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Augmentation and proliferation of T lymphocytes and Th-1 cytokines by Withania somnifera in stressed mice
International Immunopharmacology 6 (2006) 1394 – 1403 www.elsevier.com/locate/intimp
Augmentation and proliferation of T lymphocytes and Th-1 cytokines by Withania somnifera in stressed mice Beenish Khan a,1 , Sheikh Fayaz Ahmad a,1 , Sarang Bani a,⁎, Anpurna Kaul a,1 , K.A. Suri b,1 , N.K. Satti b,1 , M. Athar c , G.N. Qazi a a
c
Cell Biology Laboratory, Department of Pharmacology, Regional Research Laboratory, Jammu Tawi 180001, India b Natural Product Chemistry Division, Regional Research Laboratory, Jammu Tawi 180001, India Department of Medical Elementology and Toxicology; Faculty of Science Jamia Hamdard (Hamdard University) Hamdard Nagar, New Delhi 110062, India Received 18 October 2005; received in revised form 8 January 2006; accepted 3 April 2006
Abstract Stress has been associated with reports of both greater severity and prolongation of diseases in patients with the infectious origin as well as other immune-mediated diseases. Withania somnifera, an Indian medicinal plant used widely in the treatment of many clinical conditions in India, was investigated for its anti-stress properties using BALB/c mice subjected to chronic stress. The study aimed to investigate chronic stress-induced alterations on Th1 lymphocyte subset distribution and corresponding cytokine secretion patterns. Oral administration of chemically standardized and identified aqueous fraction of W. somnifera root (WS) at the graded doses of 25, 50, 100 and 200 mg/kg p.o. caused significant increase in the stress-induced depleted T-cell population and increased the expression of Th1 cytokines in chronically stressed mice. © 2006 Elsevier B.V. All rights reserved. Keywords: Chronic stress; Immunophenotyping; Intracellular cytokines; Flow cytometry; Polymorphonuclear leucocytes
1. Introduction Stress is defined as a constellation of events that begins with a stimulus (stressor), which precipitates a
⁎ Corresponding author. Tel.: +91 191 2549051; fax: +91 191 2573765; 2548607; 2543829. E-mail addresses: [email protected] (B. Khan), [email protected] (S.F. Ahmad), [email protected] (S. Bani), [email protected] (A. Kaul), [email protected] (K.A. Suri), [email protected] (N.K. Satti), [email protected] (M. Athar), [email protected] (G.N. Qazi). 1 Tel.: +91 191 2549051. 1567-5769/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2006.04.001
reaction in the brain (stress perception) that subsequently results in the activation of certain physiologic systems in the body (stress response). Stress generally refers to the condition where coping with various actual or perceived stimuli alters the homeostatic state of the organism, including behavioral, endocrine and immunological changes. The immune system is particularly sensitive to stress [1,3,26] and the suppression, thus induced, depresses the immune functioning and increases susceptibility to diseases [5,6,10,13]. Numerous studies have shown that stress can be immunosuppressive in nature and, hence, damaging to health, resulting in diseased/clinical conditions. Stress-induced immunologic alterations
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have been considered as the major cause of increased risk of immune-related diseases such as cancer, autoimmune disorders and infections. Acute stress generally has positive effects, while chronic stress typically provokes immunosuppression [8,9,14,16,23]. Exposure to chronic stress suppresses the T-cells and the molecular mediators that include cytokines. Animal models provide support to link between stress and infectious diseases, e.g., stress such as crowding prior to and following tuberculosis infection affects the outcome of the infection [22]. It has been shown that social disruption in mice causes reactivation of latent herpes simplex virus [18]. Herbal drugs in recent years have gained importance because of their efficacy and cost-effectiveness. The corroborative and tonic plants generally known as the Rasayana drugs in Ayurveda are known to prevent aging, increase longevity and offer resistance to disease by augmenting the immune system [21,22]. Ayurveda remains one of the most ancient and yet living traditions practised widely in India, Sri Lanka and other countries having a sound philosophical and experimental basis. A considerable research on pharmacognosy, chemistry, pharmacology and clinical therapeutics has been carried out and the Ayurvedic database has detailed descriptions of over 700 medicinal plants [19,24]. Withania somnifera (Hindi–Ashwagandha, English– Indian ginseng/winter cherry) is mentioned in the Indian Herbal Pharmacopoeia [12], is widely used as a remedy for several ailments and is an ingredient in many formulations prescribed for a variety of musculoskeletal conditions (e.g., arthritis, rheumatism). It is also used as a general adaptogen to increase energy, improve overall health and longevity, and prevent diseases in athletes, the elderly and pregnant women [4,7]. The use of W. somnifera as a general tonic to increase energy and prevent disease may be related to its immunostatic effect. It has been reported to have anti-inflammatory, antitumor, antioxidant, immunomodulatory and rejuvenating properties. Can chronic stress and its potential impact on the immune system be reduced by this Indian ginseng? Its chemistry has been extensively studied and over 35 chemical constituents have been identified, extracted, and isolated. The biologically active chemical constituents include steroidal lactones (withanolides, withaferins), saponins and withanolides with a glucose at Carbon 27 (sitoindoside IX and X) and alkaloids, isopelletierine, and anaferine [15]. In the present study we investigated relationships between stress-associated T cell depletion and the stress busting effect of the aqueous fraction of W. somnifera
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root (WS). We found WS to have significant anti-stress activity in experimental animals with the potential of modulation of the populations of Th1 cells and the corresponding Th1 cytokines. Thus, a detailed study of WS was taken up to establish its immunostatic effect on the altered immune system. 2. Materials and methods 2.1. Test material W. somnifera roots were obtained from the botanical garden of Regional Research Laboratory (RRL) Jammu, a premier research institute in India, correctly identified and authenticated by routine pharmacognostic procedures. A voucher sample is retained and deposited at RRL Herbarium, Jammu, J&K state, India. The material was ensured to be free from pathogens, aflatoxins, pesticidal residues and heavy metals to meet WHO guidelines of purity and safety [25]. The test material was prepared as fresh suspensions using 1% sterile gum acacia. 2.2. Chromatography Withanolides and glycowithanolide were separated and quantified by using Shimadzu HPLC system consisting of Pump LC-10ATVP, an automatic sampling unit (Autosampler), SIL-10ADVP, a Column oven CTO-10ASVP, a diode array detector SPD-M10AVP and system controller SCL10AVP version 5.40. Shimadzu.Class VP software version 6.10 was used for data analysis and data processing .The samples were analysed at 30 °C on a Phenomenex C18 column (5 μm, 250 × 4.0 mm I.D.) by UV detection at 237 nm. The mobile phase consisted of methanol/water (60:40) and was delivered at a flow rate of 0.7 ml/min. The withanolides and glycowithanolides were quantified by using the external standard method (Fig. 1A–F). 2.3. Isolation of markers Dried and powdered roots of W. somnifera were percolated four times with ethanol/water (1:1) at room temperature and these extracts were combined, centrifuged and finally concentrated to 1/8 of the original volume under reduced pressure at 50 ± 5 °C. The concentrated solution was extracted thoroughly with chloroform followed by n-BuOH. The chloroform extract was distilled under reduced pressure to give a residue. Withanoliodes WS-1, WS-2, WS-3, WSCO, and WSC were obtained in pure form from the chloroform extract and WSG-3 from the n-BuOH extract by using different separation techniques such as column chromatography (CC), preparative TLC and centrifugal adsorption chromatography (Chromatotron). Structures of all the compounds (Figs. 1–5) were finally purified by crystallisations and identified with the help of 1H, 13C NMR [20].
