1. INTRODUCTION:

Depression is generally known to cause mental and/or physical lethargy in individuals, and is representative of social illnesses that a large part of the modern society suffers from¹. In a society with an increasing aging population due to the increase in national income, improvement of living standards, improvement in medicines and health hygiene, and expansion of welfare policies, depression is on the rise and has become a social issue.

The elderly population, which has increased with the increase in population, has led to a rise in aging-related problems. The elderly individuals suffer from chronic degenerative diseases, decreased physical function, deterioration of mental health, loss of economic independence, and social isolation. In a society where nuclear families are becoming prevalent, the role of elderly individuals has become weak and consequently, their sense of alienation has increased².

The following theories have been proposed regarding the causes of depression: the lack of noradrenaline or serotonin in the brain causes the symptoms of depression³; depression is caused by the abnormality of the Hypothalamic-Pituitary-Adrenal (HPA) axis stimulating the secretion of cortisol by increasing the secretion of CRF⁴; and depression is caused by the abnormal secretion of cytokines such asINF-α, and IL-1by macrophages, which in turn cause the abnormal secretion of CRF, ACTH, prolactin, and cortisol⁵. The theory that depression is caused by decreased levels of noradrenaline and serotonin, has received considerable support. However, despite the progress in related research, the underlying cause of depression has not yet been elucidated.

Treatment approaches for depression can be largely divided into two groups: psychotherapy and drug-based therapy. Psychotherapy is mainly used in the treatment of mild depression, while drug-based therapy is used in the treatment of severe depression⁶. Chemically synthesized antidepressant agents commonly used in drug-based therapy includeserotonin, noradrenergic reuptake inhibitors (SNRIs), noradrenergic reuptake inhibitor (NARI), selective serotonin reuptake inhibitor (SSRI), and tricyclic antidepressants (TCA), and monoamine oxidase inhibitor (MAOI)^7-11. However, it is considered that these may not be suitable to be used for treating depression in the elderly individuals exhibiting a decline in physical function, because of side-effects such as low reactivity and recovery rate, fatigue, sleeping disorder, cognitive disorder, and sexual dysfunction^12-13. Therefore, it is necessary to develop safer natural therapeutic antidepressant agents with minimal adverse effects. Consequently, active research is being conducted in this field.

In the terms of oriental medicine, depression is called Uljeung, and its causes include gan-ul-bi-heo, gan-ul-gi-che, dam-mi-sim-kyu, sim-bi-yang-heo and bi-sin-yang-heo^14-15. Based on the oriental medicine theory, Shingi, a word that encompasses spirit and energy, weakens as aging progresses. Therefore, depression in elderly individuals may be attributed to bi-sin-yang-heo. Thus, depression in elderly individuals could be alleviated by prescribing medication that is both mentally and physically rejuvenating.

Shinkiwhan is a medicine prescribed to facilitate rejuvenation, used as a treatment for kidney disease, as described in the DonguiBogam. Moreover, Eucommiasp.¹⁶, Amomiamari fructus¹⁷, Bombycis batryticatus¹⁸, Acanthopanax¹⁹, Rubus coreanus²⁰, and Lycii fructus²¹ are sources of herbal medicines that reduce fatigue in kidney disease. Therefore, Gami-Shinkiwhan (GS), the combination of these herbal medicines with Shinkiwhan, has been hypothesized to be effective in treating depression in elderly individuals. It would further improve depression in aging people. In order to establish the role of Gami-Shinkiwhan as an antidepressant agent, this study combines tail suspension test and immunohistochemical analyses in aging mice, with depression being induced by immobilization stress.

