Molecular mechanism underlying antidepressant activity of curcumin
Name of dispatched researcher（派遣研究者名）
AfÀliation of Instructor（所属）
Host Collaborator（受入研究者）

Dr. Yaowared Chulikhit
Faculty of Pharmaceutical Science, Khon Kaen University,
Khon Kaen, Thailand
Professor Kinzo Matsumoto
Division of Medicinal Pharmacology
Institute of Natural Medicine,
University of Toyama

Abstract
Curcumin, a yellow pigment extracted from rhizomes of the plant Curcuma longa, has been
widely used as food additive and also as herbal medicine throughout Asia. It has been used toeffectively manage mental stress and depression-related disorder. Unpredictable chronic mild
stress (UCMS) has long been used as a model of depression. Antidepressant agents can reverse
most effects of UCMS. We hypothesized that curcumin may alleviate stress induced depression. Thus in present study we assessed whether curcumin treatment affect behavior in UCMS
treated ICR mice. Mice were exposed to UCMS for 5 weeks and anhedonia was evaluated by
weekly monitoring of sucrose consumption. curcumin (10 and 20 mg/kg/day, i.p.) or imipramine(20 mg/kg/day, i.p.) or vehicle were continuously administered the last two weeks of UCMS. Behavioral tests were performed over the last week of UCMS. We found that subjecting animals to
the UCMS protocol resulted in decreasing sucrose intake and increasing immobility time in tailsuspension test (TST), the tests have been used to detect helpless behavior. The results showedthat cucrucmin 20 mg/kg and imipramine 20 mg/kg signiÀcantly decrease the immobility time inboth test. Only imipramine exhibited the decreasing in locomotor activity in open Àeld test. In
addition, the anxiety test was performed in this study to determine the effect of curcumin in anxiolytic activity. In light/dark preferences test, the percentage of time reduction in dark zone andinduction time in light zone of mice were treated with curcumin and imipramine compared to thestressed-control mice. Curcumin and imipramine treatment increased the spending time in open
arm and decreased the spending time in close arm in elevated plus maze test. Moreover, we also
found that UCMS procedure decreased the BDNF mRNA expression in the frontal cortex whencompare with non stress mice and curcumin and imipramine treatment signiÀcantly reverse theeffect of UCMS by increasing the BDNF mRNA expression. There are many studies reported that
antidepressant treatment can block the UCMS-induced down regulate BDNF mRNA expression.
These results provide compelling evidence that the behavioral effects of curcumin in chronicallystressed animals may be related to their modulating effects on other organ. In addition some evidence has hypothesis that, curcumin has an property of MAO inhibitor that was effect on Catecholamine neurotransmitter(6) such as Serotonin, Epinephrine, Dopamine etc. these are regulate−53−the function of mood. Furthermore, curcumin treatment exhibited the antidepressant activity
via increasing the BDNF mRNA expression in frontal cortex similar to imipramine, a reference
antidepressant. Although BDNF gene we have selected in this study but many gene are relevanton the depression not only BDNF gene and also should be study further gene expression on theother organs.
Keywords; Curcumin, unpredictable chronic mild stress, antidepressant, BDNF

