Abstract
Purpose: Hypoxia is a condition of decreased availability of oxygen. To adapt hypoxia, some changes in blood cells occur in the body. The aim of this study was to evaluate the effect of ghrelin on different types of blood cell in normobaric hypoxia situation.
Methods: Thirty-two animals were divided in 4 groups (n=8): control (C), ghrelin (G), hypoxia (H), and hypoxic animals that received ghrelin (H+G). Hypoxia (11%) was induced by an Environmental Chamber System GO2 Altitude. Animals in ghrelin groups received a subcutaneous injection of ghrelin (150 μg/kg/day) for 14 days.
Results: Our results show that ghrelin significantly (p<0.05) increased RBC and Hct levels, whereas it significantly (p<0.05) decreased lymphocytes in the blood. RBC, Hct, Hb concentration, platelet and MCV increased significantly (p<0.05) in hypoxic conditions but lymphocytes, monocytes and Polymorphonuclears did not show any significant changes. Platelets had a significant (p<0.05) decrease in hypoxic conditions and ghrelin administration in hypoxic conditions could increase lymphocyte levels significantly (p<0.05).
Conclusion: Effect of ghrelin on blood cells could be related to blood oxygen level. Ghrelin in normal oxygen conditions increases RBC and Hct levels but decreases lymphocytes, whereas in hypoxic conditions, ghrelin increases blood lymphocytes.
Keywords: Blood cells, Ghrelin, Hypoxia, Rat
Introduction
Hypoxia, a condition of decreased availability of oxygen, contributes to the regulation of pathophysiology in various kinds of cells and tissues.1 When a cell or an organism is in an abnormal condition starts series of mechanisms to adapt or response at the cellular and molecular levels as strategies to minimize serious effects of the condition.2 To adapt hypoxia, the cell should reduce energy consumption or increase oxygen supply by inducing a change in red blood cells.2,3 Other blood cells including white blood cells and platelets also undergo some changes in hypoxic conditions.4-6 However, accomplished studies in hypoxic conditions demonstrated that hypoxia causes reduction in proliferation of lymphocytes.4 Whereas, another study has shown that hypoxia increases blood lymphocytes.7 One study done on the effects of hypoxia on platelet indicated that increase in altitude could reduce platelets.8 Another study showed that hypoxia, in the early-onset, causes thrombocytosis, however, in the late-onset, it causes thrombocytopaenia.6,9
In addition to hypoxia, endocrine factors such as growth hormone and glucocorticosteroids can also affect the production of blood cells.10,11
Ghrelin is a 28-amino acid peptide hormone that is considered by researchers for its physiological effects. It is found in the secretory granules of X/A-like cells in gastric mucosa.12 Today, studies have shown that ghrelin is also produced by other tissues such as kidney, pancreas, placenta, testis, pituitary, lung, and hypothalamus.13,14 The acylated form of ghrelin has a serum half life of only 30 minutes because of rapidly change to a deacylated form that is more stable. Acylated ghrelin binds to the growth hormone secretagogue-receptor1a (GHSR-1a) in many tissues to produce its effects.15 Among the known physiological actions of ghrelin are; glucose homeostasis, growth hormone secretion, appetite stimulation and adipogenesis, cell proliferation and survival, increase in GI motility.13,15 It has also anti-inflammatory, cardiovascular, sleep regulation, and reproduction effects.13,15-17
In studies examining the impact of the ghrelin on blood cells we faced with contradictory results. One study has shown ghrelin has no effect on red blood cell (RBC), hemoglobin (Hb) concentration, and hematocrit (Hct), mean corpuscular volume (MCV).18 But, another study has shown that ghrelin increases RBC, Hct and decreased MCV.19 Several studies have demonstrated that ghrelin dose dependently inhibits the proliferation of lymphocytes and expression of cytokines, while another study has reported an increase in blood lymphocytes.18,20-22 It has also been reported that ghrelin increases monocytes, eosinophils and basophils.20 Whereas, Narin et al showed ghrelin decreases neutrophils and it has no effect on monocytes, eosinophils and basophils.18
Therefore, in the present study we decided to investigate the influence of ghrelin on blood cell types in hypoxia.
