-7148), anti-caspase-9 (1:500, sc-81663) and β-actin (sc-130656). Then, membranes were located in ECL prime Western blotting detection reagent (Amersham, UK) and Generated Signals by exposure to autoradiography film were visualized (Kodak, USA). Finally, bands were quantified using Image J v.1.62 software (US National Institutes of Health, Bethesda, MD, USA).

Statistical Analysis

All data were presented as mean ± standard error of the mean (SEM) and analyzed by Graph Pad Prism 6.01 (Graph Pad Software Inc., La Jolla, CA, USA). Two-way or one-way analysis of variance (ANOVA) and post hoc Tukey tests was used to detect the statistically significant difference between groups. The day and group were the variables of interest in the Two-way analysis (Lashley III maze); however, outcomes were compared between groups in corresponding days. A p-value < 0.05 was considered statistically significant.

Results

The Effect of Sericin on the Escape Latency of Shuttle Escape Phase

In all groups, mice were tested for learned helplessness by comparing their latencies to escape from a foot shock by the shuttle box. The results of one-way ANOVA demonstrated significant differences in the mean latency time among the study groups. As shown in Fig. 2, the post-hoc analysis revealed that latency time markedly increased in the NS mice compared with the control group. On the other hand, pretreatment with sericin at doses of 100 (p < 0.01), 200, and 300 mg/kg (for both, p < 0.001) significantly reduced latency time in the LH mice compared with the NS group.

Fig. 2
figure 2

Mean latency time as a learned helplessness index. Data are presented as mean ± S.E.M. (n = 12). ***p < 0.001 vs. control group, #p < 0.05, ##p < 0.01, vs. NS group. (NS: normal saline; Ser: sericin; LH: learned haplessness)

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The Effect of Sericin on the Lashley III Maze Task

The Lashley III Maze task as a low-stress behavior paradigm that relies on route learning is used to evaluate spatial learning and memory (Seyedaghamiri et al. 2021). As shown in Fig. 3A, mice in the NS group had a significantly higher latency time in the Lashley III maze task on the 2nd, 3rd, and 4th days of training (at least p < 0.05 for all of these days) compared with the control group. Administration of sericin at the 200-mg/kg dose reduced latency time on days 3 and 4 of testing (p < 0.01, for both days). In addition, sericin at the dose of 300 significantly reduced latency on the 2nd, 3rd, and 4th test days (p < 0.05 for all of these days). Subjecting the animals to LH increased the mean value of the number of errors in the NS group on days 2, 3, 4, and, 5 (p < 0.05 for all days) compared with the control group. Administration of sericin at 200 mg/kg doses significantly reduced the number of errors on the 4th and 5th days of training (p < 0.05, for both days) compared with the NS group. In addition, sericin at the dose of 300 mg/kg significantly decreased the number of errors on the 2nd, 3rd, 4th, and 5th of training days compared with the Ns group (at least p < 0.05 for all days) (Fig. 3B).

Fig. 3
figure 3

(A) Latency times, and (B) the number of errors in different groups. Differences among groups were analyzed for each day. Data are presented as mean ± S.E.M., (n = 12). **p < 0.01, ***p < 0.001 vs. control group, #p < 0.05, ##p < 0.01, vs. NS group. (NS: normal saline, Ser: sericin)

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The Effect of Sericin on the Social Interaction Test

Social interaction test was conducted to evaluate sociability in the study. The results of one-way ANOVA demonstrated that the discrimination index significantly reduced in the NS group compared with the control group (p < 0.001). The post-hoc analysis demonstrated that LH-subjected animals in the ser 100 group were not significantly different in the discrimination index. Nevertheless, pretreatment with sericin at 200 and 300-mg/kg doses significantly increased this ratio compared with the NS group (for both, p < 0.001) (Fig. 4).

Fig. 4
figure 4

Discrimination index of SIT test. Data represent the mean ± S.E.M., (n = 12). Significant differences tested by one-way ANOVA followed by Tukey’s post hoc test. Data are presented as mean ± S.E.M., (n = 12). **p < 0.01, ***p < 0.001 vs. control group, #p < 0.05, ##p < 0.01, vs. NS group. (Ser: sericin, NS: normal saline)

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The Effect of Ser Pretreatment on Locomotor Activity of LH Mice

As Fig. 5 shows, neither LH nor administration of different doses of the Ser had any significant effects on the locomotor performance of mice.