Introduction
A Major Depression disorder (MDD) manifests with the symptoms at the psychological, behavioral and physiological levels. An episoderequires the presence for at least 2 weeks of one or
two core symptoms: dysphoricmood and anhedonia (a loss of interest or pleasure in activities thatusually would beenjoyed). In addition, four of the following symptoms must be present (three if
bothcore symptoms are present): disturbances of sleep, feelings of worthlessness or guilt,inability
to concentrate or think, increased or decreased psychomotor activity,decreased sexual drive, appetite disturbance or weight change and suicidal thoughts.
A Major Depression disorder preference is increasing. The World Health Organization estimated that in year 2020 this disorder will become the second largest health problem in humaninferior to heart disease. There are three groups of antidepressant that are tricyclic antidepressants (TCAs), Monoamine oxidase inhibitors (MAOIs), both of TCA and MAOI are the first ofantidepressant shown their ability facilitated to noradrenergic and/or serotonergic neurotransmission, which correlated with behavioral excitation. But in the mid-1970s, the third group of
antidepressant was found by related to serotonin, serotonin reuptake inhibitors (SSRIs). Despite
the last group is commonly prescribed in the present, there are many undesirable adverse effectsof the antidepressants. Some of the various side effect from the different antidepressants are drymouth, urinary retention, blurred vision, constipation, sedation (can interfere with driving or
operating machinery), sleep disruption, weight gain, headache, nausea, gastrointestinal disturbance/diarrhea, abdominal pain, inability to achieve an erection, inability to achieve an orgasm(men and women), loss of libido, agitation, anxiety etc Woelk, H. (2000). Thus the herbal medicine is the
major sources of drug, which may relate to depression therapy.
Curcumin, a yellow pigment extracted fromrhizomes of the plant Curcuma longa, has been
widely used as food additive and also as herbal
medicine throughout Asia. In China, curcumin
is a major constituent of Xiaoyao-san, the traditional Chinese medicine. This regimen has been
Figure 1: The yellow pigments of curcumin thatextracted from Curcuma longa

used to effectively manage mental stress and−54−

depression-related disorder.
Unpredictable chronic mild stress (UCMS) has long been used as a model of depression. In
the UCMS model, rats or mice are exposed sequentially, over a period of weeks, to a variety ofmild stressors, and the measure most commonly used to track the effects is a decrease in consumption of a palatable sweet solution. The model has good predictive validity (behavioral changes are reversed by chronic treatment with a wide variety of antidepressants), face validity (almost
all demonstrable symptoms of depression have been demonstrated), and construct validity (UCMS
causes a generalized decrease in responsiveness to rewards, comparable to anhedonia, the core
symptom of the melancholic subtype of major depressive disorder)(Willner,1997). Animal models areindispensable in clarifying the pathophysiology that underlies depression, depression–cognitioninteractions, and in searching for new antidepressants. Several animal models have been established, such as forced swimming test (FST), tail suspension test (TST), learned helplessness (LH)and chronic mild stress (CMS). These models have been used as reliable research tools to screen
effective antidepressants and to further research into pathophysiology of depression. However,the value of these animal models in deÀning the impact of depression producing stressors on spatial learning and memory remains uncertain.(L. Song et al,2006). The UCMS animal model has been also
used to evaluate anxiety-like behaviors which is an important element in the development of depression by some behavioral assay such as the elevated plus maze and ligh/dark paradigm as theevaluation of anxiety-like effects.(Yann S. M.,2006). And Genetic level may involve with depression, fromour review literature shown that Brain-derived neurotrophic factor (BDNF) is a neurotrophin
important for neuronal development and synaptic plasticity. However, it has also been recently
implicated in the etiology and treatment of psychiatric disorders, including depression (F Angelucci etal, 2005). And a number of Àndings suggest that BDNF action could be impaired in depression andstress-related affective disorders, and that BDNF is involved in the etiology of these illnesses.
Chronic administration of several antidepressants, including selective serotonin reuptake inhibitors, increases BDNF expression in the hippocampus. Another study demonstrated that centrally
administered BDNF produces antidepressant-like activity in learned helplessness paradigms andthe forced swim test. Therefore, up regulation of BDNF in response to antidepressant treatment
could have similar behavioral effects. This is further supported by an animal study demonstrating that environmental stressors, such as immobilization, decreased central BDNF mRNA. Indeed, chronic stress down regulates neurotrophin synthesis causing atrophy.
Materials and Methods
Animals
Male ICR mice, weight about 20 – 25 g, were obtained at the age of 3 weeks. They werehoused in groups of 6 – 7 per cage. Housing conditions were thermostatically maintained at ambient temperature (22 ± 1℃ ) and 12 hours light/dark cycle. They were fed with standard dietand water ad libitum and were allowed to acclimate 7 daysbefore they were used. In case of sucrose preference, animals were fasted for 18 hours before they were test. They were randomly di-−55−

