Author's Accepted Manuscript

Modulation of T -helper cytokines and in-flammatory mediators by Atropa accuminata.Royle in adjuvant induced arthritic tissues

Albeena Nisar, Nayeema Akhter, GurudarshanSingh, Akbar Masood, Akhter Malik, BasharatBanday, Mohammed Afzal Zargar

PII: S0378-8741(14)00600-XDOI: http://dx.doi.org/10.1016/j.jep.2014.08.008Reference: JEP8970

To appear in: Journal of Ethnopharmacology

Received date: 23 January 2014Revised date: 5 July 2014Accepted date: 9 August 2014

Cite this article as: Albeena Nisar, Nayeema Akhter, Gurudarshan Singh, AkbarMasood, Akhter Malik, Basharat Banday, Mohammed Afzal Zargar, Modulationof T -helper cytokines and inflammatory mediators by Atropa accuminata.Royle in adjuvant induced arthritic tissues, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2014.08.008

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Modulation of T -helper cytokines and inflammatory mediators by Atropa

accuminata.Royle in adjuvant induced arthritic tissues

Albeena Nisar a , Nayeema Akhterb , Gurudarshan Singhc , Akbar Masood a,Akhter Malik a,

Basharat Banday c, Mohammed Afzal Zargar a*

aDepartment of Biochemistry, University of Kashmir, Srinagar-190006, J&K, India bIndian Institute of Integrative Medicine, Srinagar, India cPK-PD division, Indian Institute of Integrative Medicine, Jammu, India *Corresponding author. Tel.: +0 91 9419463306. Email:�zargarafzal01@gmail.com�

Abstract Ethnopharmacological relevance

Atropa acuminata has been widely used in traditional medicine against arthritis and several

associated inflammatory disorders.

Aim of the study

The present study was undertaken to investigate the anti-arthritic activities of ethanolic extract of

Atropa accuminata (AAEE) and to explore the probable mechanism of action.

Materials and Methods

The anti-arthritic activity of AAEE was evaluated within a dose range of 125-500 mg/kg b.w. in

adjuvant induced-arthritis in male wistar rats .An array of pro-inflammatory mediators (PGE2

NO , IL-1� and LTB4) and T-cell-mediated cytokines (IL-2,TNF-a, IFN-c, IL-4, IL-10, IL-12,

IL-17, IL-6 ) were assayed in arthritic paw tissue homogenate of the treated animals. In addition

the effects on arthritic lesions, changes in body weight ; haematological (Hb, ESR, WBC and

RBC) and biochemical parameters (SOD, GSH, GR) and the serum markers (CRP, RF) were

also observed.

Results

Significant anti-arthritic activity was observed for AAEE in the polyarthiritis test both in the

developing and developed phase of the disease. This was associated with dose dependant

suppression of pro-inflammatory mediators (PGE2 , NO , IL-1� and LTB4 ) ., Th1-Th17

cytokines (IL-2, TNF-�, IFN-�, IL-12, IL-17, IL-6) and upregulation of Th2 cytokines (IL-4

and IL-10). AAEE was also observed to protect rats against the primary and secondary arthritic

lesions, body weight changes and haematological perturbations. In addition, inhibitory effects of

AAEE on biochemical parameters and the serum markers further confirmed that it reduced

signs on chronic inflammatory responses.

Conclusion

The present investigation therefore suggested that AAEE is a potent anti-arthritic agent. The

multipronged attack on the inflammatory mediators and T-helper cytokines and strong potency

of AAEE may have relevance for inhibition of the chronic inflammatory responses in arthritis.

Keywords

Atropa accuminata; Herbal medicine; NO; PGE2; LTB4; TNF-�; IL-1� ; IL-2; IFN-�; IL-4; IL-

10,IL-17,IL-6

1. Introduction

Rheumatoid Arthritis is a systemic, inflammatory autoimmune disorder which can lead to long

term joint damage, loss of function and disability (Alamanos et al., 2006).The etiology of this

disease remains largely unclear. It is proposed that the disease represents a typical T-cell

mediated disease . Once T cells are activated in RA, it consequently leads to multiple effects,

including activation and proliferation of synovial lining and recruitment of pro-inflammatory

cells into the synovium (Cope, 2008., McInnes, 2001).

Cytokines are considered to play a pivotal role in the pathogenesis of arthritis and exhibit

pleiotropic effects that contribute to the inflammatory process. TNF-� and IL-1� both escalate

the pro-inflammatory cellular actions like stimulating production of cascade of pro-inflammatory

cytokines and increased leukocyte extravasation . IL-4 and IL-10 act antagonistically to

decelerate the inflammatory response in RA by inhibiting the synthesis of pro-inflammatory

cytokines ( IL-1 and TNF-� ) by macrophages and monocytes. Any imbalance between pro and

anti-inflammatory cytokine activities favours the induction of autoimmunity, chronic

inflammation and thereby joint damage as is the case in RA (McInnes and Schett, 2007)

Therefore, modulation of Th1/Th2 cytokine balance has become a new paradigm for employing

immune modulatory therapy in rheumatoid arthritis. In addition, PGE2 and LTB4 in concert act

as potent contributors to the pathogenesis of RA and mediate vasodilatation and the infiltration

of leukocytes into the RA joint (Chizzolini et al., 2008). NO also is reported to induce the

production of cytokines such as TNF-�, IL-1� and IFN-�, in arthritic patients (Weinberg et al.,

2007). Whereas eicosanoids and NO mediate much of the early phase in inflammatory diseases,

cytokines are involved in the later destructive phase in rheumatoid arthritis (Brennan and

McInnes, 2008).

