Adaptogens stimulate neuropeptide Y and Hsp72 expression and

ORIGINAL RESEARCH ARTICLE

published: 01 February 2012

doi: 10.3389/fnins.2012.00006

Adaptogens stimulate neuropeptideY and Hsp72

expression and release in neuroglia cells

Alexander Panossian

*, Georg Wikman

, Punit Kaur

and Alexzander Asea

Department of Research and Development, Swedish Herbal Institute Research and Development, Åskloster, Sweden

Division of Investigative Pathology, Scott & White Memorial Hospital and Clinic, The Texas A&M Health Science Center College of Medicine,Temple, TX,USA

Edited by:

Maria M. Malagon, University of

Cordoba, Spain

Reviewed by:

James A. Carr, TexasTech University,

USA

Charlier Dominique Thierry, University

of Liege, Belgium

*Correspondence:

Alexander Panossian, Department of

Research and Development, Swedish

Herbal Institute Research and

Development, Spårvägen 2, SE-432

96 Åskloster, Sweden.

e-mail: alexander[email protected];

Alexzander Asea, Division of

Investigative Pathology, Scott & White

Memorial Hospital and Clinic, The

Texas A&M Health Science Center

College of Medicine, 1901 South 1st

Street, Temple, TX 76504, USA.

e-mail: [email protected]

The beneﬁcial stress–protective effect of adaptogens is related to the regulation of home-

ostasis via mechanisms of action associated with the hypothalamic–pituitary–adrenal axis

and the regulation of key mediators of the stress response, such as molecular chaperones,

stress-activated c-Jun N-terminal protein kinase, forkhead box O transcription factor, cor-

tisol, and nitric oxide (NO). However, it still remains unclear what the primary upstream

targets are in response to stimulation by adaptogens. The present study addresses this

gap in our knowledge and suggests that an important target for adaptogen mediated

stress–protective effector functions is the stress hormone neuropeptide Y (NPY). We

demonstrated that ADAPT-232, a ﬁxed combination of adaptogens Eleutherococcus senti-

cosus root extract, Schisandra chinensis berry extract, Rhodiola rosea root extract SHR-5,

and its active constituent salidroside, stimulated the expression of NPY and 72 kDa heat

shock protein (Hsp72) in isolated human neuroglia cells. The central role of NPY was val-

idated in experiments in which pre-treatment of human neuroglia cells with NPY-siRNA

and HSF1-siRNA resulted in the signiﬁcant suppression of ADAPT-232-induced NPY and

Hsp72 release. Taken together our studies suggest that the stimulation and release of the

stress hormones, NPY and Hsp72, into systemic circulation is an innate defense response

against mild stressors (ADAPT-232), which increase tolerance and adaptation to stress.

Keywords: ADAPT-232, adaptogen, Eleutherococcus senticosus, heat shock proteins, neuropeptide Y, salidroside,

Schisandra chinensis, Rhodiola rosea

INTRODUCTION

Adaptogens were initially deﬁned as substances that enhance the

“state of non-speciﬁc resistance” in stress (Brekhman and Dard-

ymov, 1969; Panossian, 2003), a physiological condition that is

linked with various disorders of the neuroendocrine–immune

system (Stratakis and Chrousos, 1995). This deﬁnition has been

updated as “a new class of metabolic regulators which increase the

ability of an organism to adapt to environmental factors and to avoid

damage from such factors”(Panossian et al., 1999). The term adap-

togen was used as a functional claim for certain botanicals and

herbal medicinal products in Europe and the USA and the adapto-

gen concept is now a generally accepted concept (Samuelsson and

Bohlin, 2009). A number of clinical trials clearly demonstrated

that adaptogens exert an anti-fatigue effect that increases mental

work capacity against a background of stress and fatigue, partic-

ularly in tolerance to mental exhaustion and enhanced attention

(Panossian and Wikman, 2010). Studies on animals and isolated

cells have revealed that adaptogens exhibit neuroprotective, anti-

fatigue, antidepressive, anxiolytic, nootropic, and CNS stimulating

and tonic effects (Panossian and Wagner, 2005; Panossian and

Wikman, 2010). In contrast to conventional stimulants such as

Abbreviations: APC, antigen presenting cells; CFS, chronic fatigue syndrome; JNK1,

c-Jun N-terminal protein kinase; FoxO, forkhead box O; HPA, hypothalamic–

pituitary–adrenal; NO, nitric oxide; NPY, neuropeptide Y; siRNA, short interfering

RNA.

sympathomimetics (e.g., ephedrine, fenﬂuramine, phentermine,

prolintane) and general tonics, adaptogens do not possess addic-

tion, tolerance and abuse potentials, or impair mental function,

or lead to psychotic symptoms with long term use (Panossian

and Wikman, 2010). Recent pharmacological studies of a number

of adaptogens have provided a rationale for these effects also at

the molecular level (Panossian and Wikman, 2010). The stress–

protective activity of adaptogens is associated with regulation of

homeostasis via several mechanisms of action which are linked

to the hypothalamic–pituitary–adrenal (HPA) axis and the regu-

lation of key mediators of the stress response, including cortisol,

nitric oxide, stress-activated protein kinase c-Jun N-terminal pro-

tein kinase (JNK; Panossian et al., 2007), forkhead box O (FoxO)

transcription factor (DAF-16; Wiegant et al., 2009), and molecular

chaperones (Chiu and Ko, 2004; Panossian and Wikman, 2010).

However, it still remains unclear what the primary upstream tar-

gets are in response to stimulation by adaptogens. In this study,

we investigate whether heat shock factor 1 (HSF1) and Neuropep-

tide Y (NPY) might be one of the primary upstream targets of

adaptogens in neuroglia cells.

Neuropeptide Y is a stress–responsive hormone widely distrib-

uted in the central and peripheral nervous system (Tatemoto et al.,

1982; Irwin, 2008). In the brain the concentrations of NPY are sig-

niﬁcantly higher than other neuropeptides, and is found mainly

in the limbic system, including the amygdala and the hypothal-

amus, which are areas of the brain involved in the regulation of

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Panossian et al. Adaptogens stimulate NPY and Hsp72

emotional behaviors and stress response (Dumont et al., 1993;

Smialowska et al., 2007). In the peripheral nervous system, NPY

is concentrated in sympathetic nerve endings (Irwin, 2008). Sym-

pathoadrenal activation during the stress response results in NPY

release from the sympathetic nerve endings either alone or with

catecholamines (Morris et al., 1986). NPY release follows stressors

including strenuous exercise (Karamouzis et al., 2002), panic dis-

orders (Boulenger et al., 1996), cold exposure (Kellogg, 2006), and

chronic fatigue syndrome (CFS; Fletcher et al., 2010). The eleva-

tion of NPY in blood of CFS patients is associated with severity of

stress,negative mood, and clinical symptoms (Fletcher et al., 2010).