Materials and Methods
Animals and chronic hypoxic protocol
All experiments were performed in agreement with guidelines of the Tabriz University of Medical Sciences for care and use of laboratory animals. Male adult Wistar rats (200–250 gr) were housed in cages in a temperature- and light-controlled environment. Food and water were available ad libitum. Animals were randomly divided in 4 groups (n=8) including control (C), ghrelin (G), hypoxia (H), and hypoxia with ghrelin (H+G). In hypoxic groups (H and H+G), hypoxia (O2 11%) was induced by an Environmental Chamber System GO2 Altitude (Biomedtech Australia, Pty. Ltd). Animals were kept in the chamber continuously for two weeks except for 20 min/day to clean the cages and perform daily injections.
Drug administration
Ghrelin was obtained from the Tocris Bioscience Co. (Bristol, UK). Rats in ghrelin groups (G and G+H) received a subcutaneous injection of ghrelin (150 μg/kg/day).23 G and H+G rats continued to receive daily injections of ghrelin for 2 weeks.
Blood cells count, hemoglobin and hematocrit measurement
Animals were deeply anaesthetized with ketamine (100mg/kg) and trunk blood was collected immediately after decapitation of the animal. Then, RBC and WBC count were performed. Hemoglobin (Hb) concentration and hematocrit were measured using cyanmethemoglobin and microhematocrit methods, respectively.
Statistical analysis
Results are reported as Mean±SEM. Data were analyzed by ANOVA to test for differences between groups. For statistically significant comparisons, post-Hoc analyses were performed using Tukey tests and P<0.05 were used as the level of significance for all statistical analyses.
Results
Effects of hypoxia and ghrelin treatment on RBC, hemoglobin concentration and hematocrit
There was a significant (p<0.05) increase in RBC level in Ghrelin group (G) compared to the control group(C). Also hypoxia (H) and hypoxia + ghrelin groups (H+G) had significant (p<0.05) increase in RBC level compared to control. Hematocrit in the G group was significantly (p<0.05) more than the C group. There was also a significant difference (p<0.05) between H+G, H and control groups. Hemoglobin concentration showed no significant difference between groups C and G. There were significant (p<0.05) increase in hemoglobin concentration in H and H+G groups compared to C and G groups. Whereas there was not a significant difference between groups H and H+G. But H+G group had not significant difference in RBC, hematocrit and hemoglobin concentration compared to H group.
Hypoxia and H+G groups had significant (p<0.05) increase in MCV compared to control group. While, there were no significant differences in MCV either between control and ghrelin or H and H+G groups (Table 1).
Table 1. Effects of hypoxia and ghrelin treatment after 2 weeks on RBC, Hb, Hct and MCV .
| Groups | Control | Ghrelin | Hypoxia | Hypoxia + Ghrelin |
| RBC (× 106) | 5.4167±0.22 | 6.018±0.08* | 6.81±0.17* | 6.97±0.08*# |
| Hb (g/dl) | 13.66±0.17 | 14.73±0.22 | 22.07±0.29* | 22.53±0.54*# |
| Hct (%) | 39.33±0.88 | 45.66±0.61* | 67.14±0.51* | 67.87±1.32*# |
| MCV (fL) | 72.72±1.35 | 75.91±1.05 | 98.62±0.57* | 97.44±0.83*# |
Red blood cell (RBC), Hemoglobin (Hb), Hematocrit (Hct), Mean Corpuscular Volume (MCV). Data are expressed as mean ± SEM for 8 animals. * p<0.05 vs the control group, # p<0.05 vs the ghrelin group.
Effects of hypoxia and ghrelin treatment on polymorphonuclears, mononuclears and lymphocytes counts
There were no significant differences in intergroup comparisons in polymorphonuclear and monocyte counts (data are not shown). Ghrelin could significantly (p<0.05) reduce lymphocytes compared to control group. Neither hypoxia nor H+G groups did show significant difference in lymphocyte count compared to control group. Hypoxia group that received ghrelin showed a significant (p<0.05) increase in lymphocyte level compared to hypoxia group (Figure 1).
Figure 1 .