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Fig. 1. Composition of the extract—Marker compounds. (A) Withanoside-IV (WSG-3); (B) withaferin-A (WS-3); (C) withanolide-A (WS-1); (D) withanone (WS-2); (E) withastramonolide (WSC); (F) 12-deoxywithastramonolide (WSCO).
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Fig. 2. (A) Flow cytometry scatter plots for T-cell populations (CD4+ T-helper cells subset and CD8+ cytolytic/cytotoxic T-cells). The plots represent events for one representative mouse from each group of naive-control, RST-control and WS-treated mouse. The gating and the quadrants are set according to the standard procedures of the BD-LSR flow cytometer. (B) Flow cytometry scatter plots for T-cell populations (CD3+ T-cells surface markers which represents the total population of the T-cells). The plots represent events for one representative mouse from each group of naive-control, RST-control and WS-treated mouse. The gating and the quadrants are set according to the standard procedures of the BD-LSR flow cytometer. (C) The histograms represent the comparative analysis of the T cell subsets (CD4+, CD8+ and CD3+ T-cells) under the normal (naive-control), restrained stress conditions (RST-control) and the WS-treated mice subjected to chronic stress. Each column is the mean±S.E. of 6 mice per group. Asterisks indicate a significant difference between the stressed mice (RST-control) and the WS-treated groups.
2.4. Sample preparation The accurately weighed quantity of the dried aqueous extract was dissolved in known volumes of HPLC-grade methanol/water (1:1). The sample was filtered through a Millipore micro-filter (0.45 μm) and then injected into the HPLC system. Stock solutions of the pure reference
compounds were prepared in HPLC-grade methanol and stored in refrigerator at 4 °C. From the stock solutions working solutions in the concentration range of 17.5 μg/ml to 35.0 μg/ ml for each reference compound were prepared by dilution with HPLC-grade methanol. These working solutions of all the reference compounds were mixed together in equal volumes for further analysis (Table 1).
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2.5. Quantification The compounds exhibited a linear response in the concentration range of 17.5 μg/ml to 35.0 μg/ml and the calibration curves were prepared by using the multipoint calibration curve method. Working solutions after mixing were injected in different concentrations. Excellent calibration curves were obtained for WS-3 (r2 = 0.999845), WSCO (r2 = 0.9997759),
WS-1 (r 2 = 0.999783), WS-2 (r 2 = 0.99767), WSC (r2 = 0.999849) and WSG-3 (r2 = 0.99897). Calibration curves were determined on the basis of six levels of concentration of each standard in the mixture. Quantification of the aqueous extract was developed by using these calibration curves. All the solvents were of HPLC grade. HPLC-grade methanol (Rankem make) was purchased from Ranbaxy Chemicals Ltd. (Mohali, Punjab, India) and HPLC-grade
Fig. 3. (A) Flow cytometry histogram representation for Th-1 cytokine IL-2, the growth factor for the CD4+ T cell subsets. The histogram represents the counts for one representative mouse from each group of naive-control, RST-control, and WS-treated mouse. The histograms are acquired according to the standard procedures of the BD-LSR flow cytometer. (B) Flow cytometry histogram representation for IFN-γ, the signature cytokine of the Th1 response. The histogram represents the counts for one representative mouse from each group of naive-control, RST-control and WS-treated mouse. The histograms are acquired according to the standard procedures of the BD-LSR flow cytometer. (C) The histograms represent the comparative analysis of the two Th1 cytokines—IL-2 and IFN-γ under the normal (naive-control), the restrained stress conditions (RST-control.) and the WS-treated restrained mice. Each column is the mean ± S.E. of 6 mice per group. Asterisks indicate a significant difference between the stressed mice (RST-control) and the restrained WS-treated groups.
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water was prepared from Milli-Q water purification system. All the five withanolides were isolated from Water/Ethanol (50:50), extract, purity ≤98% (HPLC). 2.6. Safety studies The acute oral toxicity studies were carried out after approval from the Institutional Animal Ethics Committee following OECD guidelines No. 423 [17]. The test material was found to be safe up to 2000 mg/kg with no adverse effects on general behavior in treated BALB/c mice. 2.7. Methods 2.7.1. Animals and stress exposure Male BALB/c mice 10–12 weeks old and weighing 18– 22 g obtained from the animal house of the Regional
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Research Laboratory, Jammu, in groups of six were employed for the study. These were maintained at a room temperature of 22 ± 2 °C with 12 h light/dark cycle and free access to pellet food and water. According to ethical regulations on animal research, all animals used in experimental work received humane care. Mice were restrained in well-ventilated horizontal 50-ml conical polypropylene tubes for 12 h during the dark cycle (2000–0800 h) during experimental periods of 14 days, one group served as the vehicle control to which drug was not administered but was subjected to restrained stress (RSTcontrol), treated groups were subjected to restrain stress as well as drug administration for 14 consecutive days. One group of mice was taken as a non-treated naive control group without any restrained conditions and was give full access to food and water (naive-control). The animals were not physically compressed and, therefore, did not experience pain. Restrained
Fig. 4. (A) Flow cytometry scatter plots for polymorphonuclear cells (neutrophils). The plots represent events for one representative mouse from each group of naive-control, RST-control and WS-treated mouse. The gating and the quadrants are set according to the standard procedures of the BD-LSR flow cytometer. (B) The histograms represent the effect of ‘WS’ on polymorphonuclear cells in (naive-control), chronic stress-induced mice (RSTcontrol.) and the drug-treated mice. Each column is the mean ± S.E. of 10 mice per group. Asterisks indicate a significant difference between the stressed mice (RST-control) and the WS-treated groups.