On the other hand, psychological stress elevated plasma NPY in

healthy subjects (Morgan et al., 2001). In the periphery, sympa-

thetic nerve- and platelet-derived NPY act in a stimulatory fashion;

synergizing with glucocorticoids and catecholamines to potentiate

the stress response, induce vasoconstriction and increase vascular

smooth muscle cell proliferation. However, in the brain NPY acts

as an anxiolytic and inhibits sympathetic activity which results in

lowering blood pressure and heart rate (Morris et al., 1986; Kuo

et al., 2007), and inhibiting the production of cortisol in human

adrenal cells (Kempna et al.,2010). NPY can regulate both immune

cells and neuronal cells, e.g., NPY strongly inhibits NO synthesis

through Y(1) receptor activation, which prevents IL-1β release and

thus inhibits nuclear translocation of NF-κB in microglia (Fer-

reira et al., 2010). NPY plays a protective role in viral infections

associated with glial cell activation and the production of pro-

inﬂammatory cytokines in the CNS (Du et al., 2010). It has been

suggested that the stimulation of NPY gene expression is related to

food deprivation and its overexpression causes disordered energy

balance leading to increased eating (Yang et al., 2009). Within cells,

NPY decreases the expression of mitochondrial uncoupling pro-

tein, thereby promoting ATP formation (Billington et al., 1994).

NPY stimulates the corticotrophic axis (Small et al., 1997), mod-

ulates the secretion of various hypothalamic neuropeptides and

cognition (Redrobe et al., 1999). Administration of NPY reduced

cortisol secretion during night hours in healthy subjects (Antoni-

jevic et al., 2000). In addition, NPY is known to play a role in the

pathophysiology of depression (Heilig et al., 1988). It has been

shown that NPY displayed antidepressant-like activity in the rat

forced swimming test (Stogner and Holmes, 2000; Redrobe et al.,

2002). Human studies have revealed a role for NPY in adaptation

to stress (“buffering” the harmful effects of stress; Morgan et al.,

2000, 2001; Morales-Medina et al., 2010). There is a plethora of

pre-clinical and clinical evidence suggesting a mood and cogni-

tive performance improving action for NPY (Morgan et al., 2000;

Fletcher et al., 2010). Higher levels of NPY have been observed

in soldiers who either present with reduced psychological dis-

tress or belong to the elite Special Forces branch (Morgan et al.,

2000, 2001). In contrast, decreased levels of NPY were observed in

depression and in brain tissues of suicide victims (Morales-Medina

et al., 2010).

The 72 kDa heat shock protein (Hsp72) plays an important role

in pharmacological effects of adaptogens (Panossian et al., 2010).

Hsp72 functions both as a chaperone by binding important anti-

genic peptides for delivery to antigen presenting cells (APC), and as

a cytokine by stimulating pro-inﬂammatory cytokine release. This

dual function of Hsp72 is termed the chaperokine activity (Asea,

2003, 2006, 2007, 2008;

Hecker and McGarvey, 2011).

Recently,

we demonstrated the stimulating effect of ADAPT-232, a ﬁxed

combination of extracts of three most efﬁcient adaptogens (Rho-

diola rosea, Eleutherococcus senticosus, and Schisandra chinensis),

is associated with signiﬁcant increase of circulated Hsp72 in rats

(Panossian et al., 2009). However, the target cells for ADAPT-232-

induced effects or the cells releasing Hsp72 have not yet been

identiﬁed.

MATERIALS AND METHODS

TEST ARTICLE

ADAPT-232 forte is a proprietary name of a ﬁxed combina-

tion of three genuine (native) extracts of E. senticosus (Rupr. et

Maxim) Harms root, S. chinensis (Turcz.) Baill. root, R. rosea

L., root, characterized for the content of eleutherosides E and

B (0.17%), schisandrin and γ-schisandrin (0.85%), salidroside

(0.33%), tyrosol (0.07%), rosavin (0.37%), triandrin (0.01%;

Panossian et al., 2009). The amounts of the active markers salidro-

side, rosavin, tyrosol, triandrin, eleutheroside B, eleutheroside E,

schizandrin, and γ-schizandrin were determined by analytical

RP-HPLC using an acetonitrile–water gradient system as mobile

phase. Peaks were detected by UV-PAD and analytes quantiﬁed

at 221 nm (rhodioloside and tyrosol), 252 nm (rosavin), 262 nm

(triandrin), 220 nm (eleutheroside B), 210 nm (eleutheroside E,

schizandrin, and γ-schizandrin). Analytical methods were vali-

dated for selectivity, peak purity, precision (RSD < 5%), and accu-

racy in the range 50–150% of the target amounts of analytes in the

tablets in accordance with ICH guidelines using Efﬁ Validation 3

software (version 1.03) for testing and calibration laboratories sub-

ject to EN ISO/IEC 17 025:2001. Samples used in experiments were

prepared by dilution of stock solutions of ADAPT-232 (10 mg/ml)

or salidroside (3 mg/ml, 10

−2

M) with PBS. Fifty microliters of

working solutions were added to 10 ml of cell culture in order to

obtain ﬁnal concentrations of 0.5, 1, 5, and 10 μM salidroside and

0.005, 0.05, 0.5, and 5 μg/ml of ADAPT-232 (containing 0.05, 0.5,

5, and 50 nM salidroside) in culture media.

REAGENTS

Control peptide, PB1 is a synthetic peptide containing an

N-terminal cysteine (CTRSRHSSYPNEYEEDEEMEEEL); the

sequence is derived from mouse Bad (New England Biolabs,

Ipswich, MA, USA). The NPY peptide (Sequence H-Tyr-Pro-Ser-

Lys-Pro-Asp-Asn-Pro-Gly-Glu-Asp-Ala-Pro-Ala-Glu-Asp-Met-

Ala-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-

Arg-Gln-Arg-Tyr-NH

acetate salt) was purchased from Bachem

(Torrance, CA, USA). NPY-siRNA and HSF1-siRNA were pur-

chased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Hsp72-siRNA and control-siRNA were purchased from Dharma-

con RNA Technologies (Lafayette, CO, USA).