Fig. 5
figure 5

Locomotor activity. Data are presented as mean ± S.E.M., (n = 12). (NS: normal saline; Ser: sericin: LH: learned haplessness). (NS: normal saline; Ser: sericin: LH: learned helplessness)

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The Effect of Ser on the BDNF and Synaptic Proteins Levels in the Hippocampus of the LH Mice

As shown in Fig. 6A, the hippocampal BDNF level in the NS mice markedly decreased compared with control group. Also, sericin pretreatment at doses of 200 and 300 mg/kg significantly increased BDNF levels (p < 0.01 and p < 0.001, respectively) compared with NS mice. The results of one-way ANOVA revealed significant differences in the hippocampal synaptophysin levels (F (4, 10) = 16.4, p < 0.0002), synapsin-1 levels (F (4, 10) = 19.1, p < 0. 001), and PSD-95 levels (F (4, 10) = 16.1, p < 0.0002) among the study groups. As Fig. 6B shows, LH significantly decreased synaptophysin levels in the hippocampus of the NS mice compared with the control group (p < 0.01). However, sericin at doses of 200 and 300 mg/kg markedly increased synaptophysin levels (p < 0.01 and p < 0.001, respectively) in comparison to the NS group. Post-hoc analysis of synapsin-1 levels indicated that induction of LH significantly decreased synapsin-1 levels in the NS mice compared with the control group (p < 0.001, Fig. 6C). Also, sericin at the dose of 300 mg/kg increased hippocampal synapsin-1 levels (p < 0.01) compared with the NS group. As shown in Fig. 6D, induction of LH significantly decreased PSD-95 levels in the hippocampus (p < 0.001) compared with the control group. However, sericin pretreatment at doses of 200 and 300 mg/kg markedly increased PSD-95 levels (p < 0.01) compared with the NS group.

Fig. 6
figure 6

Sericin pretreatment effect on hippocampal BDNF and synaptic proteins in the LH subjected mice. (A) Fold of control of BDNF, (B) Fold of control of SYP, (C) Fold of control of SYN-1, and (D) Fold of control of PSD-95. Representative images of corresponding protein expressions. Data are presented as mean ± S.E.M. (n = 3). ***p < 0.001 vs. control group. #p < 0.05, ##p < 0.01 vs. NS group (NS: normal saline; Ser: sericin; LH: learned helplessness; SYP: synaptophysin, SYN-1: synapsin-1)

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The Effect of Ser on the Apoptotic Proteins Levels in the Hippocampus of the LH Mice

The results of one-way ANOVA demonstrated significant differences in the hippocampal levels of Bax (F (4, 10) = 11.4, p < 0.0010) and Bcl-2 (F (4, 10) = 24.2, p < 0.0001) protein levels and the ratios of cleaved caspase-3/procaspase-3 (F (4, 10) = 28.6, p < 0.0001) and cleaved caspase-9/procaspase-9 (F (4, 10) = 22.0, p < 0.0001) among the study groups. Post-hoc analysis showed that Bax levels significantly increased in the NS group compared with the control group (p < 0.001, Fig. 7A). Nevertheless, sericin at 200 and 300-mg/kg doses reduced Bax protein levels in the LH mice (p < 0.05 and p < 0.001, respectively) compared with the NS group. As shown in Fig. 7B, Bcl-2 levels significantly reduced under the LH effect in the NS mice’s hippocampus compared with the control group (p < 0.001). On the other hand, pretreatment with sericin at the dose of 300 mg/kg resulted in a significant increase in the hippocampal Bcl-2 level compared with the NS group (p < 0.05). Also, LH significantly increased cleaved caspase 9/pro-caspase 9 (p < 0.001, Fig. 7C) and cleaved caspase 3/pro-caspase 3 (p < 0.01, Fig. 7D) ratios in the hippocampus of the NS group compared with the control group. However, sericin at the dose of 300 mg/kg significantly lowered cleaved caspase 9/pro-caspase 9 (p < 0.001) and cleaved caspase 3/pro-caspase 3 (p < 0.05) ratios compared with the NS group.

Fig. 7
figure 7

Sericin pretreatment effect on hippocampal pro-apoptotic markers in the LH subjected mice. (A) Fold of control of Bax, (B) Fold of control of Bcl-2, (C) Fold of control of cleaved caspase-3 to pro-caspase-3 ratio, and (D) Fold of control of cleaved caspase-9 to pro-caspase-9. (E) Representative images of corresponding protein expressions. Data are presented as mean ± S.E.M. (n = 3). **p < 0.01, ***p < 0.001 vs. control group. #p < 0.05, ##p < 0.01, ###p < 0.001 vs. NS group (NS: normal saline; Ser: sericin, LH: learned haplessness)

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Discussion

This study showed that LH-induced stress led to mice’s cognitive impairment, depression, and anxiety. It was also found that the level of hippocampal BDNF, synaptic proteins including synaptophysin, synapsin-1, and PSD-95 were down-regulated by LH-induced stress. Besides, pro-apoptotic markers (Bax, Caspase-3, and caspase-9) increased and Bcl-2 decreased. Impaired cognition, mood, self-worth, depression, and anxiety are seen as a consequence of LH (Raps et al. 1980). Some areas of the brain, such as the hippocampus, amygdala and medial prefrontal cortex have important roles in clinical depression and depression-like behavior in the animal models (Hasler et al. 2004; Wang et al. 2014; Kim et al. 2016).