Effect of ghrelin on lymphocyte after 2 weeks in control (C), ghrelin (G), hypoxia (H) and hypoxia plus ghrelin (H+G) groups. Data are expressed as mean ± SEM for 8 animals.* p<0.05 vs the control group, # p<0.05 vs the ghrelin group, ○ p<0.05 vs the hypoxia group.
Effects of hypoxia and ghrelin treatment on platelet count
There was not a significant difference between control and ghrelin groups. Blood platelets decreased in both hypoxia and H+G groups significantly (p<0.05), although the difference between H and H+G groups was not significant (Figure 2).
Figure 2 .
Effect of hypoxia and ghrelin on Platelet after 2 weeks in control (C), ghrelin (G), hypoxia (H) and hypoxia plus ghrelin (H+G) groups. Data are expressed as mean± SEM for 8 animals. * P< 0.05 vs the control group, # p<0.05 vs the ghrelin group.
Discussion
Our study showed that ghrelin increases RBC and Hct levels, whereas it decreases lymphocytes. RBC, Hct, Hb, MCV increased and platelets decreased in hypoxic conditions but lymphocytes, monocytes and polymorphonuclears did not show any significant changes. In hypoxia conditions, ghrelin treatment could increase lymphocytes but had no effect on other parameters.
Taati et al.’s study showed that ghrelin increases RBC and Hct, decreases MCV but it has no effect on Hb concentration.19 Some studies have suggested that ghrelin directly affects cell division and replication in bone marrow.19,24 Stimulation of the growth hormone secretion is the most known action of the ghrelin.15 Ardizzi et al. also demonstrated that growth hormone has a stimulatory effect on erythropoietin in mammals.25
Akarsu et al. stated that there was a positive relation between Hb concentration and peripheral ghrelin level. They demonstrated in their study that patients with iron deficiency anemia had low peripheral ghrelin levels.26 Vander Lely et al. also suggested that low blood ghrelin levels could be the cause of anemia.27
Our results about RBC, Hct and Hb concentration are consistent with Taati's study but according to Hb concentration it is not compatible with Akarsu et al.’s study.
Narin et al. in their study showed that ghrelin does not affect RBC, Hb and Hct.18 Aghdam et al. also suggested that intracerebrovascular injection of 0.5 or 1.0 mg ghrelin/kg at 21 days of age did not have any significant effect on the measured erythropoietic indicators including RBC, Hb, Hct, MCV, MCH and MCHC.28 Narin et al and Aghdam et al.’s findings are inconsistent with our study. These differences can be attributed to the type of animal, method and dose of drug administration, and time interval between treatment and sampling.
Taati et al. has pointed that ghrelin has no effect on lymphocytes, Whereas, Narin et al. reported increased lymphocyte.18,19 Other studies showed that ghrelin dose dependently has positive increasing effect on polymorphonuclears and decreasing effect on lymphocytes.20,21 It should be noted that all the above studies were performed in the normal oxygen conditions.
Szigligeti et al.’s study showed that hypoxia decreases lymphocytes proliferation.4 Wang et al. in their study suggested that 12% hypoxia increases entry lymphocytes to blood.7 They expressed it could be due decreased antioxidants and stress oxidative.
Hypoxia induces oxidative stress in blood and leads to aging and apoptosis of lymphocytes.7 Hypoxia also inhibits voltage-dependent potassium channels (Kv1.3). These channels are essential for the activation and proliferation of lymphocytes.4 Therefore hypoxia could reduce the number of lymphocytes.
Ghrelin acts as an antioxidant in various tissues such as ovary, stomach, kidney, and neurons.29-32 It also has antiapoptotic effects in hippocampus, adrenal tumor and cardiovascular system.33-35
A significant increase in lymphocyte in hypoxia despite receiving ghrelin may be attributed to the effect of ghrelin on decreasing oxidative stress and apoptosis also its effect on Kv1.3 channels. Thus it is noted that the environmental conditions such as hypoxia is effective on the activity of lymphocytes and the production and secretion lymphokin and probably ghrelin effect.5 But the proof of this hypothesis requires further investigation.