vided into 5 groups, which are non-stressed control group and the other 4 groups of stressed micewith unpredictable chronic mild stress.
Drugs and drug administration
After 21 days of continuous to the UCMS sequence of the below-described, mildly stressful situation, when sucrose consumption was reduced signiÀcantly in stressed animals to levels
not signiÀcantly different among them, the four groups of stressed animals were assigned randomly to one of the following treatment: (1) vehicle control, carboxymethlycellulose solution, (2)curcumin(MP Biomedicals), 10 mg/kg body wt.; (3) curcumin, 20 mg/kg body wt.; (4) Imipramine(Nacalaitesque), 20 mg/kg body wt. All drugs were administered intraperitoneally (i.p.) once daily
for last two weeks of UCMS.
Unpredictable chronic mild stress
At the start of the experiment, the animal were Àrst trained to consume a 2% sucrose solution for a 48-h period in their cages with no food or water following food and water deprivation for
18 h. They were given sucrose for 1 h per day on the Àve consecutive days at the end of training
in order to group the mice. The mice in the experimental groups were then subjected to UCMS
for 5 weeks. The UCMS procedure consisted of a variety of unpredictable mild stressors including
two periods of tilted cage 45° (12h), two periods of 1 h restricted access to food (5 micropellets),
two periods of exposure to empty bottle (3h), one period of 21 h wet cage (200 ml water in 100 g−56−

sawdust bedding), two periods of light exposure (36h), two periods of intermittent sound (3h, 5h),
two periods of paired caging (2h) and food and water deprivation for 18 h before 2% sucrose solution consumption. These stressors were randomly scheduled over a one-week period and repeatedthroughout the 5-week experiment.
Sucrose consumption
Sucrose intake were measured once weekly after the end of UCMS, 18 h of food and water
deprivation. Consumption was measure by weighing the pre-weighed bottle at the end of the test.
Baseline was measured at the week 0 before started of UCMS.
Tail suspension test
The tail suspension test allows fast evaluation of drugs psychotropic effects. The animals
were subjected to the short-term, inescapable stress of being suspended by their tail and develop
an immobile posture. Animals were suspended 50 cm above the Áoor by means of an adhesivetape, placed approximately 1 cm from the top of the tail. The time during which mice remained
immobile was measured during a test period of 6 min. Mice were considered immobile only when
they hung passively and completely motionless. Immobility time is defined as the activity ofstressed mice.
Forced swimming test
The mice were individual to swum for 15 min in glass cylinder (height: 27 cm, diameter: 20cm) containing 10 cm of water at 25℃ for pretest. A 24-h after pretest, mice were placed in glass
cylinder again for 6 min test and was recorded the last 4 min of testing period. A mouse wasjudged to be immobile when it discontinued struggling and remained Áoating motionless in thewater, making only a small movement necessary to keep its head above water.
2SHQÀHOGDQDO\VLV
In the last week of UCMS and drug exposure, the open Àeld analysis was performed. The
open Àeld consist of a black walls and base divided into 16 (4 × 4) identical sectors by white stripes.
The animals were placed in the central sector and measured the movement of mice for 5 min
manually. The open Àeld arena was thoroughly cleaned between each test. Motility was scoredwhen animals crossed a sector border with both its hind limbs or rearing and grooming. This test
can determine the effect of drug on motor function.
Light/Dark preference
The light dark (LD) test is used to evaluate the relative anxiety status of mice. The lightdark paradigm in mice is based on a conÁict between the innate aversion to brightly illuminatedareas and the spontaneous exploratory activity. If given a choice between a large brightly compartment versus a small dark compartment, mice spontaneously prefer the dark. Anxiolytic com-−57−

