|Year : 2020 | Volume
| Issue : 1 | Page : 46-52
Influence of phytomelatonin on immune cell function in male golden hamsters
Dipanshu Kumar Vishwas
Department of Botany, Brahmananda Keshab Chandra College, Kolkata, West Bengal, India
|Date of Submission||27-Dec-2019|
|Date of Decision||11-Feb-2020|
|Date of Acceptance||25-Feb-2020|
|Date of Web Publication||08-Jul-2020|
Dr. Dipanshu Kumar Vishwas
Department of Botany, Brahmananda Keshab Chandra College, 111/2 B.T. Road, Bonhooghly, Kolkata - 700 108, West Bengal
Source of Support: None, Conflict of Interest: None
BACKGROUND: An assortment of plant animal groups incorporates phytomelatonin, shows the similar pattern as in animals recommending its capacity can be closely resemble animals as in plants.
AIMS AND OBJECTIVES: An in vivo research work needed to explore the impact of dietary phytomelatonin, i.e., the cabbage and carrot, on immune cell functions in a male rodent golden hamster (Mesocricetus auratus).
MATERIALS AND METHODS: In this study, we examined the general immune response (spleen weight, total leukocyte count, and % lymphocyte), delayed type hypersensitivity (DTH) response, the blastogenic response of peripheral blood mononuclear cell (PBMC), splenocytes, and bone marrow mononuclear cell proliferation in terms of granulocyte macrophage colony forming unit (GM CFU) count, as an indicator of bone marrow macrophages.
RESULTS: An increased circulatory melatonin level due to melatonin supplemented diet enhanced the total leukocyte count, % lymphocyte count, a proliferation of splenocytes, PBMCs, GM CFU count, % DTH response, and serum cytokine (interleukin 2 and interferon γ) production.
CONCLUSION: A correlation study recommended that incited serum melatonin is emphatically corresponded with the improvement of the safe status of hamsters. In this way, our information infers that dietary melatonin may impact resistance on account of its high selectivity and sensitivity. Thus, it could be suggested that the dietary phytomelatonin might be one of the strong restorative procedures to enhance the immune responses for better well being of a person at its any age of life.
Keywords: Bone marrow mononuclear cells, cytokines, immunity, phytomelatonin, T-cells
|How to cite this article:|
Vishwas DK. Influence of phytomelatonin on immune cell function in male golden hamsters. BLDE Univ J Health Sci 2020;5:46-52
Melatonin is believed to be an animal-exclusive hormone. However, later findings demonstrate that it is additionally present in various plants or parts of plants including roots, stems, leaves, blossoms, fruits, and seeds. These findings have created a new area of investigation for melatonin research. The scientific reports of melatonin in plants (phytomelatonin) have expanded drastically in the last few years. One of the primary speculated functions of phytomelatonin is to protect plants from unfriendly natural affront, including endogenously generated free radicals, since it acts as a potent free radical scavenger. Melatonin enters into the animals from the plants via the diet. When animals get melatonin-rich foodstuffs as their feed, the increased physiological melatonin levels contribute significantly to the total antioxidant capacity of human serum. Hence, it is likely that dietary phytomelatonin from the medicinal plants/melatonin-rich foodstuffs may be necessary for protecting against oxidative damage in animals. Besides, the synthesis of melatonin in plants follows the pattern as in animals, i.e., most elevated levels during the night, proposing that the function of phytomelatonin can be similar to animals as in plants.
Most of the edible plants including many fruits and vegetables such as banana, pineapple, strawberry, carrot, cabbage, corn, cucumber, ginger, onion, rice, tomato, and so on ubiquitously contain a significant level of melatonin. Epidemiological researches indicate that consumption of fruit and vegetables can prevent a variety of diseases which might be due to the preventative properties of vitamins C and E, b carotene, and flavonoids. However, serum melatonin content might be increased in addition to endogenously produced melatonin after feeding phytomelatonin-rich products, and such phytomelatonin can bind to melatonin receptors in rabbit brain, which opens a new avenue of investigations. Therefore, it has been clear that vertebrates can supplement their endogenous melatonin according to the plant material they consume. However, still, there was a question whether such phytomelatonin has any immunomodulatory function in an individual.