Most of the conventional therapies employed for rheumatoid arthritis are directed against

non specific suppression of the inflammatory and immunologic processes .On account of such

limitations , there has been a growing interest in the recent past to use herbal therapies based on

traditional medicine as an adjunct therapy against arthritis (Ahmed et al., 2005). The present

study was designed to investigate the anti-arthritic effect of Atropa accuminata and delineate the

probable mechanism of action.

Atropa accuminata (AA) belongs to the family Solonaceae and is a tall perennial sub-

alpine plant native to Asia. It is an Indian medicinal plant, widely distributed across north

western Himalayas (Dhar and Kachroo, 1983). In Kashmir, rhizome of A.accuminata has been

traditionally used since ages for the treatment of arthritis related inflammatory disorders, muscle

and joint pain, muscle spasms (Kaul, 1997). Aerial parts of this plant have been used in

traditional medicine to treat innumerable ailments as muscle and joint pain, inflammation,

pancreatitis ,peritonitis, scarlet fever, parkinsons disease and neuoro disorders (Kahn et al., 1991;

King, 1966) .The roots of the plant has also been used as sedative (Rhodes et al., 1978) and also

against sore throat, ulcerative colitis (Shanafelt et al., 2002) whooping cough (Walach et al.,

2001). Chemically A.accuminata has been found to contain tropane alkaloid and highly

oxygenated triterpenes (Mehmood et al., 2002).

2. MATERIALS AND METHODS

2.1. Chemicals

Complete Fruend’s adjuvant (CFA), DMSO, ibuprofen (IBP), phenyl methylsulfonyl

fluoride (PMSF), aprotinin, (Sigma Chemical), Tween 20 (Santa Cruz), Gumacacia (Hi Media),

PGE2 and LTB4 ELISA (Cayman chemicals), IL-1�, IL-2, IFN-�, IL-4, IL-6 , IL-17 , TNF-a and

IL-10 ELISA kits (R&D Systems). All the other chemicals were purchased from Hi-Media and

Merck, India. Standard drugs were purchased from Sigma-Aldrich chemicals co. (St. Louis O.,

USA) and all solvents used were of HPLC grade (Ranbaxy Chemicals Ltd., Mohali, Punjab,

India).

2.2. Test material

2.2.1 Collection of plant

Atropa accuminata (whole plant) was collected from the higher reaches of Kangdoori,

Khilanmarg, Afarwarth areas of Gulmarg and Thajwas glaciers of Sonamarg. The plant was

correctly identified by Centre of Biodiversity and Toxonomy, University of Kashmir, Srinagar,

India and authenticated by a botanist, Dr. Irshad Wanchoo. A voucher sample is retained and

deposited at Central Herbarium, Department of Botany, University of Kashmir, India as 1758

KASH (16/05/2009).The material was ensued to be free from pathogens, aflatoxins, pesticidal

residues and heavy metals according to (WHO, 1998).

2.2.2. Preparation of extracts

The authentically identified whole part of the plant material was shade dried and then

powdered. About 1.6 kg of A.accuminata was subjected to Soxhlet extraction separately with

absolute ethanol (EtOH). The solvent was removed under reduced pressure on a vacuum rotary

evaporator to get a darkish green extract for A.accuminata referred from here as AAEE (yield:

32.2 %). The crude extract was stored at 4°C for experimental use.

2.2.3. Determination of total polyphenolic content :

The concentration of total phenolic compounds in the test material was determined by the Folin-

Ciocalteau method with a few modifications (Singleton and Ross, 1965). The concentration of

flavonoids was determined according to the method of Ordon et al 2006. Total Flavanols in the

test material was estimated on the method of Kumaran and Karunakarn, 2006.

2.3. Animals

Studies were carried out using Male Wistar Albino rats of either sex weighing 150-180

grams. They were procured from IIIM, Jammu and kept in animal house, KU. The animals were

grouped in polypropylene cages with not more than six animals per cage and maintained under

standard laboratory conditions (temperature 25±2°C, relative humidity 60% ±15% and with dark

& light circle 12hrs /14hrs).They were allowed free access to standard dry pellet (Hindustan

lever, Kolkata, India) and water ad libitum (CPCSEA, 2003). The animals were acclimatized to

laboratory conditions before commencement of the experiment. All animal experiments were

approved by the University animal ethical committee vides No: 80/03/CA/CPCSEA.

2.4. Acute toxicity studies

The effect of test extracts on general behaviour and safety was evaluated in mice using

the up and down procedure (Organization of Economic Co-operation and Development (OECD),

Guideline No. 423, 1996). Mice of either sex (three females); weight: 20–25 g; age: 4–6weeks)

were administered graded doses of AAEE (single dose) upto 2500mg/kg orally by gavage. The

animals were observed for toxic symptoms continuously for the first 4 hours after dosing.

Finally, the number of survivors was noted after 24 hours and these animals were then

maintained for a further 13 days with observations made daily. The animals were observed for

any changes in general behaviour, weight, mortality or other physiological activities.