CELL LINES AND CULTURE CONDITIONS

The human neuroglia cell line T98G (ATCC, CRL-1690) has a

hyperpentaploid chromosome count that was derived from a 61-

year-old Caucasian male with glioblastoma multiforme. Human

neuroglia T98G cells were cultured from ATCC-formulated Eagle’s

Minimum Essential Medium (Catalog No. 30-2003) with fetal

bovine serum to a ﬁnal concentration of 10%, 100 U/ml peni-

cillin, 100 μg/ml streptomycin (ICN, Aurora, OH, USA). To avoid

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Panossian et al. Adaptogens stimulate NPY and Hsp72

stress induced by cell overgrowth, cultures were maintained in a

37˚C incubator in humidiﬁed air with 5% CO

atmosphere. Cells

were maintained at a density of 2 × 10

cells/ml and passaged with

fresh complete medium every 3–4 days. Cell viability was assessed

using trypan blue exclusion test and routinely found to contain

<5% dead cells.

PROTEIN SEPARATION AND WESTERN BLOT ANALYSIS

Following various treatment protocols cells were washed once with

complete medium, centrifuged, and pellets lysed with 100 μlof

lysing buffer containing a cocktail of protease inhibitors (antipain,

bestain, chymostatin, E-64, pepstatin, phosphoramidon, pefabloc,

EDTA, aprotinin; Complete Protease Inhibitor Cocktail Tablets

Roche Diagnostics). Cells were then incubated for 30 min on ice

and sonicated (Brandson 1510) for 15 min. The cell suspension

was passed through a 26-gage needle and protein quantiﬁcation

was performed using the Bradford method. Proteins were sepa-

rated in a precast 10% SDS-PAGE Novex

Tricine gel (Invitrogen)

by carefully placing 3 μg of protein in each lane. Nitrocellulose

membrane (GIBCO BRL) was used to transfer the proteins and

the membrane blocked with 5% skim milk (in TBS 1% pH 7.4 and

0.01% Tween-20) and incubated for 1 h at room temperature with

appropriate primary antibody; anti-Hsp72 (Stressgen Biotech-

nologies, BC, Canada), anti-NPY (Assay Design, BC, Canada), or

anti-β-actin (Oncogene, San Diego, CA, USA). Blots were incu-

bated 50 min at room temperature with 0.5 μg of appropriate

species matched anti-peroxidase and the reaction was detected

using the Luminol reagent for chemiluminescence (Santa Cruz

Biotechnology). The intensity of the bands were analyzed by

densitometry with a video densitometer (Chemilmager™ 5500;

Alpha Innotech, San Leandro, CA, USA) using the AAB software

(American Applied Biology).

MEASUREMENT OF LACTATE DEHYDROGENASE RELEASE

Lactate dehydrogenase (LDH) is a stable cytosolic enzyme that is

released upon cell lysis. LDH released into cell culture media by

dead cells and total LDH contained in living cells was measured

using the CytoTox 96 Non-Radioactive Cytotoxicity Assay accord-

ing to the manufacturer’s instructions (Promega, Madison, WI,

USA). Brieﬂy, after various treatment protocols, culture medium

(500 μl) was removed and the remaining cells lysed by adding

500 μl of 5% Triton X-100 solution. After 30 min at room tem-

perature, cell lysate was recovered and incubated for an additional

30 min in the dark with a buffer containing NAD

, lactate, and

tetrazolium. LDH converts lactate to pyruvate, generating NADH

which reduces tetrazolium (yellow) to formazan (red), which is

detected by ﬂuorescence (490 nm). LDH release, a marker for cell

death was expressed as a percentage of the LDH in the medium

over the total LDH (lysate).

ENZYME LINKED IMMUNOSSORBANT ASSAY

After treatment the human neuroglia cell line T98G was cen-

trifuged to discard ﬂoating cells and cellular debris and the total

protein content was determined by Bradford analysis using bovine

serum albumin as a standard. The supernatant was aliquoted

and treated with or without 1% Triton X-100 or 1% Lubrol

WX or 0.5% Brij 98 for 10 min at 4˚C with gentle rocking and

Hsp72 content measured by standard sandwich Enzyme linked

immunossorbant assay (ELISA). Brieﬂy, 96-well microtiter plates

(Nunc Immunoplate Maxisorp; Life Technologies) were coated

with murine monoclonal anti-human Hsp72 (clone C92F3A-5;

Stressgen) in carbonate buffer, pH 9.5 (2 μg/mL) overnight at 4˚C.

Plates were then washed with PBS containing 1% Tween-20 (PBS-

T) and blocked by incubation with 1% bovine serum albumin in

PBS-T. Supernatant was added and bound Hsp72 was detected

by the addition of rabbit polyclonal anti-Hsp72 antibody (SPA-

812; Stressgen). Bound polyclonal antibody was detected with

alkaline phosphatase-conjugated murine monoclonal antibody to

rabbit immunoglobulins (Sigma Chemical Co), followed by p-

nitrophenyl phosphate substrate (Sigma Chemical Co). The resul-

tant absorbance was measured at 405 nm with a Bio-Rad Benmark

Plus plate reader. Standard dose–response curves weregenerated in

parallel with Hsp72 (0–20,000 ng/mL; Stressgen), and the concen-

trations of Hsp72 were determined by reference to these standard

curves with ASSAYZAP data analysis software (BIOSOFT). The

interassay variability of the Hsp72 immunoassays was <10%.

NPY ENZYME IMMUNOASSAY

Human neuroglia cells were grown to 75% conﬂuence. Twenty-

four hours prior to measuring NPY release, cell culture medium

was replaced with serum-free DMEM, then incubated with

ADAPT-232, or salidroside, or exposed to heat shock (41˚C,

60 min) and incubated for 24 h in a 37˚C incubator. Supernatant

was recovered in quadruplicates,centrifuged to clear cellular debris

and NPY concentration was measured using the NPY enzyme

immunoassay kit (assay sensitivity of 0.09 ng/ml) according to the

manufacturer’s instructions (Phoenix Pharmaceuticals,CA, USA).

Brieﬂy, cleared supernatant was added to 96-well plates contain-

ing primary antibody and biotinylated peptide and incubated at

room temperature for 2 h. Plates were washed and SA–HRP solu-

tion was added to the well and incubated for a further 1 h at room

temperature. After washing the plates TMB substrate was added

and incubated for an additional 1 h at room temperature. The

test was terminated by adding 2 N HCl to the wells. The resultant

absorbance was measured at 450 nm with a Bio-Rad Benmark

Plus plate reader. The concentration of NPY was determined by

reference to these standard curves with ASSAYZAP data analy-

sis software (BIOSOFT). The interassay variability of the NPY

enzyme immunoassays was <10%.

siRNA-MEDIATED GENE SILENCING ASSAYS

The hsp72 gene silencing was achieved by using a vector-based sys-

tem to produce hairpin RNA. The criteria that were used to silence

the hsp72 gene have previously been established. To design the

siRNA duplexes a search for 23-nucleotide motif with AA (N19)