Thus, research needs for high melatonin levels measured in different plants for medicinal purposes such as immunomodulatory effect on animals which could help explain their therapeutic action. Hence, the presentin vivo study aims to explore the impact of phytomelatonin present in cabbage and carrot for the modulation of the rodent immune system. The parameters are the proliferation of peripheral blood mononuclear cells (PBMCs), splenocytes, and bone marrow mononuclear cells (BM-MNCs) for granulocyte-macrophage colony-forming unit (GM-CFU) as an indicator of bone marrow macrophage cells in male golden hamsters.
| Materials and Methods|| |
Animals and housing conditions
We purchased outbred strain of golden hamsters from Central Drug Research Institute, Lucknow, India. The hamsters state was kept up in a room having temperature 25°C ± 2°C with elective light/dim cycle (12.5 h light, 11.5 h dull; for example, lights on 06:30–19:00 h) to keep up the photoperiod. Hamsters were kept in polypropylene confines (475 mm × 350 mm × 200 mm) in a gathering of five. Water and business rat pellet nourishment were given in ad libitum. During every one of the examinations directed, we pursued the institutional rules in agreement to the structure of reexamined Animal (Specific Procedure) Act of 2002, Government of India, for animal welfare.
Hamsters of comparable age and body weight (80 ± 10 g) were haphazardly partitioned and doled out to three gatherings (n = 5 in each): Gathering I with business mice feed just and filled in as control (Cont), Group II with business mice feed enhanced with cabbage, and Group III with mice feed enhanced carrot during night hour (between 18:00 and 18:30 h; ~1 h before nightfall). Every hamster was housed in a different enclosure. Every g of cabbage (Brassica oleracea) and carrot (Daucus carota) was picked as wellspring of phytomelatonin, which contains 0.309 and 0.494 ng/g of plant material, respectively. Animals were kept in single pens and investigation was completed for 30 days. Carrot and cabbage was enhanced with business mice feed and day-by-day utilization/hamster was determined. The normal load of cabbage and carrot devoured by every hamster/day of each gathering was 17.39 g and 18.153 g separately. Along these lines, the normal measure of phytomelatonin devoured by every hamster/day was 5.373 ng from cabbage and 8.967 ng from carrot. We yielded the test hamsters toward the finish of the investigations and lymphoid tissues and trunk blood was prepared for additional examinations.
Following the most recent day of treatment, all the three gatherings of hamsters (n = 5 in each) brought into the medical procedure room each in turn, weighed, and anesthetized to death during night time between 22:30 h and 23:30 h under diminish red light (>2 lux). The blood serum was disconnected and kept at −20°C till the hormone and cytokine estimations were performed. Bone marrow was detached from femur bone and tallied the absolute number of BM-MNCs per femur bone and prepared for GM-CFU culture. Spleen was dismembered out and used for lymphocytes culture in the term of percent stimulation ratio (%SR).
Measurement of delayed-type hypersensitivity response
Following 30 days of phytomelatonin treatment, delayed-type hypersensitivity (DTH) was incited by utilization of the antigen DNFB (Spectrochem, Pvt. Ltd., Mumbai, India) to the ear pinna of hamster following starting inoculation by applying 20 μL of 0.2% DNFB in acetone on to the shaved abdominal area for two continuous days (1st and 2nd day). On day 6, ear thickness was estimated with a Vernier caliper to decide standard thickness and DNFB immune reaction was tested by applying 20 μL of 0.2% DNFB in acetone to the skin of the inward and external ear surfaces of the right pinna. Left ear was treated with vehicle alone. Ear swellings were estimated after each 24 h for the successive 3 days to record most extreme DTH reaction. On 2nd day, most extreme reaction was observed and it is being appeared in the diagram. All measurements were made on a similar relative region of the ear pinna of the exploratory animals. Percent thickness was determined by looking differences among treated and nontreated ear pinna.