2.5. Induction of arthritis

Adjuvant induced arthritis is the best available model having nearest approximation to

human rheumatoid arthritis (Pearson and Wood, 1963). Briefly, on day 0, rats were weighed and

were marked directly above the ankle joints to provide an anatomical marker to minimize

variability when measuring paw volume. Next day, on Day 1 adjuvant arthritis was induced by

the sub-plantar injection of 100 μl of CFA containing heat killed and dried mycobacterium

tuberculosis (strain H37Ra, ATCC-25177) into the dorsum of the right hind paw between the

third and fourth digits of each rat. The animals were divided into groups of 6 animals each.

Group I, (Normal control), received 1% gum acacia, Group II, received 1% gum acacia (Arthritic

control); Group III: AAEE (125 mg/kg b.w); Group IV: AAEE (250 mg/kg b.w); Group V:

AAEE (500mg/kg b.w); Group VI: Ibuprofen (50mg/kg b.w).All the test extracts and standard

drugs were dissolved in 1% guma acacia and were given orally one day (Day 0) before the

adjuvant injection and then once daily for 21 days (Newbould, 1963). Ten days after injection,

secondary inflamed lesions were detected on the left hind paw, which began to increase in

thickness and also in the fore-paws, ears and tail. All groups were induced with arthritis by

injection of CFA except group I (Naïve Control). The dosage of the test extracts was fixed as

described in Results Section.

2.6. Evaluation of Severity of arthritis

To evaluate the arthritic progression of CFA-induced arthritis in rats, three different

parameters were measured: paw volume (primary and secondary lesions), squeaking score in the

ankle flexion test and arthritic score.

2.6.1 Assessment of mean paw volume (primary and secondary lesions)

The volume of the injected hind paws (“primary” lesions) and un-injected hind paws

(“secondary” or immune-mediated response) (Arrigoni-Martelld and Bramm, 1975) were

measured using plethysmometer (UGO, Basile, Italy) on alternate days from day 1 to day 21

after the adjuvant injection. Any changes in paw volume were calculated as the difference

between volumes on day 0 and volumes recorded on days 3, 5, 7, 9, 11, 13, 15, 17 and 19. The

paw volume was measured as relative values to that of Day 1, when CFA was injected.

2.6.2. Assessment of ankle flexion score

The squeaking test (ankle flexion test) involved gentle flexion and extension of CFA injected

hind limb(Kwon et al., 2001) .This elicited vocalization (squeaking) that were scored on a scale

(squeaking score). The procedure of flexion and extension was repeated 10 times in every 5 secs

and the rating of 0 (null) or 1 (vocalization) was given to each hind limb. This test was

performed only once a day in each animal.

2.6.3. Assessment of arthritic score

The rats were assessed daily for signs of arthritis between days 7 and 25 post-CFA using a well-

established, widely-used scoring system developed to evaluate the severity of adjuvant-induced

arthritis. Paws were examined and graded for severity and loci of erythema, swelling and

induration using a 5-point scale: 0 = no signs of disease, 1 = signs involving the ankle/wrist, 2 =

signs involving the ankle plus tarsal of the hind paw and/or wrist plus carpals of the forepaw, 3 =

signs extending to the metatarsals or metacarpals and 4 = severe disease involving the entire hind

or fore paw (Durai et al., 2004). The maximum arthritic score per rat was set at 16 (4 points × 4

paws).

2.7. Measurement of body organ weights

During the experimental period, the body weight was measured using a digital weighing

balance on day 0 and day 21st. At the end of 21 days after sacrificing the animals, thymus and

spleen once removed were also weighed.

2.8. Local tissue collection

On Day 21 post-injection, blood was collected from the animals from retro-orbital plexus

in EDTA containing tubes for serum preparation which was stored at -20°C till further analysis

and on the same day, the rats were deeply anesthetized with sodium pentobarbital (80 mg/kg,

i.p.). The paw tissue was collected, weighed and frozen.Homogenization of tissue:Tissue from

frozen paws containing bony tissue were weighed,broken into pieces on dry ice and

homogenized with an extraction buffer containing 1 mM phenyl methyl sulphonyl fluoride

(PMSF), 1μM/ ml Apopritin and 0.05 % TWEEN in PBS (4 ml/g of tissue) using polytron

homogenizer. The homogenate was centrifuged at 5000g for 15 mintues (Magari et al., 2004)

and the supernatants obtained were stored at -80ºC analysis. Some of the retained paw tissue was

washed with normal saline, fixed in 10 % formalin and stored at 4°C for histopathological

studies.The supernatant from homogenized paw samples and serum samples were subjected to

further biochemical and haematological examination. Liver, thymus and spleen were removed

from the sacrificed animals and weighed (Hu et al., 2005).

2.9. Haematological examination

Haematological parameters., WBC, RBC, Hemoglobin and erythrocyte sedimentation

rate (ESR) were assessed in serum samples using the routine laboratory methods in serum

samples on day 21.

2.10. Biochemical studies

C-Reactive protein (CRP) and Rhuematoid Factor (RF) were estimated in serum using

kits according to manufactures instructions (Monzyme India Private Limited).

2.11. Measurement of anti oxidant enzymes:

The experimental animals of CFA induced model from respective groups were sacrificed.

Liver was isolated on day 21st as described earlier and whole tissue after rinsing in ice cold

normal saline was immediately placed in normal saline (double this volume) and homogenized at

4ºC. The homogenate was centrifuged at 12000 rpm for 5 min and the supernatant stored at-20ºC

was used for assay of several anti-oxidant enzymes. The different biochemical parameters

assessed were as : a) Estimation of superoxide dismutase (SOD ): Beauchamp and Fridovich,

1971. b) Estimation of Glutatione peroxidase (GPX ):Sharma et al., 2001. c) Estimation of

Glutathione Reductase activity(GRX): Carlberg and Mannervik et al., 1985.