2. MATERIALS AND METHODS:

2.1. Experimental animals:

The experimental animals consisted of 24-month-old male ICR mice (Samtako, Korea), andwere provided sufficient feed and water, we adapted to the laboratory environment for 7 days before the study. Mice were divided into the following groups: control group (without any treatment); depressed group (DS group), which were subjected to immobilization stress but without any treatment; Gami-Shinkiwhan-treated group (GS group), in which the mice were treated with GS (0.56 g/kg/day) and subjected to immobilization stress for 10 weeks. This study was performed with the approval of the Animal Experiment Ethics Committee of Semyung University (SMECAC 16-05-05).

2.2. Depression induced by immobilization stress:

An acrylic box, with multiple holes on the top, side, and front surfaces, of the dimensions 40 × 30 × 70 mm (width × length × length), was manufactured. Depression was induced by placing the mice from the relevant groups in the acrylic box at a predetermined time for 1 hour every day, for a total period of 10 weeks, and exposing them to the long-term immobilization stress.

2.3. Production of GS extract:

For production of the extract, GS(94 g) and 1000 ml of distilled water were mixed (shown in table 1). The mixture in a round flaskwas heated for 3 hours at 100°C. The extract was concentrated using a vacuum evaporator (Eyela, Japan) and lyophilized to obtain 8.9 g of powder.

Table 1: Composition of Gami-Shinkiwhan (GS) per pack

Common Name of Herb	Scientific Name	Weight (g)
Sukjihwang	Rhizomarehmanniae	16
Sanyak	Rhizomadiscoreae	8
Sansuyu	Fructuscorni	8
Omija	Schisandrachinensisfructus	8
Taeksa	Rhizomaalismatis	6
Mokdanpi	Cortex moutanradicis	6
Baekbokryung	Poria cocas	6
Duchoong	Eucommiae cortex	6
Ikjiin	Amomiamarifructus	6
Baekgamjang	Bombycisbatryticatus	6
Ogapi	Acanthopanax cortex	6
Bokbunja	Rubuscoreanus	6
Gugija	Lyciifructus	6
Total		94

2.4. Measurement of the effects of GS on depression:

2.4.1. Tail suspension test (TST):

Tail suspension test was conducteded by using a tape to wrap half of the experimental mice's tail and fixing the tail end to a box (Height: 54 cm, Width: 30 cm, Depth: 47 cm). The mice were suspended upside down for a total of 6 minutes and their behavior was recorded using a video camera. The first 2 minutes were not considered during analysis as this period was required for adaptation. The “immobility time” was measured during the last 4 minutes based on the behavior of the experimental animal. Suspended mice which remained in completely still were considered to be in the immobile state.

The measurement was performed twice before the treatment of Immobilization stress and Gami-Shinkiwhan and for 10 weeks after the administration of immobilization stress and Gami-Shinkiwhan. The durations of “immobility time” obtained from the tests performed before and after the treatment were compared to assess the effect of GS.

2.4.2. Immunohistochemistry:

2.4.2.1. Production of hippocampal tissue samples:

The mice were euthanized using ether, and their brains were dissected. The extracted brain tissues were fixed in 10% NBF at 36.5°C for 24 hours. The fixed tissues were embedded in paraffin a conventional manner and then formed into 5 µm-thick continuoussection.

2.4.2.2. Immunohistochemistry:

To investigate the effect of GS on depression, immunohistochemical staining was performed. Hippocampus sections were treated with proteinase K (20 µg/ml) to undergo proteolysis for 5 minutesand then the samples were incubated in 10% normal goat serumwhich is a blocking solution for 2 hours. The sample were then incubated with the following primary antibodies: 1:100 mouse anti-serotonin (Santa Cruz Biotechnology, USA), 1:100mouse anti-cleaved Caspase-3 (Santa Cruz Biotechnology, USA), 1:100mouse anti-histone deacetylase (HDAC)-3 (Santa Cruz Biotechnology, USA), 1:100 mouse anti-SHH (Santa Cruz Biotechnology, USA) at 4°Cfor 72 hours in a humidified chamber. Then the sample was incubated with a secondary antibody, 1:100 biotinylated goat anti-mouse IgG (DAKO, USA), at room temperaturefor 24 hours, followed by incubation with streptavidin from the avidin biotin complex kit (VectorLab, USA) at room temperaturefor an hour. Finally, the sample was incubated in a 0.05 M tris-HCl buffer at pH 7.4 containing 0.05% 3,3'-diaminobenzidine (DAB) and 0.01% HCl, followed by incubation in hematoxylin dye, used for contrast.