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Fig. 5. (A) The histograms represent the effect of ‘WS’ on cortisol levels in naive-control, chronic stress-induced mice (RST-control) and the drugtreated mice. Each column is the mean ± S.E. of serum samples assayed in triplicates. Asterisks indicate a significant difference between the stressed mice (RST-control) and the WS-treated groups.
stress procedure is believed to be largely psychological in nature due to the feeling of confinement by the animal [11]. 2.8. Drug administration Test drug was prepared in distilled water and was administered orally daily once a day for the duration of experiment at 25, 50, 100 and 200 mg /kg p.o. dose. The nonstressed naive-control group (naive-control) and stress control group (RST-control) was given vehicle daily. 2.9. Blood collection On day 14, 1 h after subjecting the mice to restrained stress, the blood was collected from the retro-orbital plexus of the animals from all the groups, i.e., naive-control, RST-control and drug treated. Blood was collected in tubes with EDTA as an anticoagulant. 2.10. Lymphocyte immunophenotyping Immunophenotyping focuses on lymphocyte populations involved in acquired immunity and specific molecules present on the cell surface that defines characteristics of lymphocytes such as state of activation or functional capabilities. Lymphocyte subsets were measured by immunofluorescent antibody staining of whole blood and subsequently analyzed using twocolor flow cytometry (Becton & Dickinson, UK). Murine monoclonal antibodies conjugated to a fluorochrome and directed against receptors CD3, CD4 and CD8 were used for the study. CD3 surface receptor is present on all the T lymphocytes present in the whole blood FITC-labeled antimouse CD3 monoclonal antibody that reacts with the CD3 differentiation antigen expressed on MHC class I as well as
MHC class II T helper cells, was used for the determination of total T cell populations in the blood. FITC-labeled anti-mouse CD4 monoclonal antibody that reacts with the CD4 differentiation antigen expressed on MHC class II-restricted T helper cells and PE-labeled CD8 monoclonal antibody that reacts with CD8 differentiation antigen present on MHC class-I-restricted cytotoxic T lymphocyte cells were used to determine the percentage of CD4+ and CD8+ T-cells in all the groups of animals. These fluorochrome-labeled monoclonal antibodies were added directly to 100 μl of whole blood, which was then lysed using whole blood lysing reagent (BD Biosciences). Following the final centrifugation, samples were resuspended in phosphate-buffered saline (pH, 7.4) [2] and analyzed directly on the flow cytometer (LSR, BD Biosciences) using Cell Quest Pro Software (BD Biosciences). A fluorescence
Table 1 Body weights (g) in naïve, stressed (RST-control) and drug-treated mice Sample Group number
Dose Body weights (gm) (mg/ Day 0 Day 14 kg)
1 2 3 4 5 6
– – 25 50 100 200
Naive-control RST-control WS WS WS WS
16.42 ± 0.87 17.20 ± 0.97 18.92 ± 0.73 19.18 ± 0.56 17.05 ± 1.08 16.92 ± 0.98
25.71 ± 0.80 (9.29 g ↑) 21.00 ± 0.44 (3.8 g ↑) 24.05 ± 1.43 (5.13 g ↑) 24.81 ± 1.61 (5.63 g ↑) 23.57 ± 1.11 (6.52 g ↑) 22.10 ± 1.05 (5.18 g ↑)
Values shown are mean ± S.E. for six mice. Percent change against the naive-control is in parenthesis; ↑: increase. Stressing was by immobilization for 12 h (2000–0800 h) from Day 1 to Day 14 in consecutive days.
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trigger was set on the FITC (FLI) parameter to analyze CD3 + and CD4+ and PE (FL2) parameter to collect CD8+ events. 2.11. Intracellular cytokine estimation The blood was collected in heparinised tubes from retroorbital plexus as mentioned above. FITC-labeled anti-mouse CD4+ monoclonal antibody and phycoerythrin (PE)-labeled IL-2 monoclonal antibodies were used in one set and PElabeled CD8+ monoclonal antibody along with FITC-labeled IFN-γ monoclonal antibody was used in the other set of experimentation.
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control (RST-control) group, where CD3+ T lymphocytes decreased from 50% ( normal control value ) to 26% CD4+ T helper cell number was reduced from 23% (normal control value) to 13% and CD8+ T cells decreased from 13% (normal control value) to 8% (p < 0.001). WS, when administered orally at 25–200 mg/kg doses, showed a dose-related increase in lymphocyte number and the most significant increase was observed at an oral dose of 100 mg/kg where CD3+ T cell count was 58% (p < 0.001), CD4+ T cells increased to 30%, and with respect to the stressed mice, the percentage of CD8+ T cells significantly increased from 8% to 22% (Fig. 2A–C). 3.2. Intracellular cytokine estimation
2.12. Body and organ weights: thymus, spleen, axillary lymph node After the last stress session, the body weights of the animals were weighed, following which the animals were sacrificed by ether inhalation; the thymus, spleen, and axillary lymph nodes were removed and weighed. 2.13. Analysis of blood leucocytes (polymorphonuclear leucocytes) Polymorphonuclear cells were separated from whole blood by Histopaque gradient method (Histopaque-Sigma procedures No. 1077). 200 μl of the sample was run. The analysis of cell count and event percentages was carried out in a BD LSR Flow cytometer after the removal of any traces of the red blood cells by FACS Lysing Solution. The gating of the required cell population was carried out according to the standard procedures of the flow cytometer. 2.14. Cortisol assay Cortisol levels were assessed by competitive immunoenzymatic method (Adaltis Italia EIAgen CORTISOL) for determining the amount of cortisol concentration in the serum. Blood for serum was collected from the retro-orbital plexus of the animals from all the groups, i.e., naive-control, RST-control and drug-treated. All samples were assayed in triplicates at a wavelength of 450 nm. 2.14.1. Statistical analysis Fluorescence compensation, data analysis and data presentation was performed using Cell Quest Pro software, and statistical evaluation (drug-induced data) was done by Student's t-test. 3. Results 3.1. Lymphocyte immunophenotyping The percentage of the CD3+, CD4+ and CD8+ T lymphocytes cells was significantly decreased in the restraint
IL-2 and IFN-γ were assayed, as the change in relative proportion of the lymphocyte subsets is proportional to the change in the cytokine expression patterns. It was found that both the IL-2 as well as IFN-γ cytokines counts decreased in stressed animals from 11% (naive-control) to 6.5% (RSTcontrol) for IL-2 and 7.8% (naive-control) to 2.55% (RSTcontrol) for IFN-γ, respectively, i.e., in both cases, about 50% decrease was observed. In the mice treated with WS, a significant change was observed as both Th1 cytokines IL-2 and IFN-γ increased from 6.5% (naive-control) and 2.55% (RST-control) to 18.6% and 8.1% in the WS-treated group (100 mg/kg p.o.), respectively (Fig. 3A–C). 3.3. Body and organ weights: thymus, spleen, axillary lymph node Chronic immobilization stress decreased the weights of the immune-specific organs. The thymus weight decreased from 56.70 mg to 38.80 mg (Table 2). Spleen and axillary lymph node weights decreased significantly in the stressed group. Spleen weight decreased from 84.50 mg in the group of control animals (naive-control) to 42.50 mg in the group of stressed animals (RST-control) and for lymph nodes the weight decreased from 40.60 mg (naive-control) to 17.10 mg (RSTcontrol) (P⁎⁎, respectively, Table 2). In the animals that were administered with the drug, the weight observed was 49.10 mg for thymus and 65.76 mg and 34.57 mg were observed for the spleen and axillary lymph nodes. Body-weight recovery was observed in animals treated with different dose levels of WS when compared to the stress control group (RST-control) (Table 1). 3.4. Estimation of polymorphonuclear leucocytes count The analysis of the polymorphonuclear cells showed that the cell number decreased significantly in the RST-control group, however, from 16 × 104 cells to 8.7 × 104 cells. However, in the animals that are treated with WS, a marked increase in the cell number of plymorphonuclear cells was observed which increased to 12 × 104 as compared to the RSTcontrol group (Fig. 4A and B).
