Camel whey protein enhances lymphocyte survival by modulating the expression of survivin, bim/bax, and cytochrome C and restores heat stress-mediated pathological alteration in lymphoid organs

Document Type : Original Article


1 Zoology Department, Faculty of Science, Assiut University, 71516 Assiut, Egypt

2 Animal Health Research Institute, Assiut Branch, 71526 Assiut, Egypt

3 Laboratory of Immunology & Molecular Physiology, Zoology Department, Faculty of Science, Assiut University, 71516 Assiut, Egypt

4 Deanship of Scientific Research, King Saud University, Riyadh, Saudi Arabia

5 Food Science and Nutrition Department, National Research Center, Dokki, 12622 Cairo, Egypt


Objective(s): Heat stress (HS) is a catastrophic stressor that dampens immunity. The current study investigates the effect of dietary administration with camel whey protein (CWP) on apoptotic pathway caused by HS.
Materials and Methods: Forty-five male mice were divided into three groups: a control group; HS group; and HS mice that were orally supplemented with CWP (CWP-HS group).
Results: We found that reactive oxygen species (ROS), pro-inflammatory cytokines (IL-6), and C reactive protein (CRP) were elevated in the HS group along with a significant increase of caspase-9 and -3 and decrease of total antioxidant capacity (TAC). HS mice revealed impaired phosphorylation of Bcl-2 and Survivin, as well as increased expression of Bax, Bim and cytochrome C. Additionally, we observed an aberrant distribution of HSP-70 expressing lymphocytes in the spleen and thymus of HS mice. Moreover, histopathological examination showed alterations on the architectures of immune organs. In comparison with CWP-HS group, we found that CWP restored the levels of ROS, IL-6, TAC and CRP induced by HS. Furthermore, CWP restored the expression of Bcl-2/Bax, improved the histopathological changes in immune organs and HSP-70 distribution in the spleen and thymus.
Conclusion: Our findings revealed the possible ameliorative role of CWP supplementation against damages induced by exposure to HS.