TT was performed in which the target sites were selected from a

gene sequence beginning at 50–100 nucleotides downstream of the

start codon. Hits with ∼50% G/C-content were selected and anti-

sense RNA was synthesized as the complement to position 1–21 of

the 23-nucleotide motif. Finally, symmetric 3



-overhangs that help

to ensure that siRNA are formed with approximately equal ratios

of sense and anti-sense target RNA-cleaving siRNA. The 21-base

pair product, Hsp72-siRNA was cloned into the pSuper vector

under the restriction of BglII and HindIII. This construct was

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Panossian et al. Adaptogens stimulate NPY and Hsp72

then co-transfected with a green ﬂuorescent protein (GFP) plas-

mid into the THP1 human monocytic cells. Splicing of the hairpin

resulted in 21-mer long dsRNA. Psuper vector (Oligoengine, Seat-

tle, WA, USA) was used to introduce a 64-base oligomer to form

the hairpin with Hsp72 speciﬁc sequence. A chemically synthe-

sized 21-mer-nucleotide DNA to silence the hsp72 gene was used

and cloned into the pSuper vector-based silencing (Oligoengine).

The primers were designed using web-based proprietary soft-

ware from Oligoengine and three 21-mer gene sequences were

selected that predicted it would successfully silence the hsp72

gene. The sequence used in this study was Hsp72 sequence 5



AGCCCGAGCTGGGAACCATT-3



(GenBank Accession Number

LO7577). This 21-base was synthesized with deﬁned sequences to

provide two restriction sites (BglII and HindIII) and on transcrip-

tion form a hairpin, in order to initiate gene silencing. The total

length of the primer was 64-mer including the speciﬁc sequences

of Hsp72. A complementary oligonucleotide was also synthesized

from Oligoengine (Seattle,WA, USA). The primers were incubated

in annealing buffer (100 mM potassium acetate, 30 mM HEPES–

KOH pH 7.4, 2 mM magnesium acetate) at 95˚C for 5 min and

incubated overnight at room temperature. After digesting double

stranded oligo and pSuper vector with HindIII and BglII restric-

tion enzymes, primers were annealed and cloned into the pSuper

vector. Positive clones were sequenced.

TRANSFECTION AND CELL SORTING

THP1 cells were transfected with pSuper-Hsp72 and pGFP using

the lipid transfection reagent, Effectene,according to the manufac-

turer’s instructions (Qiagen, Valencia, CA, USA). Brieﬂy, 3 × 10

exponentially growing cells were seeded in 60 mm tissue culture

plates and a mixture of 1 μg GFP plasmid DNA and 1 μgpSuper-

Hsp72 plasmid in Effectene was added to the cells and incubated

for 18 h at 37˚C. After 48 h cells were harvested and immedi-

ately sorted into GFP positive and negative subpopulations using

a MoFlow cytometer (Dakocytomation, Carpinteria, CA, USA).

Brieﬂy, individual cells were gated on the basis of forward scatter

(FSC) and orthogonal scatter (SSC). The photomultiplier (PMT)

for GFP (FL1-height) was set on a logarithmic scale. Cell debris

was excluded by raising the FSC-height PMT threshold. The ﬂow

rate was adjusted to <200 cells/s and at least 10

cellsweresorted

for each sample group.

STATISTICAL ANALYSIS

Data obtained from Western blot was analyzed according to the

relationship between the signal intensity and the area and pre-

sented as optical density (OD). Data from ﬂow cytometry was

shown as histograms and ﬂuorescent arbitrary units and the mean

ﬂuorescent intensity was obtained to compare between groups.

Data management and statistical analyses were performed using

GraphPad (San Diego, CA, USA) and Prism software (version 3.03

for Windows). The signiﬁcance of the between-group differences

(at 95% conﬁdence intervals) were determined using one-way

parametric analysis of variance (ANOVA) with Tukey’s multiple

comparison post test. The F test statistic was found by dividing

the between-group variance by the within group variance (mean

squared deviations from the mean, MSB/MSW, obtained by divid-

ing of the Sum of Squares on the degrees of freedom (df) for the

between group and within group). Data were shown in absolute

(ng/ml, μM) or normalized (as percentages) values, mean and SD,

and p values < 0.05 were considered statistically signiﬁcant.

RESULTS

In this study, we demonstrate that neuroglia cells are one pos-

sible target cell that mediates ADAPT-232-induced effects. We

further demonstrate that ADAPT-232 stimulates the expression

of NPY and Hsp72 release from neuroglia cells in a concentration-

dependent fashion. Our present study suggests NPY might be

one of the primary upstream targets of adaptogens. Our present

study suggests that HSF1 and NPY might be the primary upstream

targets of adaptogens.

ADAPT-232 STIMULATES THE EXPRESSION OF NPY AND HSP72 IN

HUMAN NEUROGLIA CELLS

Human neuroglia cells were treated with ADAPT-232 and the

expression of Hsp72 and NPY was measured by Western blot

analysis. We demonstrated that treatment of human neuroglia cells

with ADAPT-232 or salidroside dose-dependently increased NPY

expression and the expression the stress protein, Hsp72. Maxi-

mum effect of ADAPT-232 required 0.5 μg/ml of the genuine

plant extracts containing salidroside in the ﬁnal concentration

of 5.5 nM (p < 0.01) (Figure 1). Interestingly, the treatment of

human neuroglia cells with salidroside induced a similar dose–

response curves, albeit, requiring 1,000 times higher concentra-

tion of salidroside (5 μM) to achieve maximum NPY expression

(p < 0.01; Figure 2).

HUMAN NEUROGLIA CELLS RELEASE NPY AND HSP72 IN RESPONSE TO

STIMULATION WITH ADAPT-232

We previously demonstrated that ADAPT-232 strongly augments

endurance of mice in a forced swimming experiment (a model

that combined physical and emotional stresses), and that there

FIGURE 1 | ADAPT-232 dose-dependently increases NPY and Hsp72

expression in human neuroglia cells. ADAPT-232 and salidroside was

admixed with 10

human neuroglia cells at the indicated concentrations and

incubated for 24 h at 37˚C in 5% CO

atmosphere. Cells were lysed and

NPY Hsp72 protein expression was determined using Western blot analysis

as described in detail in the Section “Materials and Methods.” The intensity

of the bands were analyzed by densitometry with a video densitometer

(Chemilmager™ 5500; Alpha Innotech, San Leandro, CA, USA) using the

AAB software (American Applied Biology). Data are the percentage (%)

change in protein density expression as compared to control (± SD).