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Table 2 Organ weights (mg) in unstressed, stressed and drug treated mice Organ Thymus Spleen Axillary lymph node
Naive-control
RST-control
Drug treated
70.70 ± 2.0 84.5 ± 3.2 40.60 ± 2.1
38.80 ± 2.2⁎⁎ 42.30 ± 3.4⁎⁎ 17.10 ± 3.2⁎⁎
49.10 ± 4.2⁎⁎ 65.76 ± 3.2⁎⁎ 34.57 ± 1.2⁎⁎
Values shown are means ± S.E. for six mice. Stressing was by immobilization for 12 h (2000–0800 h) for 14 consecutive days. Asterisks indicate a statistically significant decrease (⁎⁎P < 0.001) between the three groups.
3.5. Cortisol assay The analysis of the cortisol levels showed that the cortisol concentration increased in mice subjected to restrain stress (RST-control) which was observed to be 27% as compared to the naive-control group which was 17%. However, in the animals that are treated with WS, a decrease in the cortisol concentration was observed in which a maximum decrease was observed at a dose level of 200 mg/kg in which the cortisol concentration was 14% (p < 0.001), respectively, followed by a dose of 100 mg/kg, the cortisol concentration observed was 15% (Fig. 5A).
4. Discussion WS shows to ameliorate the chronic stress-induced decrease of the T lymphocyte population and prevent the decrease in the expression of the Th1 cytokines. Chronic stress-induced significant decrease of CD3+, CD4+ and CD8+ T cells (Fig. 2A–C). This relates to the decrease in the expression of Th1 cytokines IL-2 and IFN-γ. WS at graded doses exhibited recovery of CD3+, CD4+ and CD8+ T-cell counts with the most significant effect observed at the oral dose of 100 mg/kg. CD4 and CD8 are the T-cell surface markers for Th1/Th2 and Tc subsets of T lymphocytes, respectively, and CD4+ molecules are considered central to regulation of classical cell-mediated functions such as delayed-type hypersensitivity (DTH) response and B cell activation. Immune response is usually polarized to give predominantly either a Th1 or a Th2 response and fine-tuning of inhibitors, activators and regulatory signals ensures immunostasis. Th1 response primarily promotes cytolytic T cells (CTLs), which are important in responding to infections. Therefore, stress-induced decrease in the lymphocyte subsets and associated Th1 cytokines IL-2 and IFN-γ is possibly a factor that renders the immune system vulnerable to clinical conditions. WS primarily induced a Th1 response, involving the cytokines IL-2 and IFN-γ and showed a significant recovery and increase in the lymphocyte populations as compared to the restrained non-treated group of experimental
animals. WS showed dose-related potentiation of IL-2 secreted by stimulated helper T cells (CD4+) and cytotoxic T-cells (CD8+). IL-2 promotes proliferation and differentiation of additional CD4+ cells, B cells, and is a central regulator of immune response. It also stimulates the synthesis of IFN-γ. The increased expression of IL-2 in groups treated with WS possibly is responsible for increased expression of IFN-γ as well (Fig. 3A–C). The recovery in the population of polymorphonuclear leucocytes in the treated groups and recovery in the body weights and a lesser decrease in the organ weights of the animals treated with WS when compared to those in the RST-control group (Tables 1 and 2) show the stress busting potential of the test material. The results of the present study also showed a decrease in the cortisol concentration of the animals that were restrained and subsequently administered with WS. This study establishes unique immunoregulatory potential of the test material and suggests its use in T-cell function restoration where Th1 modulation is required as a reaction to chronic stress, especially in patients suffering from diseases that cause long-term stress that in turn induces suppression of the immune system. Taken together, our results suggest an impact of chronic stress on T-cell function and in turn alters T-cell response, and the immunoregulatory effect of aqueous fraction of the root of W. somnifera is effective in reversing this process. These findings may help to elucidate the physiological mechanisms through which stress plays a role in the etiology of many diseases. References [1] Ayala A, Herdon CD, Lehman DL, Ayala CA, Chaudry IH. Differential induction of apoptosis in lymphoid tissues during sepsis.Variation in onset, frequency, and the nature of the mediators. Blood 1996;87:4261–75. [2] Bani S, Kaul A, Khan B, Ahmad SF, Suri KA, Satti NK, et al. imunosuppressive properties of an ethyl acetate fraction from Euphorbia royleana. J Ethnopharmacol 2005;99:185–92. [3] Benschop R, Schedlowski MRFM. Catecholamine-induced leukocytosis: early observations, current research, and future directions. Brain Behav Immun 1996;10:77–91. [4] Bone K. Clinical applications of Ayurvedic and Chinese herbs. Monographs for the Western herbal practitioner. Australia: Phytotherapy Press; 1996. p. 137–41. [5] Borysenko M, Borysenko J. Stress, behavior, and immunity: animal models and mediating mechanisms. Gen Hosp Psychiatry 1982;4:59–67. [6] Cohen S, Tyrrell DAJ, Smith AP. Psychological stress and susceptibility to the common cold. N Engl J Med 1991;325:606–12. [7] Chatterjee A, Pakrashi SC. The Treatise on Indian Medicinal Plants 1995;4:208–212.