Main Subjects

1. Hansen PJ, Arechiga CF. Strategies for managing reproduction in the heat-stressed dairy cow. J Anim Sci 1999; 77:36-50.
2. Nardone A, Ronchi B, Lacetera N, Ranieri MS, Bernabucci U. Effects of climate changes on animal production and sustainability of livestock systems. Livest Sci 2010; 130:57-69.
3. St-Pierre N, Cobanov B, Schnitkey G. Economic losses from heat stress by US livestock industries. J Dairy Sci 2003; 86:52-77.
4. Drackley JK. Biology of dairy cows during the transition period: The final frontier? J Dairy Sci 1999; 82:2259-2273.
5. Sordillo LM, Aitken SL. Impact of oxidative stress on the health and immune function of dairy cattle. Vet Immunol Immunopathol 2009; 128:104-109.
6. Noordhuizen J, Bonnefoy JM. Heat stress in dairy cattle: major effects and practical management measures for prevention and control. Symbiosis J Vet Sci 2015; 1:103-109.
7. Tao S, Monteiro A, Thompson I, Hayen M, Dahl G. Effect of late-gestation maternal heat stress on growth and immune function of dairy calves. J Dairy Sci 2012; 95:7128-7136.
8. Ganaie A, Shanker G, Bumla N, Ghasura R, Mir N, Wani S, et al. Biochemical and physiological changes during thermal stress in bovines. J Vet Sci Technol 2013; 4:126-132.
9. Sharma S, Ramesh K, Hyder I, Uniyal S, Yadav V, Panda R, et al. Effect of melatonin administration on thyroid hormones, cortisol and expression profile of heat shock proteins in goats (Capra hircus) exposed to heat stress. Small Rumin Res 2013; 112:216-223.
10.    Dhabhar FS. Stress, leukocyte trafficking, and the augmentation of skin immune function. Ann N Y Acad Sci 2003; 992:205-217.
11.    Verghese J, Abrams J, Wang Y, Morano KA. Biology of the heat shock response and protein chaperones: budding yeast (Saccharomyces cerevisiae) as a model system. Microbiol Mol Biol Rev 2012; 76:115-158.
12.    Leite JSM, Cruzat VF, Krause M, de Bittencourt PIH. Physiological regulation of the heat shock response by glutamine: implications for chronic low-grade inflammatory diseases in age-related conditions. Nutrire 2016; 41:17.
13.    Beede D, Collier R. Potential nutritional strategies for intensively managed cattle during thermal stress. J Anim Sci 1986; 62:543-554.
14.    BV SK, Ajeet K, Meena K. Effect of heat stress in tropical livestock and different strategies for its amelioration. Journal of Stress Physiology & Biochemistry 2011; 7.
15.    El-Hatmi H, Girardet JM, Gaillard JL, Yahyaoui MH, Attia H. Characterisation of whey proteins of camel (Camelus dromedarius) milk and colostrum. Small Rumin Res 2007; 70:267-271.
16.    Badr G, Ebaid H, Mohany M, Abuelsaad AS. Modulation of immune cell proliferation and chemotaxis towards CC chemokine ligand (CCL)-21 and CXC chemokine ligand (CXCL)-12 in undenatured whey protein-treated mice. J Nutr Biochem 2012; 23:1640-1646.
17.    Badr G, Mohany M, Metwalli A. Effects of undenatured whey protein supplementation on CXCL12-and CCL21-mediated B and T cell chemotaxis in diabetic mice. Lipids Health Dis 2011; 10:203.
18.    El-Hatmi H, Levieux A, Levieux D. Camel (Camelus dromedarius) immunoglobulin G, α-lactalbumin, serum albumin and lactoferrin in colostrum and milk during the early post partum period. J Dairy Res 2006; 73:288-293.
19.    Gauthier SF, Pouliot Y, Saint-Sauveur D. Immunomodulatory peptides obtained by the enzymatic hydrolysis of whey proteins. Int Dairy J 2006; 16:1315-1323.
20.    Beaulieu J, Dupont C, Lemieux P. Whey proteins and peptides: beneficial effects on immune health. Future Medicine 2006; 3:69-78.
21.    Badr G. Supplementation with undenatured whey protein during diabetes mellitus improves the healing and closure of diabetic wounds through the rescue of functional long-lived wound macrophages. Cell Physiol Biochem 2012; 29:571-582.