About platelets, Bradford in his study showed that hypoxia has the dual effect on platelets.6 The early-onset thrombocytosis caused by hypoxia may be due to increased release of platelets from megakaryocytes and the late-onset thrombocytopaenia may be due to decreased platelet production and/or stem cell competition between erythrocytes and megakaryocytes.6 Lehmann et al. showed high-altitude leads to platelet aggregation, platelet consumption, and decreased platelet count.8 Mc Donald et al. showed long term hypoxia (6-7 days) causes decreased platelets counts and short term hypoxia (1-3 days) increased it which is in agreement with our results.9
Conclusion
Effect of ghrelin on blood cells could be related to blood oxygen level. Ghrelin in normal oxygen conditions increases RBC and Hct levels and decreases lymphocytes, whereas, in hypoxia ghrelin increases blood lymphocytes.
Acknowledgements
This study was financially supported by Tuberculosis and Lung Research Center of Tabriz University of Medical Sciences. This article is derived from PhD dissertation of Fariba Mirzaei Bavil, entitled “Effect of ghrelin on miRNA 210,424, transcription factor HIF 1α and VEGF in lung tissue in chronic hypoxic Wistar rats”.
Ethical issues
The study protocol was designed in accordance with NIH guidelines and Ethics Committee for the Use of Animals in Research at Tabriz University of Medical Sciences.
Conflict of Interest
The authors have declared that there is no conflict of interest.
References
- 1.Semenza GL, Agani F, Feldser D, Iyer N, Kotch L, Laughner E. et al. Hypoxia, HIF-1, and the pathophysiology of common human diseases. Adv Exp Med Biol . 2000;475:123–30. doi: 10.1007/0-306-46825-5_12. [DOI] [PubMed] [Google Scholar]
- 2.Clerici C, Planes C. Gen regulation in the adaptive process to hypoxia in lung epithelial cells. Am J Physiol Lung Cell Mol Physiol . 2009;296(3):L267–74. doi: 10.1152/ajplung.90528.2008. [DOI] [PubMed] [Google Scholar]
- 3.Kent BD, Mitchell PD, Mcnicholas WT. Hypoxemia in patients with COPD: cause, effects, and disease progression. Int J Chron Obstruct Pulmon Dis . 2011;6:199–208. doi: 10.2147/COPD.S10611. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Szigligeti P, Neumeier L, Duke E, Chougnet C, Takimoto K, Lee SM. et al. Signaling during hypoxia in human T lymphocytes – critical role of the src protein tyrosine kinase p56Lck in the O2 sensitivity of Kv1.3 channels. J Physiol. 2006;573(Pt 2):357–70. doi: 10.1113/jphysiol.2006.109967. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Caldwell CC, Kojima H, Lukashev D, Armstrong J, Farber M, Apasov SG. et al. Differential effects of physiologically relevant hypoxic conditions on T lymphocyte development and effector functions. J Immunol . 2001;167(11):6140–9. doi: 10.4049/jimmunol.167.11.6140. [DOI] [PubMed] [Google Scholar]
- 6.Bradford A. The role of hypoxia and platelets in air travel-related venous thromboembolism. Curr Pharm Des . 2007;13(26):2668–72. doi: 10.2174/138161207781662966. [DOI] [PubMed] [Google Scholar]
- 7.Wang JS, Lin CT. Systemic hypoxia promotes lymphocyte apoptosis induced by oxidative stress during moderate exercise. Eur J Appl Physiol . 2010;108(2):371–82. doi: 10.1007/s00421-009-1231-2. [DOI] [PubMed] [Google Scholar]