pounds have been found to increase the total duration of time spent there. Anxiogenic compounds
are observed to work in the opposite way.
Elevated plus-maze
The elevated plus-maze was constructed from black acrylic plateand elevated to a height of
50 cm. It consisted of two open arms (50 x 10 cm) and two enclosed arms (30 x 5 x 15 cm). Eachmouse was placed in the central square facing an open arm, and allowed to explore the maze for5 min. The maze was cleaned thoroughly before each test. The percentage of time spent on the
open arms (time on open arms/(time on open arms + time on closed arms) x 100), the percentage
open arm entries (open arm entries/total entries x 100), and total number of entries were determined. An entry was deÀned as three of the four paws being on the arm.
Semiquantitative revers transcription-polymerase chain reaction (RT-PCR)
Total RNA extraction
Homogenize tissue samples in 1 ml of TRIZOL reagent. Add 0.2 ml of chloroform per 1 mlof TRIZOL Reagent. Cap sample tubes securely. Vortex samples vigorously for 15 seconds andincubate them at room temperature for 2 to 3 minutes. Centrifuge the samples at no more than
12,000 x g for 15 minutes at 4 ºC. Following centrifugation, the mixture separates into lower red,
phenol-chloroform phase, an interphase, and a colorless upper aqueous phase. RNA remains exclusively in the aqueous phase. Transfer upper aqueous phase carefully without disturbing theinterphase into fresh tube. Measure the volume of the aqueous phase (The volume of the aqueous
phase is about 60% of the volume of TRIZOL Reagent used for homogenization). Precipitate theRNA from the aqueous phase by mixing with isopropyl alcohol. Use 0.5 ml of isopropyl alcoholper 1 ml of TRIZOL Reagent used for the initial homogenization. Incubate samples at 15 to 30 ºC
for 10 minutes and centrifuge at not more than 12,000 x g for 10 minutes at 4 ºC. The RNA precipitate, often invisible before centrifugation, forms a gel-like pellet on the side and bottom of the
tube. Remove the supernatant completely. Wash the RNA pellet once with 75% ethanol, adding
at least 1 ml of 75% ethanol per 1 ml of TRIZOL Reagent used for the initial homogenization. Mix
the samples by vortexing and centrifuge at no more than 7,500 x g for 5 minutes at 4 ºC. Repeat
above washing procedure once. Remove all leftover ethanol. Air-dry or vacuum dry RNA pellet
for 5-10 minutes. Do not dry the RNA pellet by centrifuge under vacuum. It is important not to
let the RNA pellet dry completely as this will greatly decrease its solubility. Dissolve RNA in 40
Pl RNase free water by passing solution a few times through a pipette tip, keep in -20 ºC refrigerator as stock RNA.
Reverse transcription
Pipette 2 Pl of 0.5 Pg/Pl RNA sample to nuclease-free microcentrifuge tube. Add 10 Pl mastermix1 [1 Pl oligo (dT) 12-18, 4 Pl 10 mM dNTP Mix (10 mM each dATP, dGTP, dCTP and dTTP atneutral pH) and RNase free water adjust to 10 Pl] Heat mixture to 65 ºC for 5 min and quick chill−58−

on ice. Add 7 Pl master mix2 [4 Pl 5X First-Strand Buffer, 2 Pl 0.1 M DTT, 0.2 Pl RNase Inhibitor, 0.8 Pl RNase free water] Mix contents of the tube gently and incubate at 37 ºC for 2 min. Add
1 Pl (200 units) of M-MLV RT, and mix by pipetting gently up and down. Incubate 50 min at 37ºC and inactivate the reaction by heating at 70 ºC for 15 min.
Table 2:6XPPDU\RIWKHSULPHUSDLUIRUKRXVHNHHSLQJJHQH ȕDFWLQ VWUHVVUHODWHGJHQHV %'1)&5(% DQG
SURGXFWOHQJWKPrimer Sequence
Gene