Subjects, who find themselves exposed to uncontrollable events, show various symptoms that change their emotion, behavior, and cognition. They usually show an experience of indifference, aggression, and difficulty in performing cognitive tasks such as problem-solving (Wortman and Brehm 1975; Roth 1980; Sullivan et al. 2012).

Latency time is defined as the time for escaping from the shocked-paired compartment toward the non-shocked paired part and used as the main parameter for confirmation of the LH induction. Mice with LH will usually have a longer latency time than non-stressed mice (Landgraf et al. 2015). Administration of the sericin (at all doses) shortened the latency time in stressed exposed groups, reflecting its ability for normalizing behavioral changes in these mice.

Our behavioral findings indicated that LH induction worsened the mice’s cognitive abilities in the Lashley III and social interaction tasks. Nevertheless, 21 days of sericin administration (200 and 300 mg/kg) reversed these impairments. Different stressors by affecting the activity of the hippocampus not only disrupt spatial learning and memory (Fekri et al. 2021; Mahmoudi et al. 2021) but also could impair sociability in mice (Salehpour et al. 2018). There is a reciprocal relationship between sociability and cognitive abilities, whereas improvement in social interaction positively affects cognition, and the risk of cognitive disabilities increases under sociability deficits (Hsiao et al. 2014). Moreover, impaired sociability is associated with depression and anxiety, indicating the important role of this phenomenon in stress-related studies (Salehpour et al. 2018).

In experiments performed on the animal models of LH, it was found that, induction of 48–72-h uncontrollable stress led to reduced swimming in water (Weiss and Simson 1986), reduced activity (Jackson et al. 1978), social interaction defects (Short and Maier 1993), and exaggerated fear conditioning (Maier 1990). BDNF as a crucial factor in the neuroprotection and neuroplasticity has a role in cognitive procedures (Hsiao et al. 2014). The hippocampal BDNF is markedly downregulated in stress exposed mice and results in marked cognitive impairments (Pizarro et al. 2004). Our results showed that induction of LH lowered BDNF expression in the hippocampus and sericin administration increased its levels. Several studies showed a decrease in the synaptic proteins associated with excitatory synapses such as synaptophysin, PSD95, and synapsin-1 in the neurological disorders (Feyissa et al. 2010; Kim et al. 2010).

Postsynaptic density protein 95 (PSD-95) is mainly expressed in the postsynaptic membrane of excitatory neurons to regulate synaptic plasticity, glutamatergic transmission, stabilization and trafficking of N-methyl-D-aspartate (NMDA) and aminomethylphosphonic acid (AMPA) receptors (Cheng et al. 2006). It was shown that reduction in PSD-95 levels led to various conditions such as schizophrenia, autism, intellectual disability, and cognitive and mental disorders (Fromer et al. 2014; Fernández et al. 2017; Coley and Gao 2018). In this study, we observed that the hippocampal level of PSD-95 significantly decreased in LH mice. However, sericin at dose of 200 and 300 mg/kg markedly increased PSD-95 hippocampal level. In line with our results, Jianhua et al. (2017) showed that social defeat stress reduces the hippocampal level of PSD-95 in mice. Also, Zhang et al. (2014) showed that exposure to 5 weeks of chronic unpredictable mild stress reduced amygdala levels of PSD-95 protein in rats. The phosphorylated form of synapsin-1 in the presynaptic terminals is associated with neuronal plasticity, neurotransmitter exocytosis, synaptic vesicle fusion, and neuronal activity (Sato et al. 2000; Eastwood and Harrison 2001). Because of these crucial functions of synapsin-1 in the brain, alteration of this protein causes a wide range of neurological disorders such as Alzheimer’s disease, autism, schizophrenia, and multiple sclerosis (Masliah and Terry 1993; Park et al. 2014). Bessa et al. (2009) demonstrated that chronic mild stress reduced synapsin-1 expression and mRNA in rats’ hippocampus and prefrontal cortex. In this study, it was found that learned helplessness reduced the hippocampal level of synapsin-1 in mice. On the other hand, treatment with sericin at the dose of 200 mg/kg significantly increased its levels in the hippocampus of mice. As with PSD-95 and synapsin-1 proteins, synaptophysin (syp) mediates synapse formation, exocytosis, biogenesis and endocytosis of synaptic vesicles (Spiwoks-Becker et al. 2001; Tarsa and Goda 2002). Various clinical and paraclinical studies showed that loss of synaptophysin was associated with neurodegenerative disorders (Raju et al. 2008; Marttinen et al. 2015). Luo et al. (2013) reported that synaptophysin expression in the amygdala of rats exposed to 5 weeks of chronic unpredictable mild stress significantly decreased. Also, previous studies showed that synaptophysin mRNA and protein in the hippocampus decreased by stress and increased by anti-depressants (Rapp et al. 2004; Han and Kim 2008). In confirmation of previous studies, we found that the hippocampal level of synaptophysin in the LH-induced mice decreased and pretreatment with 200 and 300 mg/kg markedly increased the level of this synaptic protein. Chronic stress increases the cortisol level following hyperactivity of the hypothalamic–pituitary–adrenal axis (Tsigos and Chrousos 2002). Cortisol, in turn, causes downregulation of TrkB and activities of Akt and mammalian target of rapamycin (mTOR) which are the critical components in the PI3K/Akt pathway. It is believed that activation of mTOR is associated with synaptic proteins such as SYP, PSD 95, neurexin, and neuroligin (Smith et al. 1995; Fang et al. 2013; Suri and Vaidya 2013).