22.    Ebaid H. Neutrophil depletion in the early inflammatory phase delayed cutaneous wound healing in older rats: improvements due to the use of un-denatured camel whey protein. Diagn Pathol 2014; 9:46.
23. Badr G, Abdel‐Tawab HS, Ramadan NK, Ahmed SF, Mahmoud MH. Protective effects of camel whey protein against scrotal heat‐mediated damage and infertility in the mouse testis through YAP/Nrf2 and PPAR‐gamma signaling pathways. Mol Reprod Dev 2018;85:505-518.
24.    Badr G, Sayed LH, Omar HE-DM, El-Rahim AMA, Ahmed EA, Mahmoud MH. Camel whey protein protects B and T cells from apoptosis by suppressing activating transcription factor-3 (ATF-3)-mediated oxidative stress and enhancing phosphorylation of AKT and IκB-α in type I diabetic mice. Cell Physiol Biochem 2017; 41:41-54.
25.    Chen W, Woodruff TK, Mayo KE. Activin A-induced HepG2 liver cell apoptosis: involvement of activin receptors and smad proteins. Endocrinology 2000; 141:1263-1272.
26.    MATSUDA‐MINEHATA F, Maeda A, Cheng Y, Sai T, Gonda H, Goto Y, et al. Regulation of granulosa cell apoptosis by death ligand–receptor signaling. J Anim Sci Technol 2008; 79:1-10.
27.    Oltval ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programed cell death. Cell 1993; 74:609-619.
28.    Gillies LA, Kuwana T. Apoptosis regulation at the mitochondrial outer membrane. J Cell Biochem 2014; 115:632-640.
29.    Sudo H, Minami A. Regulation of apoptosis in nucleus pulposus cells by optimized exogenous Bcl‐2 overexpression. J Orthop Res 2010; 28:1608-1613.
30.    Chipuk JE, Green DR. How do BCL-2 proteins induce mitochondrial outer membrane permeabilization?. Trends Cell Biol 2008; 18:157-164.
31.    Lalier L, Cartron PF, Juin P, Nedelkina S, Manon S, Bechinger B, et al. Bax activation and mitochondrial insertion during apoptosis. Apoptosis 2007; 12:887-896.
32.    Oshikawa T, Okamoto M, Ahmed SU, Tano T, Sato M. The relationship between gene expression of Bcl-2 and Bax and the therapeutic effect in oral cancer patients. Gan to kagaku ryoho 2006; 33:1723-1725.
33.    Badr G. Camel whey protein enhances diabetic wound healing in a streptozotocin-induced diabetic mouse model: the critical role of β-Defensin-1,-2 and-3. Lipids in health and disease 2013;12:1-
34.    Mohany M, El-Feki M, Refaat I, Garraud O, Badr G. Thymoquinone ameliorates the immunological and histological changes induced by exposure to imidacloprid insecticide. J Toxicol Sci 2012; 37:1-11.
35.    Badr G, Al-Sadoon MK, Rabah DM. Therapeutic efficacy and molecular mechanisms of snake (Walterinnesia aegyptia) venom-loaded silica nanoparticles in the treatment of breast cancer-and prostate cancer-bearing experimental mouse models. Free Radic Biol Med 2013; 65:175-189.
36.    Wang X, Yuan B, Dong W, Yang B, Yang Y, Lin X, et al. Induction of heat-shock protein 70 expression by geranylgeranylacetone shows cytoprotective effects in cardiomyocytes of mice under humid heat stress. PloS One 2014; 9:93536.
37.    Badr G, Mohany M. Maternal perinatal undernutrition attenuates T-cell function in adult male rat offspring. Cell Physiol Biochem 2011; 27:381-390.
38.    Al-Sadoon MK, Rabah DM, Badr G. Enhanced anticancer efficacy of snake venom combined with silica nanoparticles in a murine model of human multiple myeloma: molecular targets for cell cycle arrest and apoptosis induction. Cell Immunol 2013; 284:129-138.
39.    Badr G, Al-Sadoon MK, Rabah DM, Sayed D. Snake (Walterinnesia aegyptia) venom-loaded silica nanoparticles induce apoptosis and growth arrest in human prostate cancer cells. Apoptosis 2013;18:300-314.