2.12. Quantification of PGE2, LTB4, NO and IL-1�

PGE2 , LTB4 and IL-1� were estimated in serum as well as paw homogenate using

commercially available kits (Cayman chemicals) according to the manufacturer’s instructions.

NO was measured in the homogenate as previously described on RAW 246.7 macrophage cell

line in chapter 3.1.

2.13. Assay of cytokines and MMPs

Cytokines.,IL-1�, IL-2,IFN-�, IL-4, TNF-a, IL-4 ,IL-10, IL-6 and IL-17 and MMP-3 and

MMP-9 were determined in homogenate samples using enzyme linked immunosorbent assay

( ELISA) kits according to the manufacturer’s instructions (R&D Systems and Merck Millipore).

The results of cytokine assays were expressed as respective cytokine concentrations in pg/ml.

2.14. Data analysis and statistical considerations.

Data is expressed as mean ±S.E.M. Statistical significance of differences was assessed by

One way ANOVA followed by Bonferroni test for multiple comparisons. The level of

significance was set at P < 0.05. Percent inhibitory activity in different experimental groups was

derived using following method:

( )%

��

Test group Arthriticcontrol groupInhibitoryactivityArthriticcontrol group

x 100

3. RESULTS

3.1. Total polyphenolic content :

The total phenolic content in AAEE was found to be fairly high.The total polyphenolic

content in AAEE was found to be 104.19 μg Gallic acid equivalents (GAE)/gm of dry

plant material. In addition, the flavanoid and total flavanol content was found to be 1.19

and 0.97 mg quercetin (QRC)/gm of dry plant material, respectively.

3.2. Acute safety studies

There was no noticeable change in behaviour nor any mortality was observed in mice observed

upto a single maximum dose of 2500 mg/kg b.w dose of AAEE over a period of 14 days. No

adverse effects or mortality were observed in mice , which hint that doses of AAEE used in the

study are tolerable, without any noticeable side effects and was safe to use at high doses also.

3.3 Selection of doses

No toxic effects or mortality were observed in mice upto 2500 gms (LD-0 gms). Evaluation of

the test material was initiated by giving to the animals one-tenth of this dose (250 mg/kg b.w ) as

well as lower (62.5 mg/kg b.w &125 mg/kg b.w) and higher dose (500 mg/kg b.w).

3.4 Effect on Severity of arthritis

3.4.1 Effect on primary lesions (paw edema)

Edema in the injected paws (adjuvant injected paw) was apparent from the day of

adjuvant injection (DAY 1). In the uninjected paws of the same animal where adjuvant was not

injected edema is developed on Day 9 onwards and this is used to determine secondary lesions.

AAEE demonstrated significant dose dependant inhibition of swelling in the in the injected hind

paw on all alternate days through the entire period of study .Maximal suppression of paw edema

was observed at a dose of 500 mg/kg b.w with 52%inhibition on day 21st, which was statistically

highly significant. Inhibition shown by Ibuprofen on Day 21st was 51.38% .

3.4.2 Effect on secondary lesions (paw edema)

AAEE at all doses significantly reduced the edema in the uninjected paw (secondary lesions)

from Day 9 through 21 .A dose dependant increase in percentage inhibition was observed for

AAEE. At 500 mg/kg b.w dose the suppressive effects of AAEE on secondary lesions were more

effective than ibuprofen on Day21st.

3.4.3.Effect on arthritis as assessed with squeaking scores (ankle flexion score)

The vocalizations caused by flexion or extension of the injected ankle (hind limb)

reached a maximum point on day 1 after CFA injection and was sustained at a maximum level in

untreated rats (arthritic control group) through the end of the experiment. A value of 0

represented no pain and a value of 10 indicated maximum pain The assessment made over a

period of 21 days showed that the AAEE and Ibuprofen treatments had significantly reduced the

ankle flexion score in the respective treatment groups as compared with the CFA arthritic control

group as shown in Figure 1. Arthritic rats treated with AAEE demonstrated a dose dependant

decrease in the number of vocalizatitions on all days. It was observed that in case of 500 mg/kg

b.w. AAEE treated group, the squeaking scores were lower than that of Ibuprofen.

3.4.4: Effect on arthritis as assessed with arthritic scores 21 days.

Arthritic score represents the index for severity of arthritis.In the CFA-induced arthritis

control group , the adjuvant uninjected paw joints also started to show erythema, swelling and

rigidity on day 9 and the arthritic score reached the maximum level on day 21st [Figure 2 ].

Ibuprofen suppressed the arthritic score significantly from day 9 through day 21st. AAEE at all

tested doses reduced signs of arthritis development as indicated by the decrease in arthritic score.

The suppressive effects of AAEE were observed to be dose dependant and at 500 mg/kg b.w

dose, AAEE maximally reduced the arthritic score to 4.5 on day 9th and to 3 day 21st in

comparison to arthritic control group.

3.5 Effect on Body weight and organ weight changes.

The challenge with CFA showed significant decrease in the average body weight and

increase in the spleen and thymus weight in arthritis control as compared to the normal control.

Under similar conditions, Ibuprofen and all the AAEE treated groups showed average gain in

body weight.This effect was maximal in case of group treated with AAEE , 500 mg/kg b.w. Also

AAEE at all graded doses significantly reduced the net weight of spleen and thymus on 21st day

in comparison to normal control. The effect was found to be dose dependant and at 500

mg/kgb.w the effects were comparable to ibuprofen .

