FREE RADICAL LOAD IN LYMPHOID ORGANS (SPLEEN AND THYMUS) OF INDIAN GOAT CAPRA HIRCUS: ROLE OF SEX, SEASON AND MELATONIN

Objective: Lymphoid organs (i.e. spleen and thymus) are important due to functional dynamicity. As a result, the generated free radicals may limit their function. Thus, present study was aimed to note seasonal and sex dependent variation in free radical status in Indian goat Capra hircus under the aegis of melatonin which is a well-known antioxidant.Methods: Markers of oxidative stress (i.e. Super Oxide Dismutase; SOD, Catalase; CAT, Glutathione Peroxidases; GPx) were measured by standardized protocols. Total Antioxidant Status (TAS) was measured by 2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid; ABTS) radical cation method and Lipid Per Oxidation (LPO) was measured by Thiobarbituric acid reactive substances (TBARS) level. Glucocorticoid Receptor (GR) expression in lymphoid organs was noted by Western Blot analysis. The circulatory level of cortisol and melatonin were estimated by commercial ELISA kits.Results: We noted significantly high levels of SOD, Catalase, GPx activities and ABTS level in lymphoid organs during monsoon and low during winter. Malonaldehyde; MDA a marker for lipid peroxidation was significantly high during summer and was significantly low during monsoon and winter. Cortisol level was significantly high during monsoon whereas melatonin level was significantly high during winter. GR expression was significantly high in males during monsoon and winter, but the level was significantly high only during monsoon in females.Conclusion: All the results suggest that monsoon and winter are the seasons of stress and to buffer the elevated stress level, melatonin coupled both the roles of free radical scavenger (as a free molecule) and elevation of antioxidant enzymes.Â

Modulation of immunity in young-adult and aged squirrel, Funambulus pennanti by melatonin and p-chlorophenylalanine

Abstract Background Our interest was to find out whether pineal gland and their by melatonin act as modulator of immunosenescence. Parachlorophenylalanine (PCPA) – a β adrenergic blocker, is known to perform chemical pinealectomy (Px) by suppressing indirectly the substrate 5-hydroxytryptamine (5-HT) for melatonin synthesis and thereby melatonin itself. The role of PCPA, alone and in combination with melatonin was recorded in immunomodulation and free radical load in spleen of young adult and aged seasonal breeder Indian palm squirrel Funambulus pennanti. Results Aged squirrel presented reduced immune parameters (i.e. total leukocyte count (TLC), Lymphocytes Count (LC), % stimulation ratio of splenocytes (% SR) against T cell mitogen concanavalin A (Con A), delayed type hypersensitivity (DTH) to oxazolone) when compared to young adult group. Melatonin administration (25 μg/100 g body mass/day) significantly increased the immune parameters in aged more than the young adult squirrel while PCPA administration (4.5 mg/100 g body mass/day) reduced all the immune parameters more significantly in young than aged. Combination of PCPA and melatonin significantly increased the immune parameters to the normal control level of both the age groups. TBARS level was significantly high in aged than the young group. PCPA treatment increased TBARS level of young and aged squirrels both while melatonin treatment decreased it even than the controls. Nighttime peripheral melatonin level was low in control aged group than the young group. Melatonin injection at evening hours significantly increased the peripheral level of nighttime melatonin, while combined injection of PCPA and melatonin brought it to control level in both aged and young adult squirrels. Conclusion PCPA suppressed immune status more in aged than in adult by reducing melatonin level as it did chemical Px. Melatonin level decreased in control aged squirrels and so there was a decrease in immune parameters with a concomitant increase in free radical load of spleen. Decreased immune status can be restored following melatonin injection which decreased free radical load of spleen and suggest that immune organs of aged squirrels were sensitive to melatonin. Increased free radical load and decreased peripheral melatonin could be one of the reasons of immunosenescence.</p

<span style="font-size:11.0pt;font-family: "Times New Roman";mso-fareast-font-family:"Times New Roman";mso-bidi-font-family: Mangal;mso-ansi-language:EN-GB;mso-fareast-language:EN-US;mso-bidi-language: HI" lang="EN-GB">Daily variation in melatonin level, antioxidant activity and general immune response of peripheral blood mononuclear cells and lymphoid tissues of Indian goat <i style="mso-bidi-font-style:normal">Capra hircus</i> during summer and winter</span>

467-477Daily variation in circulatory melatonin level, during different seasons, has been reported to influence immune system and free-radical scavenging capacity in mammals, including human beings. Similar studies have not been carried out on small ruminant viz. goats that are susceptible to opportunistic infections, increased oxidative load and sickness during free-grazing activity and frequent exposure to agro-chemicals. Therefore, daily variation in immune status, antioxidant enzyme activity and its possible correlation with circulatory melatonin level during two different seasons, summer (long day) and winter (short day) were studied in the Indian goat, Capra hircus. The clinically important immune parameters, such as total leukocyte count, % lymphocyte count and % stimulation ratio of T-lymphocytes presented a day/night rhythm prominently in the winter. The oxidative load in terms of malonedialdehyde was always low during night while antioxidant enzymes superoxide dismutase, catalase and total antioxidant status were high during nighttime (1800 to 0600 hrs). Interestingly, the studied parameters were significantly higher during the winter in both the sexes. Rhythmometric analyses showed prominent rhythmicity in above parameters. The data presented strong positive correlation between high levels of nighttime melatonin levels and immune parameters during winter. It suggests that melatonin possesses immunoenhacing as well as antioxidative property during winter. This might be a necessity for maintenance of physiological harmony in goats to protect them from winter stress