Top line : forward primer

Product
lengthBottom line : reverse primer

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8F

Y !.!!..!...!.! Y(Y !!!..!!. Y8FY .!.!.!. Y

Semi-quantitative RT-PCR
The PCR reaction mixture; 1 Pl of cDNA, 4 Pl of 5x PCR buffer, 2 Pl of deoxyneucleoside triphosphate mixture, 2 Pl of magnesium chloride, 2 Pl of primer pair (Table 2), 0.4 Pl of Taq polymerase and 8.6 Pl of distilled water to give Ànal volume 20 Pl. In general, PCR was performed
with a preheating cycle at 95 ℃ for 2 min, denaturation, annealing, elongation and reaction cycles were carried out follow Table 3. Aliquots of PCR products were analyzed by gel electrophoresis with 10% polyacrylamide gel stained in ethidium bromide, photographed under UV light andanalyzed by GeneSnap and Gene ToolsMatch Software
Statistic Analysis
Data are expressed as the mean ± S.E.M. and analyzed by t-test between non-stress groupand UCMS group and one-way analysis of variance (ANOVA) followed by the Student–Newman–Keuls test for multiple comparisons among different groups. Differences with p < 0.05 were considered signiÀcant.

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Results
Sucrose consumption



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PLFH

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Forced swimming test and tail suspension test
The effect of administration with curcumin in forced swimming test and tail suspension testat the dose of curcumin with 10 and 20 mg/kg and imipramine with 20 mg/kg. The duration of
immobility time in forced swimming test, resulting in 21.81%, 23.88% and 14.15% immobility−61−

reduction of dose with curcumin 10, 20 mg/kg and imipramine 20 mg/kg, respectively comparedto the UCMS stressed-control mice. In the tail suspension test, these same doses of curcumin andimipramine also signiÀcantly inhibited immobility with a respective percent reduction of 19.75%,
40.90% and 43.35%. In both models of depression, the effects of curcumin were similar to thoseobserved for the classical antidepressant Imipramine (20mg/kg).
Open Àeld analysis

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Light/Dark preferenceMice receiving UCMS procedure show the signiÀcant decrease in the latency to dark zone.
After treatment with curcumin and imipramine the latency time were signiÀcantly increase. No
signiÀcant difference in spending time in dark zone and light zone between UCMS group andtreatment group. The respect percentage of dark zone timereduction were 21.75%, 31.73% and31.28% in dose of curcumin and imipramine compared to stressed-control mice. The mean oftime preference of dark zone of UCMS group was 214.34 ± 1.38. The percentage time inductionof light zone were 54.15%, 79.01% and 77.90% in dose of curcumin and imipramine compared tostressed-control mice. The mean of light time preference in the light zone of UCMS group was
85.96 ± 9.87.

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t-testYVQRQVWUHVVHGFRQWUROJURXSElevated plus maze
UCMS mice show significantly decrease in the spending time in open arm and close arm
when compare with the non stress mice. Only curcumin 20 mg/kg signiÀcantly reverse the effectof UCMS stress in both spending time in open arm and close arm. Imipramine show signiÀcantly
increase only spending time in close arm.



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Semi quantitative RT-PCR analysis

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the BDNF mRNA expression when compare with UCMS+vehicle mice.
Discussion
The UCMS model of depression involves in the presentation of a series of varied and unpredictable environment stressors, such as two periods of tilted cage 45 ° (12h), two periods of
1 h restricted access to food (5 micropellets), two periods of exposure to empty bottle (3h), one
period of 21 h wet cage (200 ml water in 100 g sawdust bedding), two periods of light exposure(36h), two periods of intermittent sound (3h, 5h), two periods of paired caging (2h) and food andwater deprivation for 18 h. Following such exposure, mice have been reported with anhedonia effect, measured by 2% sucrose consumption. To assured these results are from UCMS procedureand sucrose preferences; we performed the behavioral activities to test the effect of curcumin
substances and used the classical antidepressant, imipramine, as standard of treatment. The
forced swimming test and tail suspension test were performed for determining the antidepressant effect. Curcumin 20 mg/kg and imipramine 20 mg/kg showed the antidepressant activity
in both test. Only imipramine exhibited the decreasing in locomotor activity in open Àeld test.
In addition, the anxiety test was performed in this study to determine the effect of curcumin inanxiolytic activity. Light/dark preferences and elevated plus maze were performed to evaluate
the anxiolytic activity of curcumin. The results showed that curcumin had the anxiolytic activity−64−