Preparation of cell suspension
Subsequent to analyzing out the spleen was cleaned from its follower tissues, we washed it with chilled PBS. The tissue was minced into little pieces in a sterile watch glass in chilled RPMI-1640 (Sigma-Aldrich Chemicals, St Louis, Missouri, USA) and cell suspension was filtered with steel strainer of 400 meshes and collected in a sterile centrifuge tube and washed twice with RPMI-1640. The red blood cells present in the splenocytes cell suspension were lysed with cold ammonium chloride tris buffer (tris [hydroxymethyl] aminomethane, BDH, UK); 0.5% tris buffer and 0.84% NH4 Cl mixed in 1:10 ratio and adjusted to pH 7.2. The purity of the single-cell suspension was checked under an inverted microscope and precision of purity was examined stained with Giemsa stain. The cell viability was then checked with 1.0% Trypan blue exclusion method. This single-cell suspension of isolated splenocytes (~95%) was adjusted to 1 × 107 cells/mL in RPMI-1640, containing sodium bicarbonate, antibiotics (penicillin 100 IU/mL and streptomycin 100 μg/mL), and 10% fetal calf serum (Sigma, USA) and used for proliferative response (%SR).
Cell proliferation by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay
Cell proliferative capacity was surveyed by estimating splenocytes multiplication by the T-cell explicit mitogen Concanavalin A (Con A), utilizing a colorimetric test dependent on the decrease of tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT). Viable splenocytes (which surpassed 95%) were handled by our recently distributed protocol. 100 μL aliquots (1 × 107 cells/mL) of each cell suspension were added to the wells of sterile flat-bottom 96-well culture plates. Con-A (Sigma-Aldrich, St. Louis, USA) was added to the culture medium at the concentration of 5 μg/mL. Plates were incubated at 37°C with 5% CO2 for 69 h prior to addition of 10 μL of MTT (SRL, Mumbai, India; 5 mg/mL in phosphate-buffered saline) per well. Acidified propanol (0.04 mol/L HCL in isopropanol) was added and the optical density (OD) was determined with a microplate reader (ELx-800, Biotek Instruments, Winooski VT, USA) equipped with a 570 nm wavelength filter. Mean OD values for each set of duplicates were used in subsequent statistical analyses. Blastogenic reaction was determined as %SR representing the proportion of absorbance of mitogen stimulated cultures to control cultures.
Isolation and cell proliferation of peripheral blood mononuclear cells
Recently distributed technique was utilized for confinement of PBMC. PBMC were secluded from the heparinized blood gathered from heart in a 15 mL tube. Blood was diluted in 1:1 proportion with PBS and under layered with Ficoll Hicep™ LSM (Himedia Lab, India). White ring of mononuclear cells was collected, washed with PBS, centrifuged, and then suspended in RPMI 1640 supplemented with 10% fetal calf serum and 100 units of penicillin and streptomycin. The cell concentration was maintained in accordance with 1 × 107 reasonable cells/mL and plated in 96-well plate in copy having grouping of (2 × 106 cells/well) for the estimation of cell expansion by MTT test and OD esteems at 570 nm were taken and the %SR was determined.
Bone marrow isolation and granulocyte-macrophage colony-forming unit culture
Recently, distributed protocol was utilized for the disconnection of BM-MNCs. In short, femur bone was analyzed out and bone marrow was gathered by flushing it with PBS, washed with clean medium IMDM (GIBCO). 1.0 × 106 mononuclear cells per 35/10 mm dish were refined in a test vehicle of methylcellulose (Sigma-Aldrich Chemicals, St. Louis, USA) with fetal calf serum (heat inactivated, Sigma-Aldrich, St. Louis, Missouri, USA), IMDM 2x medium (GIBCO), and recombinant murine colony-stimulating factor (rGM-CSF; Peprotech, USA) for total 7 days at 37°C with 5% CO2 in a HERA cell CO2 incubator (Kendro Laboratory Products [India] Pvt. Ltd., New Delhi, India). The quantity of colonies (containing at least 50 cells) was checked utilizing an inverted microscope at × 20 magnification and reported.