Improvement of oxidative stress and immunity by melatonin: an age dependent study in golden hamster

Reactive oxygen species (ROS) have been proposed to play an important role in balancing the pro- and antioxidant homeostasis during aging. Melatonin has been suggested as an effective free radical scavenger that might have a role during the process of aging. We observed, that melatonin administration (25 μg/100 g body weight for 30 days) significantly augments the activity of anti-oxidative enzymes like superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) in the plasma, spleen and bone marrow (BM) of young (6 weeks), adult (30 weeks) and old aged (2.5 years) male golden hamster, Mesocricetus auratus. A sharp decline in generation of ROS was observed in peripheral blood mononuclear cells (PBMC) and splenocytes upon melatonin administration in different age group of hamsters. Reduction in the level of thiobarbituric acid-reactive substances (TBARS) and total nitrite and nitrate concentration as metabolites and indicators of nitric oxide (NO) in plasma, spleen and BM were observed along with night time (22:00 h) melatonin concentration in different age group of hamsters after administration of melatonin and compared to the control group (treated with 0.9% saline). General immune parameters like proliferation of splenocytes, PBMC and colony forming ability of GM-CFU were observed following melatonin treatment in different age group, although it was low only in aged hamsters compared to the young and adult. Our data indicates that the age related increase of oxidative load and simultaneously augments the general immunity in aged hamsters

ATR-induced ER stress response in splenocytes was ameliorated by MEL.

<p>(A) Representative immunoblot showing Calpain1 cleavage and histogram showing ratio of active to inactive Calpain1 (CF/FL, p76/p80) mean density normalized by β-actin. (B) Representative immunoblots of ATF6α, XBP-1, CREB-2, GADD153 and β-actin. Histograms show mean densities of (C) ATF-6α (CF, 70), (D) XBP-1s/XBP-1u ratio (56/32), and (E) PERK proteins (CREB-2 and GADD153) normalized by β-actin density. Data are expressed as mean ± SEM of 3 independent experiments (*<i>P</i><0.05, **<i>P</i><0.01 versus CON; ^#<i>P</i><0.05, ^##<i>P</i><0.01 versus ATR).</p

Schematic diagram showing protective action of MEL against ATR immunotoxicity.

<p>ATR treatment activates death receptor (FasL, Fas, FADD, Caspase-8) and mitochondrial (E2F-1, PUMA, Bax) apoptosis (Caspase-3 and cleaved PARP1) signals. In addition, ATR induces ER stress (ATF-6α, XBP-1s, CREB-2, GADD153) signals. MEL inhibits the Fas and mitochondrial apoptosis as well as ER stress. ATR treatment also impairs autophagy by suppressing BECN-1 and upregulating LC3B-II and p62 proteins; whereas MEL restores autophagy by reversing this dysregulation. Dotted line arrows indicate known connecting pathways that were not a part of the present study. Line arrows indicate stimulatory effect and sign T indicates inhibitory effect on the expression of corresponding proteins. Scissor symbol indicates the cleavage of target proteins.</p

ATR-induced dysregulation of autophagy in splenocytes was ameliorated by MEL.

<p>(A) Representative immunoblots of autophagy markers BECN-1, LC3B (I and II), p62 and loading control β-actin in CON, ATR and MEL+ATR groups. Histograms show (B) BECN-1, (C) LC3B-II and (D) p62 mean densities normalized by β-actin. Data are presented as mean ± SEM of 3 experiments (**<i>P</i><0.01 versus CON; ^#<i>P</i><0.05, ^##<i>P</i><0.01 versus ATR).</p

ATR-induced Fas mediated apoptosis was inhibited by MEL.

<p>(A) Representative immunoblots of splenocyte lysates from CON, ATR and MEL+ATR mice. FasL, Fas, FADD, Caspase-8 and β-actin proteins were visualized by chemiluminiscence. Corresponding histograms show (B) FasL, Fas and (C) Caspase-8 (CF/FL; p18/p57 and p12/p57) expressions as mean densities normalized by β-actin density. (D) Representative immunoblots of Caspase-3, PARP1 and β-actin. Corresponding histogram (E) shows caspase-3 (CF/FL, p17/p32) and PARP1 (CF/FL, p89/p116) mean densities normalized by β-actin. Data are presented as mean ± SEM of 3 independent experiments (*<i>P</i><0.05, **<i>P</i><0.01 versus CON; ^#<i>P</i><0.05 versus ATR). FL, full length; CF, cleaved fragments.</p