3.6 Effect on haematological parameters

In CFA induced untreated group, there was a significant decrease in RBC count and

hemoglobin and also a marked increase in WBC count and ESR. However treatment with AAEE

reversed these altered hematological parameters and the effects for AAEE were found to be dose

dependant. At 500 mg /kg b.w dose of AAEE the levels of haemglobin was upregulated from 11

in case of arthtitic control to 14 and of RBC count from 7.8 in case of arthtitic control to 9.5.

Also the WBC count was reduced from 12.2 in case of arthtitic control to 4.5 and ESR from 16

in case of arthtitic control to 13 .

3.7 Effect on C-reactive protein and Rheumatoid factor

The serum CRP and RF are markers of systemic inflammation and antibody production,

respectively against the injected adjuvant. AAEE and Ibuprofen treatments were observed to

reduce the increase in the levels of both CRP and RF in the serum (Table 1).

3.8 Effect on the levels of anti-oxidant enzymes :

In the animals of group II (CFA arthiritic control) the levels of antioxidant enzymes (SOD, GPX

and GR) were markedly reduced. Treatment with Ibuprofen and AAEE significantly up

regulated the levels of SOD ,GPX and GR in comparison to the control group .The effects of

AAEE were dose dependent and at 500 mg/kg b.w. the levels of SOD,GPX and GR were

upregulated maximally.

3.9 Downregulation of pro-inflammatory mediators of Prostaglandin E2 ( PGE2) ,

leukotriene B4 (LTB4) , Nitrite (NO) and IL-1� production in paw

homogenate supernatant

PGE2 levels were found dramatically elevated in CFA-induced untreated arthritic group

at the end of 21 days. Treatment with Ibuprofen, however demonstrated a significant inhibition

of PGE2 production. Likewise, AAEE demonstrated a dose dependant pattern for the down

regulation (51.22%) in paw homogenate PGE2 levels. AAEE also significantly decreased LTB4

levels in a dose-dependent manner, in comparison to arthritic control rats. Ibufrofen did not show

any appreciable effect on LTB4 production. The paw homogenate levels of LTB4 were reduced

to 54% at 500 mg/ kg b.w. doses of AAEE [Figure 3]. Parallel to these findings, AAEE down

regulated the nitrite levels in paw homogenate dose dependently Figure 4 (a). In the animals

receiving Ibuprofen, the standard drug nitrite levels were reduced in comparison, to untreated

CFA control rats. A dose, dependent suppression was shown by AAEE at 62.5 -500 mg/kg b.w.

doses, where nitrite levels decreased to 11% (62.5mg/kg b.w.) and 52.2% (500mg/kg b.w.) in

case of paw homogenate .A significant increase in the paw homogenate levels of IL-1�

concentration was observed on Day 21 after CFA injection. AAEE reduced the IL-1� levels in

paw tissue in a dose dependent manner showing significant inhibition of IL-1�. The results are

depicted in Figure 4 (b).

3.10 Effect on Th1 cytokines (IL-2, IFN-�, TNF-� and IL-12):

On day 21 post CFA injection, the levels of Th1 cytokines., IL-2, IFN-�, TNF-� and IL-6

in arthritic control in paw tissues were markedly elevated in comparison to that observed in non

arthritis, normal control group. However, AAEE treatment in all the groups ranging from 62.5 to

500 mg /kg b.w. significantly suppressed the levels of all the aforementioned cytokines in both

serum and paw. Maximal suppressive effects were maintained at the highest dose of 500 mg /kg

b.w for each tested cytokine. As shown in Figure 5 (a). AAEE significantly down regulated IL-

2 levels in paw tissue by 54% at 500 mg/kg b.w. AAEE in comparison to control. IFN-� levels

were decreased at all doses. However, at 500 mg/kg b.w its levels were decreased by 52% in rat

paw homogenate in comparison to untreated group and these inhibitory effects were fairly

potent than that of Ibuprofen Figure 5 (b). TNF-� and IL-12 levels were again reduced in a dose

dependant manner in both paw homogenate as well as serum as shown in Figure 5 (c) [TNF-�]

and Figure 5 (d) [IL-12]. Treatment with AAEE at 500 mg/kg b.w. maximal decreased the

levels of TNF-� and IL-12 in paw homogenate to 50 % and to 56%, respectively..

3.11 Effect on Th17 cytokines, IL-17 and IL-6:

AAEE inhibited the levels of IL-17 and IL-6 maximally at a dose of 500 mg/kg b.w . in

comparison to CFA untreated control [Figure 6 (a & b)].

3.12 Effect MMP-3 and MMP-9 :

MMP-3 and MMP-9 levels were again reduced in a dose dependant manner as shown in

Figure 7 (a)and (b) .

�

3.13 Effect onTh2 cytokines, IL-4 and IL-10:

The level of both the cytokines was raised in a dose dependent manner by the test extract,

AAEE in paw tissues in comparison to CFA untreated control [Figure 8 (a & b)].

4. Discussion

Rheumatoid arthritis is a chronic autoimmune inflammatory disorder of unknown aetiology.

Cumulative evidence suggests that multiple components of immunity and inflammation like T

and B lymphocytes, all play a role progression of the disease (McInnes, 2001). In our study, we

investigated the immune pharmacological efficacy of Atropa accuminata on chronic adjuvant

induced-arthritis and its associated immune responses.