We played out the measure adhered by the producer's guidance given on the kit (Uscn Life Science Inc. USA). The intra- and inter-examiner variety was <10% and <12% individually. The sensitivity was 4.68 pg/mL and recuperation was between 90% and 115%. The guideline of this measure depends on the competitive inhibition enzyme immunoassay procedure. Monoclonal antibody explicit for rodent melatonin has been precovered on to a microplate. A focused response was propelled between biotin marked rodent melatonin and unlabeled serum melatonin tests with the precovered counter acting agent explicit for rodent melatonin. After brooding, the unbound conjugate was washed off and avidin conjugated to horseradish peroxidase (HRP) added to each microplate well and hatched. The measure of bound HRP conjugate was switch corresponding to the concentration of melatonin in test. After expansion of substrate arrangement, the force of shading created was turn around relative to the concentration of melatonin in the example.
Sandwich ELISA was performed to measure the degree of interleukin-2 (IL-2) and interferon-γ (IFN-γ) in serum gathered from the hamsters of three gatherings as per producer's guidance for IL-2 kits (Immunotech, France). Intratest variety for IL-2 was in the range of 3.3% and 7.2%, intertest variety was in the range of 6.2% and 8.2%, sensitivity 5 pg/mL, and recuperation was in the range of 80% and 132%. Further, kits for IFN-γ were acquired from Koma biotech, Korea. Intratest variety for IFN-γ was in the range of 2.4% and 1.8%, intermeasure variety was in the range of 2.1% and 1.8%, sensitivity <21.2 pg/mL, and recuperation was in the range of 96% and 101%.
We performed the unpaired t-tests for statistical analysis. The differences were considered significant when P < 0.05. Microsoft Excel program was used for statistical analyses and documentation.
| Results|| |
The body weight of golden hamsters kept under different experimental groups did not reveal any significant difference. Cabbage- or carrot-supplemented groups of hamsters presented significantly (P < 0.01) increased spleen weight when compared with mice feed only control hamsters. Cabbage supplementation to hamsters significantly (P < 0.01) enhanced the number of lymphocyte count (LC) and also showed the significant (P < 0.05) increased total leukocyte count (TLC) when compared with control hamsters. While, a significant (P < 0.05) increased in number of both LC and TLC was noted by carrot supplemented group when compared with control hamsters [Table 1].
|Table 1: Body weights, spleen weights, lymphocyte count and total leukocyte count after 30 days of treatment of commercial mice feed supplemented with/without cabbage or carrot to male golden hamsters|
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Effect on total splenocytes and peripheral blood mononuclear cell count and its proliferative response
Cabbage supplemented group of animals showed significant (P < 0.01) increased in total number of splenocytes per mg tissue of spleen, and PBMC count per mL blood. While, carrot supplemented group of animals significantly enhanced the total number of splenocytes (P < 0.05) and PBMC count (P < 0.01) when compared with control hamsters.. Cabbage-supplemented animals presented significantly (P < 0.01) enhanced blastogenic response of splenocytes and PBMC in terms of %SR, while carrot-supplemented hamsters significantly enhanced the %SR of splenocytes (P < 0.05) and PBMC (P < 0.01) when compared with control hamsters [Figure 1]. Further, correlation between changes in circulatory melatonin level and blastogenic response of splenocytes (r = 0.585, P < 0.01) and PBMC (r = 0.763, P < 0.01) was found to be positively correlated with supplementation of cabbage and carrot [Figure 2]b and [Figure 2]c.