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Augmentation and proliferation of T lymphocytes and Th-1 cytokines by Withania somnifera in stressed mice Beenish Khan a,1 , Sheikh Fayaz Ahmad a,1 , Sarang Bani a,⁎, Anpurna Kaul a,1 , K.A. Suri b,1 , N.K. Satti b,1 , M. Athar c , G.N. Qazi a a
c
Cell Biology Laboratory, Department of Pharmacology, Regional Research Laboratory, Jammu Tawi 180001, India b Natural Product Chemistry Division, Regional Research Laboratory, Jammu Tawi 180001, India Department of Medical Elementology and Toxicology; Faculty of Science Jamia Hamdard (Hamdard University) Hamdard Nagar, New Delhi 110062, India Received 18 October 2005; received in revised form 8 January 2006; accepted 3 April 2006
Abstract Stress has been associated with reports of both greater severity and prolongation of diseases in patients with the infectious origin as well as other immune-mediated diseases. Withania somnifera, an Indian medicinal plant used widely in the treatment of many clinical conditions in India, was investigated for its anti-stress properties using BALB/c mice subjected to chronic stress. The study aimed to investigate chronic stress-induced alterations on Th1 lymphocyte subset distribution and corresponding cytokine secretion patterns. Oral administration of chemically standardized and identified aqueous fraction of W. somnifera root (WS) at the graded doses of 25, 50, 100 and 200 mg/kg p.o. caused significant increase in the stress-induced depleted T-cell population and increased the expression of Th1 cytokines in chronically stressed mice. © 2006 Elsevier B.V. All rights reserved. Keywords: Chronic stress; Immunophenotyping; Intracellular cytokines; Flow cytometry; Polymorphonuclear leucocytes
1. Introduction Stress is defined as a constellation of events that begins with a stimulus (stressor), which precipitates a
⁎ Corresponding author. Tel.: +91 191 2549051; fax: +91 191 2573765; 2548607; 2543829. E-mail addresses: [email protected] (B. Khan), [email protected] (S.F. Ahmad), [email protected] (S. Bani), [email protected] (A. Kaul), [email protected] (K.A. Suri), [email protected] (N.K. Satti), [email protected] (M. Athar), [email protected] (G.N. Qazi). 1 Tel.: +91 191 2549051. 1567-5769/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2006.04.001
reaction in the brain (stress perception) that subsequently results in the activation of certain physiologic systems in the body (stress response). Stress generally refers to the condition where coping with various actual or perceived stimuli alters the homeostatic state of the organism, including behavioral, endocrine and immunological changes. The immune system is particularly sensitive to stress [1,3,26] and the suppression, thus induced, depresses the immune functioning and increases susceptibility to diseases [5,6,10,13]. Numerous studies have shown that stress can be immunosuppressive in nature and, hence, damaging to health, resulting in diseased/clinical conditions. Stress-induced immunologic alterations
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have been considered as the major cause of increased risk of immune-related diseases such as cancer, autoimmune disorders and infections. Acute stress generally has positive effects, while chronic stress typically provokes immunosuppression [8,9,14,16,23]. Exposure to chronic stress suppresses the T-cells and the molecular mediators that include cytokines. Animal models provide support to link between stress and infectious diseases, e.g., stress such as crowding prior to and following tuberculosis infection affects the outcome of the infection [22]. It has been shown that social disruption in mice causes reactivation of latent herpes simplex virus [18]. Herbal drugs in recent years have gained importance because of their efficacy and cost-effectiveness. The corroborative and tonic plants generally known as the Rasayana drugs in Ayurveda are known to prevent aging, increase longevity and offer resistance to disease by augmenting the immune system [21,22]. Ayurveda remains one of the most ancient and yet living traditions practised widely in India, Sri Lanka and other countries having a sound philosophical and experimental basis. A considerable research on pharmacognosy, chemistry, pharmacology and clinical therapeutics has been carried out and the Ayurvedic database has detailed descriptions of over 700 medicinal plants [19,24]. Withania somnifera (Hindi–Ashwagandha, English– Indian ginseng/winter cherry) is mentioned in the Indian Herbal Pharmacopoeia [12], is widely used as a remedy for several ailments and is an ingredient in many formulations prescribed for a variety of musculoskeletal conditions (e.g., arthritis, rheumatism). It is also used as a general adaptogen to increase energy, improve overall health and longevity, and prevent diseases in athletes, the elderly and pregnant women [4,7]. The use of W. somnifera as a general tonic to increase energy and prevent disease may be related to its immunostatic effect. It has been reported to have anti-inflammatory, antitumor, antioxidant, immunomodulatory and rejuvenating properties. Can chronic stress and its potential impact on the immune system be reduced by this Indian ginseng? Its chemistry has been extensively studied and over 35 chemical constituents have been identified, extracted, and isolated. The biologically active chemical constituents include steroidal lactones (withanolides, withaferins), saponins and withanolides with a glucose at Carbon 27 (sitoindoside IX and X) and alkaloids, isopelletierine, and anaferine [15]. In the present study we investigated relationships between stress-associated T cell depletion and the stress busting effect of the aqueous fraction of W. somnifera
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root (WS). We found WS to have significant anti-stress activity in experimental animals with the potential of modulation of the populations of Th1 cells and the corresponding Th1 cytokines. Thus, a detailed study of WS was taken up to establish its immunostatic effect on the altered immune system. 2. Materials and methods 2.1. Test material W. somnifera roots were obtained from the botanical garden of Regional Research Laboratory (RRL) Jammu, a premier research institute in India, correctly identified and authenticated by routine pharmacognostic procedures. A voucher sample is retained and deposited at RRL Herbarium, Jammu, J&K state, India. The material was ensured to be free from pathogens, aflatoxins, pesticidal residues and heavy metals to meet WHO guidelines of purity and safety [25]. The test material was prepared as fresh suspensions using 1% sterile gum acacia. 2.2. Chromatography Withanolides and glycowithanolide were separated and quantified by using Shimadzu HPLC system consisting of Pump LC-10ATVP, an automatic sampling unit (Autosampler), SIL-10ADVP, a Column oven CTO-10ASVP, a diode array detector SPD-M10AVP and system controller SCL10AVP version 5.40. Shimadzu.Class VP software version 6.10 was used for data analysis and data processing .The samples were analysed at 30 °C on a Phenomenex C18 column (5 μm, 250 × 4.0 mm I.D.) by UV detection at 237 nm. The mobile phase consisted of methanol/water (60:40) and was delivered at a flow rate of 0.7 ml/min. The withanolides and glycowithanolides were quantified by using the external standard method (Fig. 1A–F). 2.3. Isolation of markers Dried and powdered roots of W. somnifera were percolated four times with ethanol/water (1:1) at room temperature and these extracts were combined, centrifuged and finally concentrated to 1/8 of the original volume under reduced pressure at 50 ± 5 °C. The concentrated solution was extracted thoroughly with chloroform followed by n-BuOH. The chloroform extract was distilled under reduced pressure to give a residue. Withanoliodes WS-1, WS-2, WS-3, WSCO, and WSC were obtained in pure form from the chloroform extract and WSG-3 from the n-BuOH extract by using different separation techniques such as column chromatography (CC), preparative TLC and centrifugal adsorption chromatography (Chromatotron). Structures of all the compounds (Figs. 1–5) were finally purified by crystallisations and identified with the help of 1H, 13C NMR [20].