40.    Badr G, Saad H, Waly H, Hassan K, Abdel-Tawab H, Alhazza IM, et al. Type I interferon (IFN-α/β) rescues B-lymphocytes from apoptosis via PI3Kδ/Akt, Rho-A, NFκB and Bcl-2/Bcl XL. Cell Immunol 2010; 263:31-40.
41.    Badr G, Garraud O, Daghestani M, Al-Khalifa MS, Richard Y. Human breast carcinoma cells are induced to apoptosis by samsum ant venom through an IGF-1-dependant pathway, PI3K/AKT and ERK signaling. Cell Immunol 2012; 273:10-16.
42.    Badr G, Al-Sadoon MK, El-Toni AM, Daghestani M. Walterinnesia aegyptia venom combined with silica nanoparticles enhances the functioning of normal lymphocytes through PI3K/AKT, NFκB and ERK signaling. Lipids Health Dis 2012; 11:27.
43.    Drury R, Wallington E. Preparation and Fixation of Tissues. Carleton’s Histological Technique 1980; 5:41-54.
44.    Al-Sadoon MK, Abdel-Maksoud MA, Rabah DM, Badr G. Induction of apoptosis and growth arrest in human breast carcinoma cells by a snake (Walterinnesia aegyptia) venom combined with silica nanoparticles: crosstalk between Bcl2 and caspase 3. Cell Physiol Biochem 2012; 30:653-665.
45.    Sayed D, Al-Sadoon MK, Badr G. Silica nanoparticles sensitize human multiple myeloma cells to snake (Walterinnesia aegyptia) venom-induced apoptosis and growth arrest. Oxid med cell longev 2012; 2012:386286.
46.    Power O, Jakeman P, FitzGerald R. Antioxidative peptides: enzymatic production, in vitro and in vivo antioxidant activity and potential applications of milk-derived antioxidative peptides. Amino Acids 2013; 44:797-820.
47.    Shin MH, Moon YJ, Seo JE, Lee Y, Kim KH, Chung JH. Reactive oxygen species produced by NADPH oxidase, xanthine oxidase, and mitochondrial electron transport system mediate heat shock-induced MMP-1 and MMP-9 expression. Free Radic Biol Med 2008; 44:635-645.
48.    Al-Humaid A, Mousa H, El-Mergawi R, Abdel-Salam A. Chemical composition and antioxidant activity of dates and dates-camel-milk mixtures as a protective meal against lipid peroxidation in rats. Am J Food Technol 2010; 5:22-30.
49.    Ozdemir G, Inanc F. Zinc may protect remote ocular injury caused by intestinal ischemia reperfusion in rats. Tohoku J Exp Med 2005; 206:247-251.
50.    Schett G, Redlich K, Xu Q, Bizan P, Gröger M, Tohidast-Akrad M, et al. Enhanced expression of heat shock protein 70 (hsp70) and heat shock factor 1 (HSF1) activation in rheumatoid arthritis synovial tissue. Differential regulation of hsp70 expression and hsf1 activation in synovial fibroblasts by proinflammatory cytokines, shear stress, and antiinflammatory drugs. J Clin Invest 1998; 102:302-311.
51.    Calabró P, Willerson JT, Yeh ET. Inflammatory cytokines stimulated C-reactive protein production by human coronary artery smooth muscle cells. Circulation 2003; 108:1930-1932.
52.    Chun OK, Chung SJ, Song WO. Estimated dietary flavonoid intake and major food sources of US adults. J Nutr 2007; 137:1244-1252.
53.    Ghiselli A, Serafini M, Natella F, Scaccini C. Total antioxidant capacity as a tool to assess redox status: critical view and experimental data. Free Radic Biol Med 2000; 29:1106-1114.
54.    Lin X, Lin CH, Zhao T, Zuo D, Ye Z, Liu L, et al. Quercetin protects against heat stroke-induced myocardial injury in male rats: Antioxidative and antiinflammatory mechanisms. Chem Biol Interact 2017; 265:47-54.
55.    Kasahara E, Miyoshi M, Konaka R, Hiramoto K, Sasaki J, Tokuda M, et al. Role of oxidative stress in germ cell apoptosis induced by di (2-ethylhexyl) phthalate. Biochem J 2002; 365:849-856.
56.    Gao HB, Tong MH, Hu YQ, You HY, Guo QS, Ge RS, et al. Mechanisms of glucocorticoid-induced leydig cell apoptosis. Mol Cell Endocrinol 2003; 199:153-163.