CFA-induced experimental model for arthritis is the most widely used chronic test model

for arthritis (Butler et al., 1992). AAEE dose dependently attenuated adjuvant induced arthritis

and also facilitated recovery as measured by the decreasing paw edema. The appearance of

secondary lesions, i.e. non-injected paw swelling is a manifestation of cell-mediated immunity

(T-cell response).The suppression of these secondary lesions by AAEE suggested

immunosuppressive activity for AAEE. It has been reported that a selective reduction in the

arthritis score distinguishes the immunosuppressive effects of a drug from its anti-inflammatory

effects (Yu et al., 2006).In our study the arthritis score was significantly reduced by AAEE

which indicates for a possible immunosuppressant effect. Ankle flexion test is commonly used to

assess the functional impairment in arthritis (Kumar et al., 2006). Parallel to the findings on paw

edema, it was found that AAEE reduced the ankle flexion score also, significantly and in a dose

dependant manner indicating for a reduction in pain.

AAEE also caused apparent increase in body weight which could be attributed

improvement of intestinal absorption of the nutrients by AAEE (Patil et al., 2004). The reduction

in spleen weight and thymus weight in AAEE treated groups may be related to a variety of

effects on the immune system (Padernera et al., 2006). And the reversal of RBC counts and Hb

levels observed in case of AAEE treated groups could be due to the protective effects on tissue

repair and deceleration of disease progression. AAEE also tended to normalize the WBC count

and ESR probably through its immune modulation effects .

Rheumatoid arthritis (RA) has been associated with aberrant expression of superoxide

dismautase (SOD), glutathione peroxidase (GPx) and glutahthione reductase (GR) (Aryaeian et

al., 2011). The significant up regulation of antioxidant enzymes., SOD, GPX and GR by AAEE

supports their role in anti-arthritic potential through effects on antioxidant parameters.

The serum CRP concentration directly reflects the intensity of the pathological process in

RA. It has been reported that inflammatory stimuli prompt the release of IL-1,IL-6, and TNF-�,

which in turn stimulates the liver to produce CRP (Gabay and Kushner, 1999). Moreover,

rhuematoid factor (RF) is considered as a potential systemic marker for autoimmune stimulation

in the pathogensis of rheumatoid arthritis (Brum-Fernandes et al., 1988). Significant reduction in

the level of these biomarkers by AAEE indicates for its inhibitory effects on inflammatory

cascade in the progression of arthritis.

Prostaglandins are the ubiquitous mediators exerting numerous vascular and inflammatory

effects in arthritis.Our results indicated that treatment with AAEE dose dependently decreased

the levels of PGs in inflamed paw homogenates. PGE2 production in arthritis is predominantly

induced by cytokines., IL-1� and TNF-� (Raz et al., 1988). In addition , AAEE at all graded

doses also significantly decreased LTB4 levels in inflamed paw tissues. LTB4 is found to be

highly elevated in arthritic joints and mediates the infiltration of leukocytes into the RA joint

(Mathis et al., 2007). Overproduction of NO is another vital in the pathogenesis of RA

(Holoshitz & Ling , 2007). The production of NO is regulated by IL-1� and TNF-�. Inhibitory

effects of AAEE on production of NO could be due to inhibitory effects on IL-1� and TNF-�

production.

To further delineate the possible mechanism AAEE was examined for effects on Th1-Th17

cytokines (IL-2, IFN-�, TNF-�, IL-17,IL-6 and IL-12) which are predominantly involved in RA

pathogenesis (Othoro et al., 1999). IL-2 produced by CD4+ T-helper cells stimulates the

synthesis of IFN-� in T cells as well as induces the secretion of pro-inflammatory cytokines such

as TNF-� by activated macrophages (McInnes and Schett, 2007). It therefore could be speculated

that the inhibition of IL-2 by AAEE is possibly responsible for reducing expression of IFN-� and

TNF-� .The inhibition of TNF-� by AAEE strongly signals anti-inflammatory effect of AAEE.

TNF-� is also reported to regulate IL-1� expression (Brennan and McInnes, 2008) which in turn

is critically important for the induction of prostanoid and MMP production (Arend and Dayer,

1995). The strong inhibitory effects of AAEE on TNF-� production therefore explains the

inhibitory effects observed on IL-1�, PGE2 and NO production .

IL-12 is the main stimulator of IFN-� production and levels of IL-12 have been elevated in

RA patients (Kim et al., 2000). IFN-� itself has been found to be critical in priming Th1 cell

development. When produced IFN-� also induces IL-12 p40 production by macrophages (Ma et

al., 1996), thus establishing an autocrine loop which the amplifies the Th1 immunity. We found

that on treatment with AAEE there was highly significant inhibition of IL-12 cytokine in dose

dependant manner which in turn possibly decreases the levels of IFN� and mediates the

significant suppression of arthritis by AAEE.