Frontiers in Neuroscience | Neuroendocrine Science February 2012 | Volume 6 | Article 6 | 4

Panossian et al. Adaptogens stimulate NPY and Hsp72

FIGURE 2 | Salidroside dose-dependently increases NPY expression in

human neuroglia cells. Salidroside was admixed with 10

human

neuroglia cells at the indicated concentrations and incubated for 24 h at

37˚C in 5% CO

atmosphere. Cells were lysed and NPY expression was

determined using Western blot analysis as described in detail in the Section

“Materials and Methods.” The intensity of the bands were analyzed by

densitometry with a video densitometer (Chemilmager™ 5500; Alpha

Innotech, San Leandro, CA, USA) using the AAB software (American

Applied Biology). Data are the percentage (%) change in protein density

expression as compared to control (± SD).

was a concomitant increase in serum Hsp72 levels (Panossian

et al., 2009). To test our working hypothesis that human neu-

roglia cells release Hsp72, cells were treated with ADAPT-232 or

salidroside for 24 h in a 37˚C incubator and the concentration

of NPY and Hsp72 in the supernatant was measured by Hsp72

ELISA. We demonstrated a dose-dependent release of NPY and

Hsp72 into the supernatant of cultured human neuroglia cells

(p < 0.0001; Ta b le 1 ). To negate the possibility that the adaptogens

were inducing Hsp72 release by stimulating necrotic cell death,

in the same experiment supernatant was subjected to LDH assay

for the determination of cell death. We demonstrated that there

was no signiﬁcant increase in cell death at the concentrations of

0.005–0.5 mg/L of ADAPT-232, as compared to control (0 mg/L;

PBS only) or exposure of cell to non-lethal heat shock treatment

(41˚C, 60 min; Table 1).

ADAPT-232-INDUCED RELEASE OF HSP72 AND NPY FROM HUMAN

NEUROGLIA CELLS IS DEPENDENT ON THE TRANSCRIPTION FACTOR

HSF1

We next explored the mechanistic basis for the induction of Hsp72,

to determine if it was primary or secondary to the ADAPT-232

response. This is important since, if the induction of Hsp72 is

a secondary event, upstream events would be of the primary

interest. The treatment of human neuroglia cells with ADAPT-

232 or salidroside signiﬁcantly increased the expression of HSF1

as judged by Western blot analysis (Figure 3A). To prove that

the adaptogens stimulate Hsp72 release by a mechanism depen-

dent HSF1, the expression of HSF1 was signiﬁcantly silenced

using siRNA directed against hsf-1 gene (HSF1-siRNA) before

treatment with the adaptogens, as compared to neuroglia cells

pre-treated with control-siRNA (Figure 3A). Control-siRNA is

a non-targeting 20–25 nucleotide siRNA designed as a negative

control, with sequences that do not target any gene product nor

has any signiﬁcant sequence similarity to mouse, rat, or human

gene sequences, and has been tested in cell-based screens and

proved to have no signiﬁcant effect on cell proliferation, viabil-

ity, or morphology, according to the manufacturer (Dharmacon

RNA Technologies, Lafayette, CO, USA). We further demonstrated

that pre-treatment of human neuroglia cells with HSF1-siRNA

strongly suppressed the expression of Hsp72 (Figure 3B) and

release of Hsp72 and NPY (Figure 5) stimulated by ADAPT-

232 and salidroside, as compared to neuroglia cells pre-treated

with control-siRNA (Figures 3B and 5A). The possibility that the

release of Hsp72 from human neuroglia cells was due to cell death

was negated by results showing that adaptogens increased signiﬁ-

cant release of Hsp72 as judged by ELISA (p < 0.05), but there was

no similar increase in cell death, as conﬁrmed by the LDH assay.

NPY PEPTIDE STIMULATES HSP72 RELEASE FROM HUMAN NEUROGLIA

CELLS IN AN AUTOCRINE LOOP

To determine if NPY peptide itself can stimulate Hsp72 expression,

human neuroglia cells were treated with NPY peptide and Hsp72

release measured in the supernatant 24 h post treatment. We

demonstrated that treatment of human neuroglia cells with NPY

peptide dose-dependently increased the expression (Figure 4A)

and signiﬁcant release of Hsp72 (p < 0.05; Figure 6). We further

demonstrated that silencing the expression of intracellular NPY

by pre-treating human neuroglia with NPY-siRNA effectively sup-

pressed the expression of Hsp72 (Figure 4A). The pre-treatment of

human neuroglia cells with NPY-siRNA signiﬁcantly suppressed

the expression of ADAPT-232- and salidroside-mediated upreg-

ulation of intracellular Hsp72 expression (Figure 4B) and con-

comitantly resulted in a signiﬁcant suppression of Hsp72 release,

as compared to neuroglia cells pre-treated with control-siRNA

(Figure 6). Cell death was excluded as a possible reason for

increased Hsp72 release by experiments in which culture super-

natant was probed for signs of cell death using LDH assay. We

demonstrated that the signiﬁcant increase in Hsp72 release in

response to NPY was not followed by a similar increase in cell death

(Figure 6). To determine the role of NPY in ADAPT-232- and

salidroside-mediated responses, we demonstrated that silencing

NPY expression in human neuroglia cells by pre-treatment with

NPY-siRNA strongly suppressed ADAPT-232- and salidroside-

induced increase in Hsp72 expression, as compared to neuroglia

cells pre-treated with control-siRNA (Figure 4C). Taken together,

these ﬁndings are consistent with a recent study which reports that

NPY promotes the expression of intracellular Hsp72 in rat renal

vascular smooth muscle (Zhong et al., 2003).

DISCUSSION

ADAPT-232 is a combination of extracts of three well studied

and efﬁcient adaptogenic plants – R. rosea, E. se nticosus, and S.

chinensis (Panossian and Wikman, 2008; Panossian et al., 2011).

Their effects on CNS have been recently reviewed (Panossian and

Wikman, 2010). Clinical efﬁcacy of single and repeated doses of

ADAPT-232 on cognitive functions and mental performance of

healthy volunteers, cosmonauts (Bogatova et al., 1997; Aslanyan

et al., 2010), and patients with pneumonia (Narimanian et al.,

www.frontiersin.org February 2012 | Volume 6 | Article 6 | 5

Panossian et al. Adaptogens stimulate NPY and Hsp72

Table 1 | Human neuroglia cells release NPY and Hsp72 in response to treatment with ADAPT-232 and its active constituent salidroside.