by decreasing the latency to dark zone in light/dark preference and decreased spending time inclose arm and increased the spending time in open arm. UCMS induced behavioral changed werereverse by chronic antidepressant medication and long-term curcumin consumption. In addition
curcumin and imipramine reversed the effect of UCMS on BNDF expression. Interestingly, we
found that curcumin increased BDNF levels quite similar to the imipramine (p > 0.05) In fact;
chronic antidepressants could produce long-term adaptation in cellular signaling mechanisms
in mice. The ability of curcumin to up-regulate BDNF mRNA expression is also considered toprove its potential mechanism as antidepressant agent. Some mechanism have an involve withcurcumin to explain, why it is involve to depression increased cell proliferation and neuronal
populations may be a mechanism by which curcumin treatment overcomes the stress-induced behavioral abnormalities and neuronal damage. Moreover, curcumin treatment, via up-regulationof 5-HT1A receptors and BDNF, may reverse or protect neurons from further damage in responseto chronic stress, which may underlie the therapeutic actions of curcumin (8). And the topic ofdose of curcumin 10 mg/kg and 20 mg/kg are not signiÀcantly different (p > 0.05) on relative gene
expression, they are seem to the dose of curcumin 10 mg/kg, 20 mg/kg gives the efÀciency to promote BDNF mRNA expression closely for each other.

Conclusion
The effect of Curcumin can reversed the decrease of Anhedonicbehavior, whichinvolves withdepressionby monitor the sucrose consumption. These changes were reversed by chronic curcumin administration (10 or 20 mg/kg, p.o.). According to 2% sucrose consumption, at the last 2
weeks of UCMS procedure, resulting in signiÀcantly differences of sucrose consumption among
their groups. In the group of Curcumin and Imipramine treated, the mice showed the inductionof sucrose preferences compared to the stressed-control mice.The effect of administration with
Curcumin in forced swimming test and tail suspension test at the dose of Curcumin with 10 and
20 mg/kg and Imipramine with 20 mg/kg. The duration of immobility time in forced swimming
test, resulting in 21.81%, 23.88% and 14.15% immobility reduction of dose with Curcumin 10,20 mg/kg and Imipramine 20 mg/kg, respectively compared to the UCMS stressed-control mice.
In the tail suspension test, these same doses of Curcumin and Imipramine also signiÀcantly inhibited immobility with a respective percent reduction of 19.75%, 40.90% and 43.35%. In bothmodels of depression, the effects of Curcumin were similar to those observed for the classical antidepressant Imipramine (20mg/kg). In case of open Àeld test, they showed no differences among
their groups but there is signiÀcantly differences between stressed-control and Imipramine miceand showed as the same result as light/dark preference. The light/dark preferences showed thepercentage of time reduction in dark zone and induction time in light zone of mice were treatedwith Curcumin and Imipramine compared to the stressed-control mice. In addition, we also
found that the unpredictable chronic stress procedure induced a down-regulation of brain-derived
neurotrophic factor (BDNF) protein levels (1), in the frontal cortex of UCMS mice. Furthermore,−65−

these stress-induced decreases in BDNF were also blocked by chronic curcumin administration (10or 20 mg/kg, p.o.). These results provide compelling evidence that the behavioral effects of curcumin in chronically stressed animals may be related to their modulating effects on other organ.
In addition some evidence has hypothesis that, curcumin has an property of MAO inhibitor thatwas effect on Catecholamine neurotransmitter(6) such as Serotonin, Epinephrine, Dopamine etc.
these are regulate the function of mood. Although BDNF gene we have selected in this study but
many gene are relevant on the depression not only BDNF gene and also should be study further
gene expression on the other organs.

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