- 8.Lehmann T, Mairbaurl H, Pleisch B, Maggiorini M, Bartsch P, Reinhart WH. Platelet count and function at high altitude and in high-altitude pulmonary edema. J Appl Physiol (1985) . 2006;100(2):690–4. doi: 10.1152/japplphysiol.00991.2005. [DOI] [PubMed] [Google Scholar]
- 9.Mcdonald TP, Cottrell M, Clift R. Effects of short-term hypoxia on platelet counts of mice. Blood . 1978;51(1):165–75. [PubMed] [Google Scholar]
- 10.Christ ER, Cummings MH, Westwood NB, Sawyer BM, Pearson TC, Sonksen PH. et al. The importance of growth hormone in the regulation of erythropoiesis, red cell mass, and plasma volume in adults with growth hormone deficiency. J Clin Endocrinol Metab . 1997;82(9):2985–90. doi: 10.1210/jcem.82.9.4199. [DOI] [PubMed] [Google Scholar]
- 11.Lechner O, Dietrich H, Wiegers GJ, Vacchio M, Wick G. Glucocorticoid production in the chicken bursa and thymus. Int Immunol . 2001;13(6):769–76. doi: 10.1093/intimm/13.6.769. [DOI] [PubMed] [Google Scholar]
- 12.Kojima M, Hosoda H, Matsuo H, Kangawa K. Ghrelin: discovery of the natural endogenous ligand for the growth hormone secretagogue receptor. Trends Endocrinol Metab . 2001;12(3):118–22. doi: 10.1016/s1043-2760(00)00362-3. [DOI] [PubMed] [Google Scholar]
- 13.Gnanapavan S, Kola B, Bustin SA, Morris DG, Mcgee P, Fairclough P. et al. The tissue distribution of the mRNA of ghrelin and subtypes of its receptor, GHS-R, in humans. J Clin Endocrinol Metab . 2002;87(6):2988. doi: 10.1210/jcem.87.6.8739. [DOI] [PubMed] [Google Scholar]
- 14.Kojima M, Kangawa K. Ghrelin: structure and function. Physiol Rev . 2005;85(2):495–522. doi: 10.1152/physrev.00012.2004. [DOI] [PubMed] [Google Scholar]
- 15.DeBoer MD. Use of Ghrelin as a Treatment for Inflammatory Bowel Disease: Mechanistic Considerations. Int J Pept 2011;189242. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Li WG, Gavrila D, Liu X, Wang L, Gunnlaugsson S, Stoll LL. et al. Ghrelin inhibits proinflammatory responses and nuclear factor-kappaB activation in human endothelial cells. Circulation . 2004;109(18):2221–6. doi: 10.1161/01.CIR.0000127956.43874.F2. [DOI] [PubMed] [Google Scholar]
- 17.Camina JP. Cell biology of the ghrelin receptor. J Neuroendocrinol . 2006;18(1):65–76. doi: 10.1111/j.1365-2826.2005.01379.x. [DOI] [PubMed] [Google Scholar]
- 18.Narin N, Çetin E. Effect of ghrelin administration on some hematological parameters in rats. Saglik Bilim Derg . 2010;19(3):202–8. [Google Scholar]
- 19.Taati M, Kheradmand A, Tarahi MJ. Effects of ghrelin on hematopoietic wistar rats. J Guilan Univ Med Sci . 2009;17(68):7–13. [Google Scholar]
- 20.Lotfi A, Aghdam Shahryar H, Narimani Rad M, Dolghari sharaf J, Tunalioglu R, Ova G. Changes in the Differential Leukocyte Count in Newly Hatched Chicks Following In Ovo Ghrelin Administration. Kafkas Univ Vet Fak Derg . 2011;17(6):949–52. [Google Scholar]
- 21.Xia Q, Pang W, Pan H, Zheng Y, Kang JS, Zhu SG. Effects of ghrelin on the proliferation and secretion of splenic T lymphocytes in mice. Regul Pept . 2004;122(3):173–8. doi: 10.1016/j.regpep.2004.06.016. [DOI] [PubMed] [Google Scholar]
- 22.Dixit VD, Schaffer EM, Pyle RS, Collins GD, Sakthivel SK, Palaniappan R. et al. Ghrelin inhibits leptin- and activation-induced proinflammatory cytokine expression by human monocytes and T cells. J Clin Invest . 2004;114(1):57–66. doi: 10.1172/JCI21134. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Alipour MR, Feizi H, Mohaddes G, Keyhanmanesh R, Khamnei S, Ansarin K. et al. Effect of exogenous ghrelin on body weight and hematocrit of male adult rats in chronic hypoxia. Int J Endocrinol Metab . 2010;8(4):201–5. [Google Scholar]