It has been reported that Th17 cells drive the chronic pathogenesis of rheumatoid arthritis

(Gaffen et al.,2004; Fouser et al., 2008). IL-17 produced by Th17 cells causes upregulation of

proinflammatory cytokines, such as IL-6, IFN-�, and TNF-� by fibroblasts, and monocytes

(Tokuda et al., 2004). In addition, IL-17 also increases the expression of chemotactic factors

inducing recruitment of neutrophils and T cells which produce IL-6, IL-1 and TNF-� (Van

Hamburg ., 2011). In addition , overproduction of IL-6 is found to be critical in the pathology of

rheumatoid arthritis (Kimuru ., 2010).IL-6 has been reported to regulate the balance between

IL-17 producing Th17 cells and T-regulatory cells .It induces the development of Th17 cells

from naïve T cells and inhibits the differentiation of T-reg cells. Blockade of IL-6 inhibits IL-17

inflammatory Th17 responses and suppresses autoimmune arthritis (Fujimoto ., 2008) and

therefore targeting IL-6 has emerged as potentially an effective approach in the treatment of

arthritis. These results are further supported by our data where AAEE significantly

downregulated the IL-17 and IL-6 levels in arthritic rats .

Th17 cells stimulate the production of matrix metalloproteinases (MMPs) and pro-

inflammtory cytokines especially in the early course of arthritis . IL-17 is reported to induce the

expression of MMP-3 and MMP-9 from multiple target cells, including epithelial and

endothelial cells, fibroblasts, neutrophils, and osteoblasts�and these enzymes degrade non-

collagen matrix components of the joints (Burrage et al., 2006; Pappu et al., 2012) .MMP-3 (

Stromyelysin ) is found to be significantly elevated in rheumatoid arthritis and is considered as

critical target for joint damage . MMP-3 further activates other MMPs including MMP-9 .

MMP-9 actively participates in joint destruction of RA through the expression of neutrophils and

monocytes-macrophages (Seki et al., 1997). The significant inhibition of MMP-3 and MMP-9 by

AAEE in all treated groups could be traced to suppression of IL-17 induced inflammatory

responses by AAEE.

AAEE not only inhibited the Th1/Th17 cytokines but also elevated the natural capacity of

the immune system to keep the Th1 cytokines under control by increasing IL-4 and IL-10.

While Th1 cytokines are responsible for initiation of disease in RA, activation of Th2 cytokines

correlates with disease regression (Agarwal and Malaviya, 2005). Our findings demonstrated that

a dosed dependant increase of IL-4 and IL-10 by AAEE . IL-4 and IL-10 inhibit the synthesis of

IL-1�, TNF-�, IL-6, IL-8 and IL-12 by macrophages and monocytes and suppress the

progression of arthritis in animal models (Katsikis et al.,1994). Also IL-10 and IFN-� are

reciprocally regulated., IL-10 possibly inhibiting IFN-� production and IFN-� regulating IL-10

(Chomart et al.,1993). Induction of the high levels of IL-4 and IL-10 by AAEE could possibly be

involved in the downregulation of Th1 cytokines. Therefore it could be suggested therefore that

A.accuminata exhibits its potent anti-arthritic activity associated with selective T cell modulating

properties causing significant inhibition of pro-inflammatory (Th1-Th17) cytokines and also a

corresponding increase in anti-inflammatory (Th2) cytokines.

The diverse mode of anti-arthritic activity of A.accuminata maybe attributed to the additive

effects of the bioactive constituents possibly present in it and also due to the high polyphenolic

and flavonoid content present in the extract . AAEE was demonstrated to have significant high

phenolic , total flavonoid and flavanol content. Many of the naturally occurring compounds such

as polyphenolics, diterpenoids, triterpenoids are reported to ameliorate chronic inflammatory

responses. Experimental data has revealed significant correlation between the high content of

phenolic compounds and the pharmacological activities of natural products (Spada et al.,2008). .

Flavonoids have anti-inflammatory effects in both proliferative and exudative phases of

inflammation such as inhibition of enzymes., lipoxygenase, cycloxygenase, etc.

In safety studies, oral dose of A.accuminata as high as 2,500 mg/kg did not cause any sign of

mortality or any observable negative symptoms in the general behavior of mice over a period of

2 weeks, indicating its apparent safety. Repeated once daily dosing up to 500 mg/kg b.w. in a

poly-arthritis test also did not cause any deviation from normal general behavior when compared

to the control group, or induce mortality in rats up to 21 days. Taken together with long standing

usage as folk medicine and no observable lethality, the herb therefore could be considered as a

safe option to explore for its therapeutic uses against arthritis.

5. Conclusion

Atropa accuminata was found to have potent anti-arthritic potential, the mode of action

of which is related modulation of T-helper cytokines causing suppression of nitric oxide,

prostaglandin and leukotriene production and subsequent inhibition of chronic inflammation.

This report is the first of its kind concerning the pharmacological properties of these plants

under study.

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LEGENDS TO FIGURES Figure 1: Effect of graded doses of AAEE on arthritis as assessed with squeaking scores

(ankle flexion score) in normal and treated rats over 21 days.

AAEE and ibuprofen were dissolved in 1% guma acacia and were given orally one day (Day 0)

before the CFA-adjuvant injection and then once daily for 21 days .Data is represented as dorsal

flexion score., the Mean ± SEM (n=6) . A value of 0 represents no indication of pain .

Statistical significance: *P < 0.05, **P < 0.01, *** P < 0.001, versus with respect to arthritic

control (AC) group on day 21st (One way repeated measures ANNOVA followed by Bonferroni

Multiple comparison test).

Figure 2: Effect of graded doses of AAEE on arthritis as assessed with arthritic scores.