|Figure 1: Effect of phytomelatonin supplement on total number of PBMC counts/mL blood, total number of splenocytes/mg tissue, and %SR of PBMC and splenocytes of hamsters. Vertical bar on histograms represents mean ± SD, n = 5. **P < 0.01 Cont versus other treated groups of hamsters, while *P < 0.05 Cont versus Carrot-supplemented hamsters only. PBMC = Peripheral blood mononuclear cell, SD = Standard deviation, %SR = Percent stimulation ratio|
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|Figure 2: Correlation analysis between the level of serum (a) melatonin and % DTH response, (b) melatonin and %SR of splenocytes, (c) melatonin and %SR of PBMCs, (d) melatonin and GM-CFU count, (e) melatonin and IL-2, and (f) melatonin and IFN-γ in hamsters being presented. Points are denoted with individual melatonin (pg/mL); % DTH response; IL-2 (pg/mL); IFN-γ (pg/mL); %SR of T cells; and GM-CFU count. The differences were considered significant when P < 0.01. Positive correlations were depicted on values of correlation coefficient = r, which measured the strength of the relationship. DTH = Delayed-type hypersensitivity, %SR = Percent stimulation ratio, PBMCs = Peripheral blood mononuclear cells, GM-CFU = Granulocyte macrophage colony forming unit, IL = Interleukin, IFN = Interferon|
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Effect on total number of bone marrow mononuclear cell count and its proliferation
We found similar result for total number of BM-MNCs count per femur bone and also its proliferation in terms of GM-CFU count. Cabbage- and carrot-supplemented hamsters showed no significant (P > 0.05) change in BM-MNCs count per femur bone, but significantly (P < 0.01) enhanced number of GM-CFU count when compared with control hamsters [Figure 3]. Further, hamsters with cabbage and carrot supplementation showed the positive correlation (r = 0.816, P < 0.01) between number of GM CFU count with serum melatonin level [Figure 2]d.
|Figure 3: Effect of dietary phytomelatonin supplementation on (a) total number of BM-MNCs/femur bone of hamsters, and its proliferation to GM-CFU. Histogram represents mean ± SD, n = 5. **P < 0.01 Cont versus Cabbage or Carrot treated group. (b) Photograph showing GM-CFU colony after 7 days of BM-MNC culture of male golden hamster Mesocricetus auratus (×20). GM-CFU = Granulocyte macrophage colony forming unit, SD = Standard deviation, BM-MNCs = Bone marrow mononuclear cells|
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Effect on delayed-type hypersensitivity response, serum melatonin, interleukin-2, and interferon-γ level
DTH response is an importantin vivo manifestation of a cell-mediated immune response in which T cells are implicated. Cabbage and carrot supplemented animals showed significant (P<0.01) increase in % DTH response when compared with control hamsters. The level of serum melatonin (night time; 23:00 h), pro-inflammatory cytokine IL-2, and IFN-γ was recorded significantly (P < 0.01) high in cabbage- and carrot-supplemented hamsters when compared with control hamsters [Figure 4]. Further, the positive correlation has been found between the % DTH response [r = 0.771, P < 0.01; [Figure 2]a, level of serum cytokine IL-2 [r = 0.899, P < 0.01; [Figure 2]e, and serum IFN-γ [r = 0.744, P < 0.01; [Figure 2]f with circulatory melatonin level following different experimental hamster groups.
|Figure 4: Effect of phytomelatonin treatment on % DTH response, level of serum melatonin (pg/mL), IL-2 (pg/mL) and IFN-γ (pg/mL) in male hamsters. Values are expressed as mean ± SD, n = 5. **P < 0.01 and *P < 0.05 Cont versus other treatment groups. DTH = Delayed-type hypersensitivity, IL = Interleukin, IFN = Interferon, SD = Standard deviation|
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| Discussion|| |
The endogenous melatonin emission in animals provides strong evidence to legitimize the clinical estimations of immunotherapy. Inspite of that, more investigation needs to understand whether the melatonin rich foodstuffs interferes with the physiological functions implicated with immunomodulation in a nocturnal male rodent golden hamsters, that may lead to modulation in the immune function. The result of our study demonstrates that phytomelatonin-rich cabbage (Brassica oleracea) and carrot (Daucus carota) supplementation significantly elevated the spleen weight, but had no significant change in hamsters' body weight, supports earlier observations noted with exogenous melatonin treatment.
We further noted that serum melatonin concentration increased significantly following phytomelatonin supplementation to animals. This increased circulatory melatonin level might play an important role in the local synthesis of melatonin hormone from lymphoid tissues such as the spleen, thymus and bone marrow, which scavenge the toxic molecules locally and shields the immune cells from oxidative stress.