57.    Cong X, Zhang Q, Li H, Jiang Z, Cao R, Gao S, et al. Puerarin ameliorates heat stress–induced oxidative damage and apoptosis in bovine Sertoli cells by suppressing ROS production and upregulating Hsp72 expression. Theriogenology 2017; 88:215-227.
58.    Li L, Tan H, Gu Z, Liu Z, Geng Y, Liu Y, et al. Heat stress induces apoptosis through a Ca 2+-mediated mitochondrial apoptotic pathway in human umbilical vein endothelial cells. PLoS One 2014; 9:e111083.
59.    Pagliari LJ, Kuwana T, Bonzon C, Newmeyer DD, Tu S, Beere HM, et al. The multidomain proapoptotic molecules Bax and Bak are directly activated by heat. Proc Natl Acad Sci U S A 2005; 102:17975-17980.
60.    Sayed LH, Badr G, Omar HM, El-Rahim AMA, Mahmoud MH. Camel whey protein improves oxidative stress and histopathological alterations in lymphoid organs through Bcl-XL/Bax expression in a streptozotocin-induced type 1 diabetic mouse model. Biomed Pharmacother 2017; 88:542-552.
61.    Li F, Ambrosini G, Chu EY, Plescia J, Tognin S, Marchisio PC, et al. Control of apoptosis and mitotic spindle checkpoint by survivin. Nature 1998; 396:580-584.
62.    Reed JC, Bischoff JR. BIRinging chromosomes through cell division—and survivin’the experience. Cell 2000; 102:545-548.
63.    Mirza A, McGuirk M, Hockenberry TN, Wu Q, Ashar H, Black S, et al. Human survivin is negatively regulated by wild-type p53 and participates in p53-dependent apoptotic pathway. Oncogene 2002; 21:2613.
64.    Zaffaroni N, Pennati M, Colella G, Perego P, Supino R, Gatti L, et al. Expression of the anti-apoptotic gene survivin correlates with taxol resistance in human ovarian cancer. Cell Mol Life Sci 2002; 59:1406-1412.
65.    Jai YY, Silke J, Ekert PG. Inhibitor of Apoptosis Proteins and Caspases.  Apoptosis, Cell Signaling, and Human Diseases: Springer; 2006. p.313-334.
66.    Kim MG, Cho EJ, Lee JW, Ko YS, Lee HY, Jo SK, et al. The heat-shock protein-70–induced renoprotective effect is partially mediated by CD4+ CD25+ Foxp3+ regulatory T cells in ischemia/reperfusion-induced acute kidney injury. Kidney Int 2014; 85:62-71.
67.    Heck TG, Schöler CM, de Bittencourt PIH. HSP70 expression: does it a novel fatigue signalling factor from immune system to the brain?. Cell Biochem Funct 2011; 29:215-226.
68.    Di YP, Repasky EA, Subjeck JR. Distribution of HSP70, protein kinase C, and spectrin is altered in lymphocytes during a fever‐like hyperthermia exposure. J Cell Physiol 1997; 172:44-54.
69.    Stankiewicz AR, Lachapelle G, Foo CP, Radicioni SM, Mosser DD. Hsp70 inhibits heat-induced apoptosis upstream of mitochondria by preventing Bax translocation. J Biol Chem 2005; 280:38729-38739.
70.    Khan VR, Brown IR. The effect of hyperthermia on the induction of cell death in brain, testis, and thymus of the adult and developing rat. Cell Stress Chaperones 2002; 7:73-90.
71.    Mosser DD, Duchaine J, Bourget L, Martin LH. Changes in heat shock protein synthesis and heat sensitivity during mouse thymocyte development. genesis 1993; 14:148-158.
72.    Ravagnan L, Gurbuxani S, Susin SA, Maisse C, Daugas E, Zamzami N, et al. Heat-shock protein 70 antagonizes apoptosis-inducing factor. Nat Cell Biol 2001; 3:839-843.
73.    Adly AA. Oxidative stress and disease: an updated review. Res J Immunol 2010; 3:129-145.
74.    Roberts GT, Ghebeh H, Chishti MA, Al-Mohanna F, El-Sayed R, Al-Mohanna F, et al. Microvascular injury, thrombosis, inflammation, and apoptosis in the pathogenesis of heatstroke. Arterioscler Thromb Vasc Biol 2008; 28:1130-1136.
75.    Brinton MR, Tagge CA, Stewart RJ, Cheung AK, Shiu Y-TE, Christensen DA. Thermal sensitivity of endothelial cells on synthetic vascular graft material. Int J Hyperthermia 2012; 28:163-174.