Treatment (concentration)

Mean Hsp72 conc. (ng/ml ± SD)

Mean NPY conc. (ng/ml ± SD)

Mean cell death (% ± SD)

ADAPT-232 (μg/ml) Salidroside (μM)

†

0035± 91.2± 0.2 8 ± 5

0.005 (0.00005)

†

65 ± 11 3.6 ± 0.1* 9 ± 4

0.05 (0.0005)

†

95 ± 10** 5.7 ± 0.3** 7 ± 6

0.5 (0.005)

†

124 ± 15** 9.5 ± 0.5** 12 ± 7

5.0 (0.05)

†

165 ± 12** 9.8 ± 1.6** 36 ± 10**

F = 76.2 F = 94.2 F = 9.9

df = 19(4/15) df = 19(4/15) df = 14(4/10)

P < 0.0001 P < 0.0001 P = 0.0017

Salidroside (μM)

040± 91.1± 0.3 10 ± 5

0.5 57 ± 6* 6.8 ± 0.5** 9 ± 3

1. 0 14 5 ± 9** 11.9 ± 0.8** 13 ± 4

5.0 189 ± 12** 21.4 ± 0.9** 15 ± 5

10.0 209 ± 13** 18.9 ± 2.1** 35 ± 11**

F = 228.5 F = 227.3 F = 9.2

df = 14(4/15) df = 14(4/15) df = 14(4/10)

P < 0.0001 P < 0.0001 P = 0.0021

Heat shock (41˚C, 60 min) 2086 ± 276** 31.5 ± 10** 23 ± 15

†

Concentration of salidroside released from ADAPT-232 in the cell culture.

Human neuroglia cells (10

) were treated with various concentrations of ADAPT-232 or salidroside or exposed to heat shock (41˚C, 60 min) and incubated for 24 h in

a 37˚C incubator.

Twenty-four hours after treatment protocols the supernatant from human neuroglia cells were recovered, centrifuged to clear cellular debris and Hsp72 concentration

was measured using the classical Hsp72 ELISA as described in detail in the Section “Materials and Methods.” Data are the mean concentration of Hsp72 (ng/ml ± SD)

and is the sum of four independent experiments performed in quadruplicates. *p < 0.05 vs control (0 μg/ml), **p < 0.001 vs control in Tukey’s multiple comparison

post test.

Human neuroglia cells were grown to 75% conﬂuence.Twenty-four hours prior to measuring NPY release, cell culture medium was replaced with serum-free DMEM,

then incubated with ADAPT-232, or salidroside, or exposed to heat shock (41˚C, 60 min) and incubated for 24 h in a 37˚C incubator. Supernatant was recovered in

quadruplicates, centrifuged to clear cellular debris and NPY concentration was measured using the NPY enzyme immunoassay kit (assay sensitivity of 0.09 ng/ml)

according to the manufacturer’s instructions (Phoenix Pharmaceuticals, CA, USA). Data are the mean concentration of NPY (ng/ml ± SD) and is the sum of four

independent experiments performed in quadruplicates. *p < 0.05 vs control (0 μg/ml genuine extracts), p < 0.001 vs control inTukey’s multiple comparison post test.

Twenty-four hours after treatment protocols the supernatant from human neuroglia cells and viability assayed using the CytoTox 96 Non-Radioactive Cytotoxicity Assay

according to the manufactures instructions (Promega), and the percentage of LDH released vs total LDH was calculated. Data are mean percentage cell death ± SD

and represent three independently performed experiments in quadruplicates. **p < 0.01 vs control (0 μg/ml genuine extracts) in Tukey’s multiple comparison post

test.

2005) have been studied. Interestingly, the long term treatment

of aged rats with ADAPT-232 diminished or prevented a range of

age-related disorders including malfunction of the central nervous

system, loss of memory, and loss of learning ability (Makarov et al.,

2007).

Recently, we demonstrated that ADAPT-232 signiﬁcantly

increased tolerance to stress, augmenting the endurance of mice in

a swimming test model. This effect was associated with a dramatic

increase in the blood level of circulating Hsp72 (Panossian et al.,

2009). However, in that study the target cell was not identiﬁed. In

the present study, we demonstrated that ADAPT-232 and its active

constituent salidroside stimulate the expression (Figures 1, 2, 3B,

and 4B,C) and release (Table 1 ; Figure 5) of NPY and the stress

protein Hsp72 from isolated human brain glioblastoma T98G

cells. There is now strong evidence that NPY are synthesized in

astrocytes, oligodendrocytes, microglia, and Schwann cells in vivo

(Ubink et al., 2003). We speculate that human normal neuroglia

cells might be one of the target cells for ADAPT-232 (and its active

constituent salidroside) as well. Further studies supporting this

hypothesis are currently ongoing in our laboratory (Kaur et al., in

preparation).

Neuropeptide Y is known to perform a wide range of biological

functions through the interaction with distinct G-protein-coupled

receptors (Wahlestedt and Reis, 1993). Tab l e 2 illustrates the

similarities in pharmacological proﬁles of NPY and adaptogens.

Table 3 exempliﬁes several NPY-mediated beneﬁcial effects and

other potential effects of adaptogens in various disorders. Taken

together,the data presented in Tables 2 and 3 suggests that a stress–

protective activity of adaptogens is mainly associated with effects

of Hsp72, while stimulating activity of adaptogens – with NPY.

Neuropeptide Y is known to play an essential role in the

basic mechanisms of morphine tolerance and opioid dependence

(Woldbyeet al., 1998). Morphine signiﬁcantly decreases NPY levels

in the hypothalamus, the striatum, and the adrenal glands (Pages

Frontiers in Neuroscience | Neuroendocrine Science February 2012 | Volume 6 | Article 6 | 6

Panossian et al. Adaptogens stimulate NPY and Hsp72

FIGURE 3 | Silencing HSF1 signiﬁcantly suppresses ADAPT-232- and

salidroside-mediated Hsp72 expression. Human neuroglia cells (10

)

were either pre-treated with PBS or transfected with either 10 μM

control-siRNA or 10 μM HSF1-siRNA or incubated for 48 h in a 37˚C

incubator. Cells were then treated with either PBS or 0.5 μg/ml ADAPT or

5 μM salidroside and incubated for a further 24 h at 37˚C. Cells were lysed

and (A) HSF1 protein expression or (B) Hsp72 protein expression was

determined using Western blot analysis as described in detail in the Section

“Materials and Methods.” β-Actin was used as a loading control. The

intensity of the bands were analyzed by densitometry with a video

densitometer (Chemilmager™ 5500; Alpha Innotech) using the AAB

software (American Applied Biology). Data are the percentage (%) change

in speciﬁc protein density as compared to control (normalized to 100%),

and are representative of three independently performed experiments with

similar results.