Apart from the diet in terms of phytomelatonin, exogenous melatonin is known for its immunomodulatory work. It stimulates the proliferation of splenocytes, thymocytes, and PBMC in bothin vivo andin vitro conditions. Similarly, we find that phytomelatonin supplementation significantly elevated the quantity of T cells in circulation, thus stimulating the proliferation of splenocytes/PBMCs underin vivo condition and thereby improved the general immunity of the hamsters. Further, it was also noted that bone marrow myeloid and lymphoid progenitor cells function significantly raised the BM-MNCs proliferation in terms of GM-CFU count (a marker of macrophages) after the melatonin-rich diet to animals. Thus, it could be explained as the bone marrow is an important tissue which differentiates into all type of immune cells that compasses to other lymphoid tissue for further maturation/differentiation, and melatonin from plant product might be playing an important role in significant enhancing of the bone marrow differentiation and potentiality of T cells of hamsters against the infection.
We also observed the DTH response, a T cell-mediated immune reaction which is characterized by antigen-specific Th1 type CD4+ T cells expansion during the initial phase and inflammatory response by Th1 cytokines discharged from CD4+ T cells during the effector phase. In the present investigation, we recorded that phytomelatonin from various sources raised the inflammatory DTH responses that might be related to the elevated Th1 cytokines (IL-2 and IFN-γ) from T lymphocytes. Once stimulated, T cells secrete a range of cytokines including IFN-γ which promote the nearby recruitment of cells from the blood. Therefore, the discharge of cytokines (s) such as IFN-γ and IL-2 might be responsible to induce the manifestations of the DTH reaction. Of these cytokines, IFN-γ is a crucial mediator for immune cell proliferation and along with IL-2 is a relevant effector of the immune response. Our information on the enhanced concentration of serum IL-2 and IFN-γ following phytomelatonin treatment underpins this clarification.
Moreover, it could be speculated that the significant increment of TLC, LC and the blastogenic response of PBMC and splenocytes after receiving daily melatonin-rich foodstuffs to hamsters, suggest a possible light of the fact that the consumed melatonin might come in a faster rate to the circulation. Thereafter, phytomelatonin and other indoles present in the diet scavenge the toxic free radicals through dependent/independent of antioxidant activity of foods. The amphipathic nature of the melatonin molecule enables it to trace pass physiological barrier to protect the cells under oxidative stress and enhances the capability of immune cell functions. The melatonin is well-absorbed after oral administration. It is evident in our experiment, oral intake of plant melatonin in an amount of ~ 5–9 ng/day/100 g body weight for 30 days may increase the serum melatonin level from ~ 12 pg/ml to 20 pg/ml during day time, as reported in human. It appears that the duration of melatonin supplementation given to the animals was less but much effective as noted in this study. The reason behind could be our animal model hamster fed only by cabbage or carrot as their preference which carries a lesser amount of melatonin, i.e., 0.309 and 0.494 ng/g plant tissue, respectively. This research work might help understand the immunomodulatory impact of phytomelatonin on different immune cells.
| Conclusion|| |
We rationale that the phytomelatonin may assume a significant role by expanding the circulatory melatonin, which has an immunomodulatory function by enhancing the bone marrow myeloid and lymphoid progenitor cell function and thereby upgrading the TH and TC cells reaction in lymphoid tissues of living beings. Our outcomes likewise proposed that customary utilization of therapeutic plant items including vegetables, foods grown from the ground and so forth containing phytomelatonin may have an immunotherapeutic property. They could improve the well-being status of a life form, including people. In this way, our information reach a conclusion that phytomelatonin daily supplementation could be one of the most strong immunotherapeutic strategies to improve immunity and along these lines endurance of people.
The author highly acknowledged to Prof. Chandana Haldar (Retired.), Pineal Research Lab., Department of Zoology, Banaras Hindu University, Varanasi - 221005, Uttar Pradesh, India, for providing the research facility in her laboratory. The author also would like to acknowledge the University Grant Commission, New Delhi, India, for financial support as NET JRF and SRF to DKV, and likewise the Instrument gift from Alexandar von Humboldt foundation, Bonn, Germany, to Professor C. Haldar is gratefully acknowledged.
Financial support and sponsorship
University Grant Commission, New Delhi, India, for financial support as NET JRF and SRF to Author.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]