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Fig. 1. Composition of the extract—Marker compounds. (A) Withanoside-IV (WSG-3); (B) withaferin-A (WS-3); (C) withanolide-A (WS-1); (D) withanone (WS-2); (E) withastramonolide (WSC); (F) 12-deoxywithastramonolide (WSCO).
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Fig. 2. (A) Flow cytometry scatter plots for T-cell populations (CD4+ T-helper cells subset and CD8+ cytolytic/cytotoxic T-cells). The plots represent events for one representative mouse from each group of naive-control, RST-control and WS-treated mouse. The gating and the quadrants are set according to the standard procedures of the BD-LSR flow cytometer. (B) Flow cytometry scatter plots for T-cell populations (CD3+ T-cells surface markers which represents the total population of the T-cells). The plots represent events for one representative mouse from each group of naive-control, RST-control and WS-treated mouse. The gating and the quadrants are set according to the standard procedures of the BD-LSR flow cytometer. (C) The histograms represent the comparative analysis of the T cell subsets (CD4+, CD8+ and CD3+ T-cells) under the normal (naive-control), restrained stress conditions (RST-control) and the WS-treated mice subjected to chronic stress. Each column is the mean±S.E. of 6 mice per group. Asterisks indicate a significant difference between the stressed mice (RST-control) and the WS-treated groups.
2.4. Sample preparation The accurately weighed quantity of the dried aqueous extract was dissolved in known volumes of HPLC-grade methanol/water (1:1). The sample was filtered through a Millipore micro-filter (0.45 μm) and then injected into the HPLC system. Stock solutions of the pure reference
compounds were prepared in HPLC-grade methanol and stored in refrigerator at 4 °C. From the stock solutions working solutions in the concentration range of 17.5 μg/ml to 35.0 μg/ ml for each reference compound were prepared by dilution with HPLC-grade methanol. These working solutions of all the reference compounds were mixed together in equal volumes for further analysis (Table 1).
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2.5. Quantification The compounds exhibited a linear response in the concentration range of 17.5 μg/ml to 35.0 μg/ml and the calibration curves were prepared by using the multipoint calibration curve method. Working solutions after mixing were injected in different concentrations. Excellent calibration curves were obtained for WS-3 (r2 = 0.999845), WSCO (r2 = 0.9997759),
WS-1 (r 2 = 0.999783), WS-2 (r 2 = 0.99767), WSC (r2 = 0.999849) and WSG-3 (r2 = 0.99897). Calibration curves were determined on the basis of six levels of concentration of each standard in the mixture. Quantification of the aqueous extract was developed by using these calibration curves. All the solvents were of HPLC grade. HPLC-grade methanol (Rankem make) was purchased from Ranbaxy Chemicals Ltd. (Mohali, Punjab, India) and HPLC-grade
Fig. 3. (A) Flow cytometry histogram representation for Th-1 cytokine IL-2, the growth factor for the CD4+ T cell subsets. The histogram represents the counts for one representative mouse from each group of naive-control, RST-control, and WS-treated mouse. The histograms are acquired according to the standard procedures of the BD-LSR flow cytometer. (B) Flow cytometry histogram representation for IFN-γ, the signature cytokine of the Th1 response. The histogram represents the counts for one representative mouse from each group of naive-control, RST-control and WS-treated mouse. The histograms are acquired according to the standard procedures of the BD-LSR flow cytometer. (C) The histograms represent the comparative analysis of the two Th1 cytokines—IL-2 and IFN-γ under the normal (naive-control), the restrained stress conditions (RST-control.) and the WS-treated restrained mice. Each column is the mean ± S.E. of 6 mice per group. Asterisks indicate a significant difference between the stressed mice (RST-control) and the restrained WS-treated groups.
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water was prepared from Milli-Q water purification system. All the five withanolides were isolated from Water/Ethanol (50:50), extract, purity ≤98% (HPLC). 2.6. Safety studies The acute oral toxicity studies were carried out after approval from the Institutional Animal Ethics Committee following OECD guidelines No. 423 [17]. The test material was found to be safe up to 2000 mg/kg with no adverse effects on general behavior in treated BALB/c mice. 2.7. Methods 2.7.1. Animals and stress exposure Male BALB/c mice 10–12 weeks old and weighing 18– 22 g obtained from the animal house of the Regional
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Research Laboratory, Jammu, in groups of six were employed for the study. These were maintained at a room temperature of 22 ± 2 °C with 12 h light/dark cycle and free access to pellet food and water. According to ethical regulations on animal research, all animals used in experimental work received humane care. Mice were restrained in well-ventilated horizontal 50-ml conical polypropylene tubes for 12 h during the dark cycle (2000–0800 h) during experimental periods of 14 days, one group served as the vehicle control to which drug was not administered but was subjected to restrained stress (RSTcontrol), treated groups were subjected to restrain stress as well as drug administration for 14 consecutive days. One group of mice was taken as a non-treated naive control group without any restrained conditions and was give full access to food and water (naive-control). The animals were not physically compressed and, therefore, did not experience pain. Restrained
Fig. 4. (A) Flow cytometry scatter plots for polymorphonuclear cells (neutrophils). The plots represent events for one representative mouse from each group of naive-control, RST-control and WS-treated mouse. The gating and the quadrants are set according to the standard procedures of the BD-LSR flow cytometer. (B) The histograms represent the effect of ‘WS’ on polymorphonuclear cells in (naive-control), chronic stress-induced mice (RSTcontrol.) and the drug-treated mice. Each column is the mean ± S.E. of 10 mice per group. Asterisks indicate a significant difference between the stressed mice (RST-control) and the WS-treated groups.