www.frontiersin.org February 2012 | Volume 6 | Article 6 | 7

Panossian et al. Adaptogens stimulate NPY and Hsp72

FIGURE 5 | ADAPT-232 and its active constituent salidroside induce the

release of Hsp72 and NPY via a mechanism dependent on the

upregulation of HSF1. Human neuroglia cells (10

) were either pre-treated

with PBS or transfected with either 10 μM control-siRNA (control) or 10 μM

HSF1-siRNA or 12 μM NPY-siRNA or exposed to heat shock (41˚C, 60 min)

and incubated for 48 h in a 37˚C incubator. Cells were then treated with

either PBS or 0.5 μg/ml ADAPT-232 (0.005 μM salidroside) or 5 μM

salidroside and incubated for a further 24 h at 37˚C. Twenty-four hours after

treatment protocols the supernatant from human neuroglia cells were

recovered, centrifuged to clear cellular debris and (A) NPY or (B) Hsp72

concentrations were measured using the NPY enzyme immunoassay kit

(assay sensitivity of 0.09 ng/ml) according to the manufacturer’s

instructions (Phoenix Pharmaceuticals, CA, USA), or the classical Hsp72

ELISA as described in detail in the Section “Materials and Methods.” Data

are the mean concentration of NPY (ng/ml ± SD), Hsp72 (ng/ml ± SD), and

is the sum of three independent experiments performed in quadruplicates.

*p < 0.05 vs control (control-siRNA). Twenty-four hours after treatment

protocols the supernatant from human neuroglia cells and viability were

assayed using the CytoTox 96 Non-Radioactive Cytotoxicity Assay according

to the manufacturer’s instructions (Promega), and the percentage of LDH

released vs total LDH was calculated. Data are mean percentage cell

death ± SD and represent four independently performed experiments in

quadruplicates. *p < 0.001 vs control (0 μg/ml genuine extracts).

FIGURE6|EffectofNPYonHsp72 release from human neuroglia cells.

Treatment of human neuroglia cells with NPY peptide dose-dependently

increased the release of Hsp72. Human neuroglia cells (10

) were

transfected with either 12 μM control-siRNA or 12 μM NPY-siRNA for 48 h

at 37˚C. Cells were then treated with either various concentrations of NPY

peptide or 40 μM PB1 (control peptide) and incubated for a further 24 h at

37˚C. Twenty-four hours after treatment protocols the supernatant from

human neuroglia cells were recovered, centrifuged to clear cellular debris,

and Hsp72 concentration was measured using the classical Hsp72 ELISA

as described in detail in the Section “Materials and Methods.” Data are the

mean concentration of Hsp72 (ng/ml ± SD) and is the sum of ﬁve

independent experiments performed in quadruplicate. *p < 0.05 vs

control-siRNA. Twenty-four hours after treatment protocols the supernatant

from human neuroglia cells and viability were assayed using the CytoTox 96

Non-Radioactive Cytotoxicity Assay according to the manufacturer’s

instructions (Promega), and the percentage of LDH released vs total LDH

was calculated. Data are mean percentage cell death ± SD and represent

three independently performed experiments in quadruplicates. *p < 0.001

vs PB1 (control peptide).

‡

p < 0.001 vs control-siRNA.

Table 2 | Similarities in pharmacological proﬁles of adaptogens and

NPY.

Activity NPY Adaptogens

Stress response modifying (protective and mimetic)

effect

Stimulation of HPA axis, modulation of cortisol

release

Stimulation of CNS system ++

Effect on endocrine system (via cortisol, insulin, etc.) ++

Stimulation of immune system (via Hsp72) ++

Effect on sympathetic and parasympathetic system ++

Effect on energy homeostasis ++

Anabolic effect ++

Effect on physical endurance, locomotor activity ++

Effect on cognitive function (attention, memory) ++

Cardiovascular effects, vasoconstrictor ++

Anti-narcotic effect ++

Cytoprotection (via Hsp72) ++

Anti-aging (via Hsp72 and FoxO) ++

+, See the references on relevant publications, reference list and the reviews on

adaptogens (Brekhman and Dardymov, 1969; Panossian, 2003; Panossian and

Wagner, 2005; Panossian and Wikman, 2008, 2009, 2010).

One of the active constituents of ADAPT is salidroside, which

also stimulates expression of NPY at concentrations approximately

100–1,000 times higher than in ADAPT. It might be speculated that

Frontiers in Neuroscience | Neuroendocrine Science February 2012 | Volume 6 | Article 6 | 8

Panossian et al. Adaptogens stimulate NPY and Hsp72

Table3|NPY-mediated beneﬁcial effects

‡

and potential effects

of adaptogens in various disorders.

Disorder/syndrome Effect Adaptogens reference NPY reference

Depression Antidepressive

‡

Darbinyan et al. (2007), Panossian et al.

(2009)

Stogner and Holmes (2000), Redrobe et al.

(2002), Morales-Medina et al. (2010, 2012)

Anxiety Anxiolytic

‡

Bystritsky et al. (2008) Boulenger et al. (1996), Trent and Menard

(2011). Morales-Medina et al. (2012)

Stress induced fatigue Anti-fatigue

‡

Brekhman and Dardymov (1969), Olsson

et al. (2009), Panossian and Wikman (2009)

Morgan et al. (2001), Karamouzis et al.

(2002)

Chronic fatigue syndrome Relief of symptoms

Panossian and Wikman (2009) Morgan et al. (2000), Fletcher et al. (2010)

Post-traumatic stress syndrome Relief of symptoms

–

Rasmusson et al. (2010)

Metabolic syndrome Relief of symptoms

–

Abe et al. (2010), Rasmusson et al. (2010)

Fibromyalgia Relief of symptoms

–

Crofford et al. (1996), Di Franco et al.

(2009), Sedlackova et al. (2011)

Anorexia nervosa Gain of body weight

‡

–

Nergardh et al. (2007), Himmerich et al.

(2010), Sedlackova et al. (2011)

Bulimia Loss of body weigh

‡

–

Morris et al. (1986)

Asthenia Anti-fatigue

‡

Brekhman and Dardymov (1969), Panossian

and Wagner (2005), Panossian and Wikman

(2008, 2009, 2010), Panossian et al. (2011)

–

ADAPT contains some other active compound(s) synergistically

increasing the expression of NPY. Interestingly tyrosol, salidro-

side, and NPY contain ﬁve tyrosine residues (Labelle et al., 1997),

and all have the same p-hydroxyl-methylene residue. The impor-

tance the tyrosine moieties for brain receptors binding and NPY

activity has been demonstrated (Martel et al., 1990). We hypoth-

esize that the p-hydroxyl-methylene residue of the tyrosine unit

(in polypeptide chain of NPY), and p-hydroxyl-ethylene residue of

tyrosol and salidroside (in ADAPT-232) can compete on the recep-

tor binding site. In support of this speculation, we demonstrated

that ADAPT-232 and salidroside dose-dependently stimulate the

expression (Figure 1) and release (Table 1 ) of Hsp72 in human

neuroglia cells. Again, salidroside alone is active in concentrations

signiﬁcantly higher than it is in ADAPT-232 reviling a similar effect

on release of Hsp72 from neuroglia cells (Figure 5). In the next

series of experiments, we demonstrated that ADAPT-232 signif-

icantly activates HSF1, a protein known to initiate the synthesis

of Hsp72 (Wu, 1995), However, ADAPT-232 is inactive in “silent”

cells transfected by HSF1-siRNA which effectively inhibits baseline

levels of HSF1 expression (Figure 3A).