2.5. Image analysis and statistical analysis:

The images obtained from immunohistochemical experiments were quantified (mean±standard error) using Image Pro Plus (Media cybernetics, USA). The region of interest (ROI) was randomly selected from the hippocampal region of each group and images were captured at 100×magnification. Image analysis was conducted with positive pixels/20,000,000 pixels. Statistical analysis for the skin damage scores and immunohistochemistry results was performed using SPSS software (SPSS 20, SPSS Inc., USA). One-way ANOVA was used to test significance (P < 0.05).

3. RESULTS:

3.1. Change of immobility time in the TST:

Tail suspension test (TST) was conducted in order to confirm the induction of depression. The immobility time for the DS group increased significantly after the mice were exposed to immobilization stress, compared to that recorded in the naïve condition. However, the immobility time for the GS group decreased significantly after exposure to immobilization stress, compared to that recorded in the naïve condition.

A comparison of the results obtained from each group after exposure to immobilization stress revealed that the immobility time was significantly greater for the DS group compared to the control group, and lower in the GS group compared to the control groupsignificantly. However, there was no significant difference between the DS and GS groups as showns in table 2.

Table 2: Change in the immobility time of mice, as recorded from the tail suspension test.

Group	Time period
Group	Before immobilization	After immobilization
Control	134.29 ± 18.4	143.29 ± 24.4
DS group	146.38 ± 21.9	165.00 ± 12.3^#
GS group	161.63 ± 18.1	136.71 ± 30.5

Values are represented as mean ± standard deviation (n = 10). ^# indicates statistical significance (P < 0.05), compared to control group.

3.2. Change in level serotonin in the hippocampus:

Immunohistochemical analysis revealed that in the GS group,serotonin level in the hippocampus increased significantly (200%) compared to the DS group in figure 1.

4. DISCUSSION:

Depression refers to an emotional disorder that involves anxiety, a sense of gloominess, failure, helplessness, and worthlessness, resulting from one's negative perception. Depression can be accurately described as a continuum from normal mood changes to pathological states²². The World Health Organization (WHO) classified depression, which affects about 15 to 25% of the world’s population, as one of the most dangerous illnesses in the modern society. With increasing age, depression correlates strongly with suicide^{8, 23}.The prevalence rate of depression in the elderly is reported to be about 1~2%. However, in the case of South Korea, the frequency of depression in the elderly is known to vary from 20 to 60%, depending on diagnostic tools. According to the results from a study conducted by Ki Bae-seok, 15% elderly individuals exhibited symptoms of depression^24-25.

Elderly individuals experience a considerable level of physical, psychological, and social stress. Aging involves several changes, not only in physical and physiological in nature, but also cognitive and behavioral. Moreover, physical and psychological degeneration associated with aging compromises cognitive function, sensory function, and emotional response, and increase the tendency toward depression^26-27.

The major clinical features of depression observed in the elderly include lack of sleep, loss of appetite or overeating, decreased attention, loss of productivity induced by a sense of helplessness, social withdrawal, decreased activity, loss of interest, pessimistic attitude toward the future, and repeated thoughts of suicideordeath. Symptoms of depression are highly correlated with suicide for this age group in particular^28-29. In other words, depression not only lowers the satisfaction with life in elderly individuals, but also increases the risk of suicide.