AAEE and ibuprofen were dissolved in 1% guma acacia and were given orally one day (Day 0)

before the CFA-adjuvant injection and then once daily for 21 days . Data is represented as

arthritic score., the Mean ± SEM (n=6) .A value of 0 represents no indication no arthritic

disease. Statistical significance: *P < 0.05, **P < 0.01, *** P < 0.001, versus with respect to

arthritic control (AC) group on day 21st (One way repeated measures ANNOVA followed by

Bonferroni Multiple comparison test).

Figure 3 : Effect of graded doses of AAEE on PGE2 and LTB4 production expressed in

supernatant from paw homogenates. AAEE and ibuprofen were given orally one day (Day 0)

before the CFA-adjuvant injection and then once daily for 21 days . Data represent Mean ± SEM

{pg/ml} (n=6) .Statistical significance: *P < 0.05, **P < 0.01, *** P < 0.001, versus with

respect to arthritic control (AC) group on day 21st (One way repeated measures ANNOVA

followed by Bonferroni Multiple comparison test).

Figure 4 : Effect of graded doses of AAEE on (a) NO and (b) IL-1� production expressed

in supernatant from paw homogenates . AAEE and ibuprofen were given orally one day (Day

0) before the CFA-adjuvant injection and then once daily for 21 days . Data represent the Mean ±

SEM {pg/ml} (n=6) .Statistical significance: *P < 0.05, **P < 0.01, *** P < 0.001, versus with

respect to arthritic control (AC) group on day 21st (One way repeated measures ANNOVA

followed by Bonferroni Multiple comparison test).

Figure 5: Effect of graded doses of AAEE on production of Th1 cytokines IL-2 (a) ,IFN�

(b), TNF-� (c) and IL-12 (d) in supernatant from paw homogenates. AAEE and ibuprofen

were given orally one day (Day 0) before the CFA-adjuvant injection and then once daily for 21

days . Data represent the Mean ± SEM {pg/ml} (n=6) .Statistical significance: *P < 0.05, **P <

0.01, *** P < 0.001, versus with respect to arthritic control (AC) group on day 21st (One way

repeated measures ANNOVA followed by Bonferroni Multiple comparison test).

Figure 6: Effect of graded doses of AAEE on production of Th17 cytokines ; IL-17 (a)

and IL-6 (b) in supernatant from paw homogenate. AAEE and ibuprofen were given

orally one day (Day 0) before the CFA-adjuvant injection and then once daily for 21 days . Data

represent the Mean ± SEM {pg/ml} (n=6) ..Statistical significance: *P < 0.05, **P < 0.01, ***

P < 0.001, versus with respect to arthritic control (AC) group on day 21st (One way repeated

measures ANNOVA followed by Bonferroni Multiple comparison test).

Figure 7: Effect of graded doses of AAEE on production of MMPs ; MMP-3 (a) and

MMP-9 (b) in supernatant from paw homogenate. AAEE and ibuprofen were given orally

one day (Day 0) before the CFA-adjuvant injection and then once daily for 21 days . Data

represent the Mean ± SEM {pg/ml} (n=6) ..Statistical significance: *P < 0.05, **P < 0.01, ***

P < 0.001, versus with respect to arthritic control (AC) group on day 21st (One way repeated

measures ANNOVA followed by Bonferroni Multiple comparison test).

Figure 8: Effect of graded doses of AAEE on production of Th2 cytokines ; IL-4 (a) and

IL-10 (b) in supernatant from paw homogenate. AAEE and ibuprofen were given orally one

day (Day 0) before the CFA-adjuvant injection and then once daily for 21 days . Data represent

the Mean ± SEM {pg/ml} (n=6) ..Statistical significance: *P < 0.05, **P < 0.01, *** P <

0.001, versus with respect to arthritic control (AC) group on day 21st (One way repeated

measures ANNOVA followed by Bonferroni Multiple comparison test).

Table 1: Effect of graded doses of AAEE on C-reactive protein and Rheumatoid factor in

adjuvant induced arthritic and treated animals. AAEE and ibuprofen were given orally one

day (Day 0) before the CFA-adjuvant injection and then once daily for 21 days . Data represent

the Mean ± SEM (n=6) ..Statistical significance: *P < 0.05, **P < 0.01, *** P < 0.001, versus

with respect to arthritic control (AC) group on day 21st (One way repeated measures ANNOVA

followed by Bonferroni Multiple comparison test).

���Table 1 Effect of graded doses of AAEE on C-reactive protein and Rheumatoid factor in adjuvant induced arthritic and treated animals. Concentration

(mg/kg) CRP

(�106/mm

3)

RF�(�103/mm

3)

Normal�Control – 1.6�0.6 18�0.9 Arthritic�Control – 9.2�1.2 76�6.7 Ibuprofen 50 2.2�0.8*** 26�3.3*** AAEE 125 6.0�0.8 50�4.2** AAEE 250 4.8�0.4* 40�3.2*** AAEE 500 2.6�0.4*** 28�4.0*** AAEE and ibuprofen were given orally one day (Day 0) before the CFA-adjuvant injection and then once daily for 21 days. Data represent the Mean±SEM (n=6). *P<0.05 Statistical significance: versus with respect to arthritic control (AC) group on day 21st (One way repeated measures ANNOVA followed by Bonferroni Multiple comparison test). **P<0.01 Statistical significance: versus with respect to arthritic control (AC) group on day 21st (One way repeated measures ANNOVA followed by Bonferroni Multiple comparison test). ***P<0.001 Statistical significance: versus with respect to arthritic control (AC) group on day 21st (One way repeated measures ANNOVA followed by Bonferroni Multiple comparison test). ���

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