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Fig. 5. (A) The histograms represent the effect of ‘WS’ on cortisol levels in naive-control, chronic stress-induced mice (RST-control) and the drugtreated mice. Each column is the mean ± S.E. of serum samples assayed in triplicates. Asterisks indicate a significant difference between the stressed mice (RST-control) and the WS-treated groups.
stress procedure is believed to be largely psychological in nature due to the feeling of confinement by the animal [11]. 2.8. Drug administration Test drug was prepared in distilled water and was administered orally daily once a day for the duration of experiment at 25, 50, 100 and 200 mg /kg p.o. dose. The nonstressed naive-control group (naive-control) and stress control group (RST-control) was given vehicle daily. 2.9. Blood collection On day 14, 1 h after subjecting the mice to restrained stress, the blood was collected from the retro-orbital plexus of the animals from all the groups, i.e., naive-control, RST-control and drug treated. Blood was collected in tubes with EDTA as an anticoagulant. 2.10. Lymphocyte immunophenotyping Immunophenotyping focuses on lymphocyte populations involved in acquired immunity and specific molecules present on the cell surface that defines characteristics of lymphocytes such as state of activation or functional capabilities. Lymphocyte subsets were measured by immunofluorescent antibody staining of whole blood and subsequently analyzed using twocolor flow cytometry (Becton & Dickinson, UK). Murine monoclonal antibodies conjugated to a fluorochrome and directed against receptors CD3, CD4 and CD8 were used for the study. CD3 surface receptor is present on all the T lymphocytes present in the whole blood FITC-labeled antimouse CD3 monoclonal antibody that reacts with the CD3 differentiation antigen expressed on MHC class I as well as
MHC class II T helper cells, was used for the determination of total T cell populations in the blood. FITC-labeled anti-mouse CD4 monoclonal antibody that reacts with the CD4 differentiation antigen expressed on MHC class II-restricted T helper cells and PE-labeled CD8 monoclonal antibody that reacts with CD8 differentiation antigen present on MHC class-I-restricted cytotoxic T lymphocyte cells were used to determine the percentage of CD4+ and CD8+ T-cells in all the groups of animals. These fluorochrome-labeled monoclonal antibodies were added directly to 100 μl of whole blood, which was then lysed using whole blood lysing reagent (BD Biosciences). Following the final centrifugation, samples were resuspended in phosphate-buffered saline (pH, 7.4) [2] and analyzed directly on the flow cytometer (LSR, BD Biosciences) using Cell Quest Pro Software (BD Biosciences). A fluorescence
Table 1 Body weights (g) in naïve, stressed (RST-control) and drug-treated mice Sample Group number
Dose Body weights (gm) (mg/ Day 0 Day 14 kg)
1 2 3 4 5 6
– – 25 50 100 200
Naive-control RST-control WS WS WS WS
16.42 ± 0.87 17.20 ± 0.97 18.92 ± 0.73 19.18 ± 0.56 17.05 ± 1.08 16.92 ± 0.98
25.71 ± 0.80 (9.29 g ↑) 21.00 ± 0.44 (3.8 g ↑) 24.05 ± 1.43 (5.13 g ↑) 24.81 ± 1.61 (5.63 g ↑) 23.57 ± 1.11 (6.52 g ↑) 22.10 ± 1.05 (5.18 g ↑)
Values shown are mean ± S.E. for six mice. Percent change against the naive-control is in parenthesis; ↑: increase. Stressing was by immobilization for 12 h (2000–0800 h) from Day 1 to Day 14 in consecutive days.
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trigger was set on the FITC (FLI) parameter to analyze CD3 + and CD4+ and PE (FL2) parameter to collect CD8+ events. 2.11. Intracellular cytokine estimation The blood was collected in heparinised tubes from retroorbital plexus as mentioned above. FITC-labeled anti-mouse CD4+ monoclonal antibody and phycoerythrin (PE)-labeled IL-2 monoclonal antibodies were used in one set and PElabeled CD8+ monoclonal antibody along with FITC-labeled IFN-γ monoclonal antibody was used in the other set of experimentation.
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control (RST-control) group, where CD3+ T lymphocytes decreased from 50% ( normal control value ) to 26% CD4+ T helper cell number was reduced from 23% (normal control value) to 13% and CD8+ T cells decreased from 13% (normal control value) to 8% (p < 0.001). WS, when administered orally at 25–200 mg/kg doses, showed a dose-related increase in lymphocyte number and the most significant increase was observed at an oral dose of 100 mg/kg where CD3+ T cell count was 58% (p < 0.001), CD4+ T cells increased to 30%, and with respect to the stressed mice, the percentage of CD8+ T cells significantly increased from 8% to 22% (Fig. 2A–C). 3.2. Intracellular cytokine estimation
2.12. Body and organ weights: thymus, spleen, axillary lymph node After the last stress session, the body weights of the animals were weighed, following which the animals were sacrificed by ether inhalation; the thymus, spleen, and axillary lymph nodes were removed and weighed. 2.13. Analysis of blood leucocytes (polymorphonuclear leucocytes) Polymorphonuclear cells were separated from whole blood by Histopaque gradient method (Histopaque-Sigma procedures No. 1077). 200 μl of the sample was run. The analysis of cell count and event percentages was carried out in a BD LSR Flow cytometer after the removal of any traces of the red blood cells by FACS Lysing Solution. The gating of the required cell population was carried out according to the standard procedures of the flow cytometer. 2.14. Cortisol assay Cortisol levels were assessed by competitive immunoenzymatic method (Adaltis Italia EIAgen CORTISOL) for determining the amount of cortisol concentration in the serum. Blood for serum was collected from the retro-orbital plexus of the animals from all the groups, i.e., naive-control, RST-control and drug-treated. All samples were assayed in triplicates at a wavelength of 450 nm. 2.14.1. Statistical analysis Fluorescence compensation, data analysis and data presentation was performed using Cell Quest Pro software, and statistical evaluation (drug-induced data) was done by Student's t-test. 3. Results 3.1. Lymphocyte immunophenotyping The percentage of the CD3+, CD4+ and CD8+ T lymphocytes cells was significantly decreased in the restraint
IL-2 and IFN-γ were assayed, as the change in relative proportion of the lymphocyte subsets is proportional to the change in the cytokine expression patterns. It was found that both the IL-2 as well as IFN-γ cytokines counts decreased in stressed animals from 11% (naive-control) to 6.5% (RSTcontrol) for IL-2 and 7.8% (naive-control) to 2.55% (RSTcontrol) for IFN-γ, respectively, i.e., in both cases, about 50% decrease was observed. In the mice treated with WS, a significant change was observed as both Th1 cytokines IL-2 and IFN-γ increased from 6.5% (naive-control) and 2.55% (RST-control) to 18.6% and 8.1% in the WS-treated group (100 mg/kg p.o.), respectively (Fig. 3A–C). 3.3. Body and organ weights: thymus, spleen, axillary lymph node Chronic immobilization stress decreased the weights of the immune-specific organs. The thymus weight decreased from 56.70 mg to 38.80 mg (Table 2). Spleen and axillary lymph node weights decreased significantly in the stressed group. Spleen weight decreased from 84.50 mg in the group of control animals (naive-control) to 42.50 mg in the group of stressed animals (RST-control) and for lymph nodes the weight decreased from 40.60 mg (naive-control) to 17.10 mg (RSTcontrol) (P⁎⁎, respectively, Table 2). In the animals that were administered with the drug, the weight observed was 49.10 mg for thymus and 65.76 mg and 34.57 mg were observed for the spleen and axillary lymph nodes. Body-weight recovery was observed in animals treated with different dose levels of WS when compared to the stress control group (RST-control) (Table 1). 3.4. Estimation of polymorphonuclear leucocytes count The analysis of the polymorphonuclear cells showed that the cell number decreased significantly in the RST-control group, however, from 16 × 104 cells to 8.7 × 104 cells. However, in the animals that are treated with WS, a marked increase in the cell number of plymorphonuclear cells was observed which increased to 12 × 104 as compared to the RSTcontrol group (Fig. 4A and B).