As for the cause of depression, it is broadly understood that a biological factor plays a crucial role, but the precise cause has not been identified yet. Among the several proposed hypotheses, the theory that depression is caused by a deficiency in monoamine neurotransmitters of the central nervous system such as serotonin, noradrenalin, and dopamine, is now considered a strong hypothesis^6-7. In line with this hypothesis, people with a tendency toward depression are known to have a relatively weak serotonergic system. Therefore, the mechanisms of action of all anti-depressants are aimed at increasing the level of serotonin present at the synapses.

Serotonin (5-hydroxy tryptamine) is one of the important neurotransmitters associated with major mental illnesses and psychopathies reported in the psychiatric field. It is present in nerve cells, platelets, and mastocytes. Serotonin present in the brain accounts for about 1 to 2% of the total amount. Serotonergic neurons and receptors are abundantly distributed in most parts of the corticolimbic circuit. Serotonin present in the cerebrum is synthesized locally by the cerebral neurons, and decrease in serotonin utilization has been known to be associated with depression, anxiety disorder, and suicide^30-31.

The hippocampus is responsible for learning, memory, emotions, and aggression. Hippocampal changes have been reported in the context of many brain disorders. Hippocampal degeneration occurs in the early stages of Alzheimer's disease, and hippocampal sclerosis often occurs in temporal lobe epilepsy. Structural and functional abnormalities of the hippocampus have been reported in diseases such as long-term stress, posttraumatic stress disorder, and depression^32-35.The neurobiological basis of the role of hippocampus in depression has not yet been elucidated, but the possibility of abnormal neural circuitry has been suggested. Many studies have reported that hippocampus volume is reduced in depression patients. Moreover, a consistent reduction in the hippocampal volume has been reported in diseases such as recurrent depression and early-onset depression^36-37.

In <Somun, Sanggocheonjinlon>, the biological changes were explained on the basis of age (for males 8 years and for females7 years), and the explanation revolves around the changes in Singi³⁸. In other words, Singi gradually weakens as the age increases. Based on this, depression can be considered to be an emotional or mental illness associated with the process of aging. Therefore, administration of rejuvenating medication that strengthens Shingi or mental health might alleviate depression in elderly individuals.

To test this hypothesis, Gami-Shinkiwhan, added with Eucommiaulmoides, Alpiniaeoxyphyllaefructus, Bombycisbatryticatus, Acanthopanax sp., Robuscoreanus, and Lyciumchinense, was administered to the experimental animals in which depression had been induced by immobilization stress. Behavioral tests (TST) and immunohistochemical analyses were performed to assess the putative antidepressive effects of Gami-Shinkiwhan.

Depression was induced in aged mice by subjecting them to immobilization stress. This is the established method for simultaneously inflicting physical and mental stress on mice when studying behavioral and biological phenomena associated with depression in humans. The behavioral symptoms observed in patients with depression such as due to altered endocrine activity, are induced in mice exposed to long-term stress³⁹. The immobility time measure during the tail suspension test was used as a read-out to verify whether the mice exhibited behavioral improvement on administration of GS. Immobility time refers to the time period during experimentation when the animal remains suspended without any movement, and is an indicator of depression. The severity of depression is considered to increase with increasing immobility time.

The significant increase in immobility time for the DS group after exposure to immobilization stress, compared to immobility time for the same animals before subjecting them to stress, indicates that depression was successfully induced. The significant reduction in the immobility time for the GS group even after exposure to immobilization stress was, compared to the immobility time for the same animals before subjecting them to stress indicates alleviation of depression due to the administration of Gami-Shinkiwhan (GS).

Furthermore, comparison of the results obtained after exposure to immobilization stress across all the groups revealed that the immobility time was significantly greater for the DS group, compared to the control group, while the it was significantly lower for the GS group, compared to the control group. However, there was no significant difference between the DS and GS groups. This indicates that although depression was induced in the DS group due to immobilization stress, the GS group exhibited a degree of depression similar to that of the control group in which animals were not exposed to stress, due to the administration of the Gami-Shinkiwhan (GS).