Interferences RNA technology (RNAi) was also used to abro-

gate the expression of HSF1 (using HSF1-siRNA) in order to clarify

what is upstream of NPY, HSF1, and Hsp72 activation. Figure 5

demonstrated that ADAPT-232 and an active constituent salidro-

side induce the release of Hsp72 via a mechanism dependent on

the upregulation of HSF1. Pre-treatment of human neuroglia cells

HSF1-siRNA, which silences/knock down the expression of intra-

cellular HSF1, before treatment with ADAPT-232 or salidroside,

resulted in a signiﬁcant suppression of Hsp72 and NPY release

(Figure 5). We further demonstrated that pre-treatment of human

neuroglia cells with NPY-siRNA, which silences/knock down the

expression of intracellular NPY, before treatment with ADAPT-

232 or salidroside resulted in a signiﬁcant suppression of Hsp72

expression and release (Figures 3A, 3B and 5).In a separate set

of experiments, we demonstrated that the treatment of human

neuroglia cells with NPY peptide dose-dependently increases the

expression and release of Hsp72 (Figure 6). These results are in

agreement with recent ﬁndings that NPY stimulates Hsp72 in rat

renal vascular smooth muscle (Zhong et al., 2003).

In this study we demonstrated that ADAPT-232-induced

expression and release of Hsp72 from glioma cells was dependent

of HSF1 or NPY. The general mechanism of activation and regu-

lation of Hsp72 by HSF1 in the brain and aging is well elucidated

(Asea and Brown, 2008; Calderwood et al., 2009). However, the

mechanism activation of Hsp72 by NPY still remains uncertain.

The expression of NPY receptors on glial cells has been demon-

strated (Canto Soler et al., 2002; Hashimoto et al., 2011). Recently,

the Y(1) receptor has been demonstrated to play an essential role

in mediating NPY-induced NO and IL-1β production in microglia

(Ferreira et al., 2010). However, further studies are required to elu-

cidate the exact mechanism of action and the signaling pathway

utilized by NPY receptors to transducer it beneﬁcial effects.

Taken together our studies suggest that the ADAPT-232 and

its active constituent salidroside act on NPY expression via a

mechanism dependent on the upregulation of HSF1 expression,

which is upstream of Hsp72 expression, which is upstream of

Hsp72 release (Figure 7). Our hypothetical model suggest that

the release of Hsp72 take place via a mechanism dependent on

the upregulation of NPY, which is upstream of Hsp72 and all

other mediators of stress response involved in effects of ADAPT-

232 (Figure 7). ADAPT-232 stimulates secretion of NPY which is

known to play an important role in regulation of HPA axis and

energy homeostasis and secretion of Hsp72, playing an impor-

tant role in cytoprotection and innate immunity. Hsp72 in turn

inhibits FoxO transcription factor, playing an important role in

adaptation to stress and longevity (Lam et al., 2006; Senf et al.,

2010). These pathways contribute the anti-fatigue effect of ADAPT,

increase attention and the improvement in cognitive function.

www.frontiersin.org February 2012 | Volume 6 | Article 6 | 9

Panossian et al. Adaptogens stimulate NPY and Hsp72

FIGURE7|Schematics representation of the hypothetical working

model for the effect of ADAPT-232 on neuroglia cells. ADAPT-232

(green star) and its active constituent salidroside activated the synthesis

and release of NPY and Hsp72 via a HSF1-dependent mechanism,

including the trimerization and nuclear translocation of HSF1 (blue

circles). Hsp72 functions intracellularly to enhance anti-apoptotic

mechanisms, protect proteins against mitochondria generated

oxygen-containing radicals, including nitric oxide (red star) and

superoxide anion (purple star). The released Hsp72 acts as an

endogenous danger signal and plays an important role in immune

stimulation. While released NPY plays a crucial role in the HPA axis and

maintains energy balance, both NPY and Hsp72 are directly involved in

cellular adaptation to stress, increased survival, enhanced longevity, and

an improvement in cognitive function.

The activation of NPY by ADAPT-232 initiates Hsp72 expres-

sion in human neuroglia cells, which are known to maintain

homeostasis of neuronal cells. Stimulation and release of these

stress hormones (NPY and Hsp72) into the blood circulating sys-

tem apparently is an innate defense response to mild stressors

(adaptogens), which increases tolerance and adaptation to stress.

This gives rise to adaptive and stress–protective effects via various

components of central nervous, sympathetic, endocrine, immune,

cardiovascular, and gastrointestinal systems. Both NPY and Hsp72

play important roles in stress, regulation of aging, and pathogen-

esis of age-related diseases (Kmiec, 2006; Calderwood et al., 2009;

Rasmusson et al., 2010). Taken together, the data presented in this

study suggests that adaptogenic activity of ADAPT-232, such as

increased mental performance, attention, endurance, tolerance to

stress, and beneﬁcial effects on age-related disorders are associated

with activation of NPY and Hsp72 gene expression in neuroglia

cells and the release of NPY and Hsp72 into the systemic blood

circulation.

ACKNOWLEDGMENTS

This work was supported in part by the Swedish Herbal Institute

(Alexander Panossian, Georg Wikman); Scott & White Memo-

rial Hospital and Clinic Research Advancement Awards (Punit

Kaur); the U.S. National Institute of Health (RO1CA91889), Scott

& White Memorial Hospital and Clinic, the Texas A&M Health Sci-

ence Center College of Medicine (Alexzander Asea), the Central

Texas Veterans Health Administration and an Endowment from

the Cain Foundation (Alexzander Asea).

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Conﬂict of Interest Statement: Punit

Kaur, Alexzander Asea, and Alexander

Panossian declare no competing ﬁnan-

cial interests. Georg Wikman is a stock-

holder in the Swedish Herbal Institute

(SHI).

Received: 21 October 2011; paper pending

published: 12 November 2011; accepted:

13 January 2012; published online: 01

February 2012.

Citation: Panossian A, Wikman G, Kaur

P and Asea A (2012) Adaptogens stimu-

late neuropeptide Y and Hsp72 expression

and release in neuroglia cells. Front. Neu-

rosci. 6:6. doi: 10.3389/fnins.2012.00006

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