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Table 2 Organ weights (mg) in unstressed, stressed and drug treated mice Organ Thymus Spleen Axillary lymph node
Naive-control
RST-control
Drug treated
70.70 ± 2.0 84.5 ± 3.2 40.60 ± 2.1
38.80 ± 2.2⁎⁎ 42.30 ± 3.4⁎⁎ 17.10 ± 3.2⁎⁎
49.10 ± 4.2⁎⁎ 65.76 ± 3.2⁎⁎ 34.57 ± 1.2⁎⁎
Values shown are means ± S.E. for six mice. Stressing was by immobilization for 12 h (2000–0800 h) for 14 consecutive days. Asterisks indicate a statistically significant decrease (⁎⁎P < 0.001) between the three groups.
3.5. Cortisol assay The analysis of the cortisol levels showed that the cortisol concentration increased in mice subjected to restrain stress (RST-control) which was observed to be 27% as compared to the naive-control group which was 17%. However, in the animals that are treated with WS, a decrease in the cortisol concentration was observed in which a maximum decrease was observed at a dose level of 200 mg/kg in which the cortisol concentration was 14% (p < 0.001), respectively, followed by a dose of 100 mg/kg, the cortisol concentration observed was 15% (Fig. 5A).
4. Discussion WS shows to ameliorate the chronic stress-induced decrease of the T lymphocyte population and prevent the decrease in the expression of the Th1 cytokines. Chronic stress-induced significant decrease of CD3+, CD4+ and CD8+ T cells (Fig. 2A–C). This relates to the decrease in the expression of Th1 cytokines IL-2 and IFN-γ. WS at graded doses exhibited recovery of CD3+, CD4+ and CD8+ T-cell counts with the most significant effect observed at the oral dose of 100 mg/kg. CD4 and CD8 are the T-cell surface markers for Th1/Th2 and Tc subsets of T lymphocytes, respectively, and CD4+ molecules are considered central to regulation of classical cell-mediated functions such as delayed-type hypersensitivity (DTH) response and B cell activation. Immune response is usually polarized to give predominantly either a Th1 or a Th2 response and fine-tuning of inhibitors, activators and regulatory signals ensures immunostasis. Th1 response primarily promotes cytolytic T cells (CTLs), which are important in responding to infections. Therefore, stress-induced decrease in the lymphocyte subsets and associated Th1 cytokines IL-2 and IFN-γ is possibly a factor that renders the immune system vulnerable to clinical conditions. WS primarily induced a Th1 response, involving the cytokines IL-2 and IFN-γ and showed a significant recovery and increase in the lymphocyte populations as compared to the restrained non-treated group of experimental
animals. WS showed dose-related potentiation of IL-2 secreted by stimulated helper T cells (CD4+) and cytotoxic T-cells (CD8+). IL-2 promotes proliferation and differentiation of additional CD4+ cells, B cells, and is a central regulator of immune response. It also stimulates the synthesis of IFN-γ. The increased expression of IL-2 in groups treated with WS possibly is responsible for increased expression of IFN-γ as well (Fig. 3A–C). The recovery in the population of polymorphonuclear leucocytes in the treated groups and recovery in the body weights and a lesser decrease in the organ weights of the animals treated with WS when compared to those in the RST-control group (Tables 1 and 2) show the stress busting potential of the test material. The results of the present study also showed a decrease in the cortisol concentration of the animals that were restrained and subsequently administered with WS. This study establishes unique immunoregulatory potential of the test material and suggests its use in T-cell function restoration where Th1 modulation is required as a reaction to chronic stress, especially in patients suffering from diseases that cause long-term stress that in turn induces suppression of the immune system. Taken together, our results suggest an impact of chronic stress on T-cell function and in turn alters T-cell response, and the immunoregulatory effect of aqueous fraction of the root of W. somnifera is effective in reversing this process. These findings may help to elucidate the physiological mechanisms through which stress plays a role in the etiology of many diseases. References [1] Ayala A, Herdon CD, Lehman DL, Ayala CA, Chaudry IH. Differential induction of apoptosis in lymphoid tissues during sepsis.Variation in onset, frequency, and the nature of the mediators. Blood 1996;87:4261–75. [2] Bani S, Kaul A, Khan B, Ahmad SF, Suri KA, Satti NK, et al. imunosuppressive properties of an ethyl acetate fraction from Euphorbia royleana. J Ethnopharmacol 2005;99:185–92. [3] Benschop R, Schedlowski MRFM. Catecholamine-induced leukocytosis: early observations, current research, and future directions. Brain Behav Immun 1996;10:77–91. [4] Bone K. Clinical applications of Ayurvedic and Chinese herbs. Monographs for the Western herbal practitioner. Australia: Phytotherapy Press; 1996. p. 137–41. [5] Borysenko M, Borysenko J. Stress, behavior, and immunity: animal models and mediating mechanisms. Gen Hosp Psychiatry 1982;4:59–67. [6] Cohen S, Tyrrell DAJ, Smith AP. Psychological stress and susceptibility to the common cold. N Engl J Med 1991;325:606–12. [7] Chatterjee A, Pakrashi SC. The Treatise on Indian Medicinal Plants 1995;4:208–212.
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