Immunohistochemical analysis of hippocampal slices indicated that the level of serotonin was significantly lower in the DS group compared to the levels in the control group, and was significantly higher in the GS group compared to the levels in the DS group. This result is consistent with several reports about a reduction in the amount of serotonin to be associated with depression. Based on these results, it can be concluded that Gami-Shinkiwhan exhibits antidepressive effects by increasing the serotonin level in the hippocampus.

Immunohistochemical analysis was also performed to study the levels of caspase-3, HDAC3, and SHH in hippocampal slices.

The results indicate that the levels of caspase-3 and HDAC3 were lower in the GS groupsignificantly, compared to the DS group, while the level of SHH was higher in the GS group compared to the DS groupsignificantly.

Twelve caspases are known to exist, and out of them, caspase-3 is known to play the most important role during apoptosis^40-41. Hence, the fact that the level of caspase-3 decreased implies inhibition of apoptotic mechanisms in hippocampus. This result is consistent with several studies that have reported decreased hippocampal volume in depressionpatients. Therefore, the results indicate that Gami-Shinkiwhan exhibits an anti-depressive effect by inhibiting caspase-3-mediated apoptosis.

Histone deacetylase (HDAC) is an enzyme that deacetylates histone, which supercoils the chromatin structure, and reduces DNA transcription levels⁴². In other words, the reduction in HDAC3 level in hippocampal tissue suggests that the HDAC3 activity was lowered, increasing the acetylation of histones, thereby promoting transcription of genes in the hippocampus. This is consistent with the results from a study⁴³ that the demonstrated antidepressiveeffect of an HDAC3 inhibitor.

Sonic Hedgehog (SHH) is one of the hedgehog proteins, and is involved in cell growth and differentiation of healthy cells. In the SHH signaling cascade, when SHH is activated by cholesterol, palmitate is secreted by the cell and binds to the cell membrane receptor PTCH expressed on reactive cells. The activity of SMO, which is otherwise inhibited by PTCH, causes structural transformation, initiating a signaling cascade which eventually leads to proliferation and differentiation of cells^44-45. Therefore, an increase in the level of SHH in hippocampus indicates that Gami-Shinkiwhan exerts an antidepressive effect by in promoting SHH-mediated proliferation in hippocampal cells.

Taken together, the results indicate that Gami-Shinkiwhan might be able to alleviate the depression in elderly individuals by increasing serotonin levels in the hippocampus, reducing the activity of caspase-3 and HDAC3 associated with apoptosis, and enhancing SHH-mediated proliferation of hippocampal cells.

5. CONCLUSION:

We studied the putative antidepressive effects of Gami-Shinkiwhan by conducting the tail suspension test and immunohistochemical analysis after administering Gami-Shinkiwhan to aged mice in which depression had been induced by immobilization stress. The following results were obtained:

1 The GS group exhibited significantly lower immobility time during the tail suspension test.

2 The GS group exhibited higher level of serotonin.

3 The GS group exhibited lower level of caspase-3.

4 The GS group exhibited lower level of HDAC3.

5 The GS group exhibited higher level of SHH.

Based on these results, we conclude that Gami-Shinkiwhan alleviates depression in aged mice by increasing the level of serotonin in the hippocampus, decreasing caspase-3 and HDAC3 activity, which are closely associated with apoptosis, and increasing SHH-mediated proliferation.

6. ACKNOWLEDGMENT:

This study was sponsored by the Korea Health Industry Development Institute, with funding from the Ministry of Health and Welfare (HI16C-0469-010016)

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Received on 12.12.2017 Modified on 11.01.2018

Research J. Pharm. and Tech 2018; 11(5):1909-1916.

DOI: 10.5958/0974-360X.2018.00354.2

Antidepressive effects of Gami-Shinkiwhanin Immobilization stressed aging Mice

Table 1: Composition of Gami-Shinkiwhan (GS) per pack

Table 2: Change in the immobility time of mice, as recorded from the tail suspension test.