From a neuroscience perspective, apoptosis is considered a vital component of neuronal development and homeostasis, neurodegeneration, and stress-related disorders (Danial and Korsmeyer 2004; De Kloet et al. 2005; Lledo and Saghatelyan 2005; Elmore 2007). Cell death following acute stress may be beneficial and lead to the integration of newly formed neurons (Lledo and Saghatelyan 2005). These neurons result from neurogenesis within neural networks and finally display increased plasticity (Schmidt-Hieber et al. 2004). However, chronic and continuous stress induces functional and physiological disorders in the brain, reduces brain ability (De Kloet et al. 2005), and increases the susceptibility of neurons to cell death (Joëls et al. 2004). Stress-induced apoptosis and DNA fragmentation in postmortem brains of depressed patients have been reported, suggesting neurological vulnerability during stress and depression (Lucassen et al. 2006). Caspases are synthesized in the pro-caspase inactive form and by proteolytic enzymes cleavage and activated. Caspases activate each other in a cascading process. For example, in the neural cells some biochemical stimuli activate caspase 3 and die (Yuan and Yankner 2000). Also, the role of caspase-3 and caspase-9 in neural apoptosis is well established (Tait and Green 2010; Fuchs and Steller 2011). Previous studies have shown that chronic stress causes an increase in caspase-3 and caspase-9 in mice hippocampus (Liu et al. 2010; Hidayat et al. 2020; Wang et al. 2020). Fan et al. (2018) reported that cleaved caspase-3 and caspase-9 expression within the vmPFC region significantly decreased in the rat model of chronic unpredictable mild stress. Bcl-2 is an endogenous anti-apoptotic membrane protein that protects neurons against cell death and enhances neurogenesis and axonal regeneration (Kowaltowski et al. 2004). It has been shown that chronic mild stress decreases the expression of BAG-1 (The gene that binds to Bcl-2 and enhances the anti-apoptotic effects) in the hippocampus (Silva et al. 2008). Kosten et al. (2008) demonstrated that unpredictable stress in the rats leads to decreased mRNA and protein levels of BCl-2 in different brain regions related to cognitive function. Bax, as a pro-apoptotic mitochondrial protein, induces the release of Cyt c into the cytosol and, Cyt c triggers the cleavage of pro-caspase 9 and pro-caspase 3 and finally induces apoptosis (Portt et al. 2011). Wang et al. demonstrated that in rats with chronic mild stress, there was a significant increase in the hippocampal level of Bax, suggesting an increase in pro-apoptotic pathways (Portt et al. 2011). Here, we showed that sericin reversed the increased ratio of Bax/Bcl-2 and inhibited the cleavage of procaspase-9 and procaspase-3 after learned helplessness in mice. With these findings, sericin pretreatment showed a great efficacy in lowering pro-apoptotic proteins and increasing anti-apoptotic factors in the hippocampus.

A limitation of this study was that we did not measure electrophysiological properties in the hippocampus to demonstrate the neuronal disconnection/plasticity reduction, etc. It is recommended that for real-time readout of neural functions and network potential and demonstrate the neuronal plasticity function in the hippocampus, these factors are measured in future studies by electrophysiology methods.