- 24.Thompson NM, Gill DA, Davies R, Loveridge N, Houston PA, Robinson IC. et al. Ghrelin and des-octanoyl ghrelin promote adipogenesis directly in vivo by a mechanism independent of the type 1a growth hormone secretagogue receptor. Endocrinology . 2004;145(1):234–42. doi: 10.1210/en.2003-0899. [DOI] [PubMed] [Google Scholar]
- 25.Ardizzi A, Guzzaloni G, Grugni G, Moro D, Calo G, Mazzilli G. et al. [The effect of GH on erythropoiesis in vivo] Minerva Endocrinol . 1993;18(2):83–5. [PubMed] [Google Scholar]
- 26.Akarsu S, Ustundag B, Gurgoze MK, Sen Y, Aygun AD. Plasma ghrelin levels in various stages of development of iron deficiency anemia. J Pediatr Hematol Oncol . 2007;29(6):384–7. doi: 10.1097/MPH.0b013e3180645170. [DOI] [PubMed] [Google Scholar]
- 27.Van Der Lely, Tschop M, Heiman ML, Ghigo E. Biological, physiological, pathophysiological, and pharmacological aspects of ghrelin. Endocr Rev . 2004;25(3):426–57. doi: 10.1210/er.2002-0029. [DOI] [PubMed] [Google Scholar]
- 28.Aghdam Shahryar H, Lotfi A, Dolghari sharaf J. Effect of intracerebrovascular injection of ghrelin on some of erythropoietic indicators in 42-day-old broiler chicken. Asian Pac J Trop Biomed 2012;S1010-2. [Google Scholar]
- 29.Kheradmand A, Alirezaei M, Birjandi M. Ghrelin promotes antioxidant enzyme activity and reduces lipid peroxidation in the rat ovary. Regul Pept . 2010;162(1-3):84–9. doi: 10.1016/j.regpep.2010.02.008. [DOI] [PubMed] [Google Scholar]
- 30.El Eter E, Al Tuwaijiri A, Hagar H, Arafa M. In vivo and in vitro antioxidant activity of ghrelin: Attenuation of gastric ischemic injury in the rat. J Gastroenterol Hepatol . 2007;22(11):1791–9. doi: 10.1111/j.1440-1746.2006.04696.x. [DOI] [PubMed] [Google Scholar]
- 31.Neamati SH, Alirezaei M, Kheradmand A, Rashidipour M, Saki M. Antioxidant enzyme activities assay and thiobarbituric acid reactive substances concentration following administration of ghrelin in the rat kidney. Int J Pharm Teach Pract . 2013;4(1):451–7. [Google Scholar]
- 32.Liu L, Xu H, Jiang H, Wang J, Song N, Xie J. Ghrelin prevents 1-methyl-4-phenylpyridinium ion-induced cytotoxicity through antioxidation and NF-kappaB modulation in MES23.5 cells. Exp Neurol. 2010;222(1):25–9. doi: 10.1016/j.expneurol.2009.11.009. [DOI] [PubMed] [Google Scholar]
- 33.Xu J, Wang S, Lin Y, Cao L, Wang R, Chi Z. Ghrelin protects against cell death of hippocampal neurons in pilocarpine-induced seizures in rats. Neurosci Lett . 2009;453(1):58–61. doi: 10.1016/j.neulet.2009.01.067. [DOI] [PubMed] [Google Scholar]
- 34.Delhanty PJ, Van Koetsveld PM, Gauna C, Van De Zande B, Vitale G, Hofland LJ. et al. Ghrelin and its unacylated isoform stimulate the growth of adrenocortical tumor cells via an anti-apoptotic pathway. Am J Physiol Endocrinol Metab . 2007;293(1):E302–9. doi: 10.1152/ajpendo.00377.2006. [DOI] [PubMed] [Google Scholar]
- 35.Baldanzi G, Filigheddu N, Cutrupi S, Catapano F, Bonissoni S, Fubini A. et al. Ghrelin and des-acyl ghrelin inhibit cell death in cardiomyocytes and endothelial cells through ERK1/2 and PI 3-kinase/AKT. J Cell Biol . 2002;159(6):1029–37. doi: 10.1083/jcb.200207165. [DOI] [PMC free article] [PubMed] [Google Scholar]