Volume 2, Issue 1 (2-2018)                   GMJ Medicine 2018, 2(1): 27-32 | Back to browse issues page

XML Print

Download citation:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:

Bakhtiari A, Aboudzadeh S, Vaziri S, Mirzaei Roozbahani M. Efficiency of Probiotic in Immobilized Rats Through Involvement of the Antioxidant and Anti-inflammatory Systems. GMJ Medicine. 2018; 2 (1) :27-32
URL: http://gmjpress.de/article-1-33-en.html
1- Department of Internal Medicine, Lorestan University of Medical Sciences, Khorramabad, Iran
2- Imam Ali Health Center, Abadan University of Medical Sciences, Abadan, Iran
3- Student Research Committee, Hormozgan University of Medical Sciences, Bandar Abbas, Iran
4- Department of General Surgery, Lorestan University of medical Sciences, Khorramabad, Iran
Full-Text [PDF 806 kb]   (807 Downloads)     |   Abstract (HTML)  (1718 Views)
Full-Text:   (759 Views)
Stress is known as a factor that causes negative effects on general physiology and leads psychology [1], heart and causes diabetes and loss weight [2]. It also causes faulted working memory, anxious and depression [3]. Studies have accepted the role of stress in suppression of immunity [4] and intestinal microflora [5]. It also causes to produce the free radicals, oxidative damages on macromolecules [6]. Stress also increases production of pro-inflammatory cytokines such as transcription factor NF-κB-mediated pathways and other inflammatory mediators [7]. In addition, stress is related with increased oxidative stress that stimulates phosphorylation of mitogen-activated protein kinases in animal models [8, 9]. Immobilization is type of physical stress that limits mobilization and increases aggression in the animal model [2] and is also used to study the stress-induced changes [10].

Experimental studies that have shown the possible effects of probiotics in the psychological status of the host are rare. Gastrointestinal tract is known as metabolic active organ which contains various microbial species. It has been accepted that decreased healthy microflora provides opportunity for pathogenic bacteria and increases inflammation [11]. It is shown that specific probiotic strains reduce production of pro-inflammatory cytokine through decreasing integrity of the tight junctional complexes between epithelial cells [12, 13]. The profitable role of probiotic lactobacilli and bifido bacteria for inflammatory condition has been reported [14]. A study has also been shown that probiotics significantly decrease the basal levels of some oxidative stress markers and increase the powerful of antioxidant enzymes; showing antioxidant properties of probiotics [15]. Immobilization is type of stress that influences inflammation and oxidative stress. On the other hand, probiotics can have positive effects on oxidative stress and inflammation. So far, no study has been conducted to evaluate the effects of probiotics on immobility stress.  This study was thus conducted to evaluate the effects of probiotics in immobilized rats through involvement of the antioxidant and anti-inflammatory systems.
Materials and Methods

Commercial kits of interlukin-10 (IL-10), tumor necrosis factor-α (TNF-α), nitric oxide (NO), corticostrone, transforming growth factor-beta (TGF-β), malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPx) were purchased from Sigma Aldrich Company. Commercial strains of Lactobacillus plantarum and Bifidubacterium B94 were selected to test.
A total number of 72Albino Wistar rats (6 weeks-age, 170±10 g) were adapted for one week before of trial and kept on basis the animal welfare laws. All the animals were maintained in an optimal temperature (25±1 ºC), and humidity (55±5%) and illumination period (12 h light and 12 h dark) were kept in the experimental period. Animals received ad libitum water and feed. To induce the immobilization stress, animals were exposed to stress as reported by others [16]. Animals were exposed to immobilization for 2h/d and for 2 weeks by a restraining chamber [17]. In days 7 and 14 of conduction of trial, animals were anesthetized; blood samples were collected and sera were collected.
Animals were divided into 3 groups (n=24) and each group was divided into 4 sub-groups. Groups were included: 1) Normal control group without stress (Control), 2) Immobilized rats without additive (Immobilized), 3) Immobilized rats given with 104 CFU probiotic.day-1(Probiotic).
Inflammatory cytokines and antioxidant status
Inflammatory and pro-inflammatory cytokines including IL-10, TNF-α, TGF-β and also NO and corticostrone were measured in days 7 and 14. The MDA, SOD and GPx concentrations in serum were evaluated by commercial kits according to the manufacturers’ instructions in days 7 and 14. 
Statistical AnalysisThe obtained data were analyzed by using Graph Pad Prism 7.0 (GraphPad Software, Inc., 7825 Fay Avenue, Suite230, La Jolla, CA, USA) and reported as mean ±standard deviation (SD). Groups were compared by Tukey test.
Corticostrone, MDA and NO concentrations
Our results for serum concentrations of corticostrone, MDA and NO in immobilized rats are presented in Table 1. As results shows, immobilization could significantly increase the serum concentrations of corticostrone, MDA and NO in days 7 and 14, as immobilized rats were compared with control rats (P<0.05). However, administration of probiotic could significantly decrease the levels of corticostrone, MDA and NO (P<0.05).

Table 1 Effects of experimental treatments on serum concentrations of corticostrone, MDA and NO in immobilized rats

Inflammatory factors
Our findings for inflammatory responses are presented in Figure 1. Results showed that exposing to immobilization increased levels of TNF-α and TGF-β and decreased levels of IL-10 in days 7 and 14 (P<0.05). Treatment with probiotic could significantly decrease TNF-α and TGF-β and increase levels of IL-10 in days 7 and 14 (P<0.05).

Figure 1 Effects of experimental treatments on inflammatory responses in immobilized rats. Superscripts (a-c) show significant differences among groups in same day.

Antioxidant enzymes
Our findings for antioxidant status are shown in Figure 2. Results showed that the serum concentrations of SOD and GPx were decreased in immobilized rats but treatment with probiotic could significantly improve antioxidant status in days 7 and 14 (P<0.05).


Figure 2 Effects of experimental treatments on antioxidant status in immobilized rats. Superscripts (a-c) show significant differences among groups in same day.
Immobility stress increased levels of MDA, NO and corticostrone. It has been accepted that stress increases production of reactive oxygen species by biological system ability [18] and increases MDA production as marker for lipid per-oxidation. MDA shows increased oxidative damage and reduced antioxidants. It has been reported relation between stress, increased corticosterone and intestinal injuries [19]. Stress changes neuroendocrine system through activation of the hypothalamic-pituitary-adrenal axis and increases production of corticosterone [20]. Stress increases corticostrone production and changes structural proteins [21]. It has been reported that MDA and NO as inflammatory biomarkers [22]. On the other hand, oxidative stress plays major role in pathogenesis of immobilized stress [23]. Improved MDA and NO were observed in probiotic groups. Improved MDA and NO could be attributed to antioxidant and anti-inflammatory responses that would be discussed. It has been reported that probiotic strains of L. rhamnosus [24], Enterococcus faecium [25] and L. acidophilus [26] improve antioxidant capacity which could be attributed to microbial metabolic activity during fermentation [27]. Increased inflammatory responses were observed in rats exposed to stress. It has been reported that stress increases corticostrone and increased corticostrone decreases mRNA expression of anti-inflammatory cytokines, i.e. IL-10 [28], as observed in the current study. IL-10 is known as one of the most important anti-inflammatory cytokines which prevents production of pro-inflammatory cytokines such as TNF-α [29].IL-10 has been reported to have anti-inflammatory properties that is formed by both T-cells and monocytes/macrophages. The TNF-α has been known as an inflammatory cytokine that initiates inflammation. It means that immobilization stress increases inflammation but probiotic decreased inflammation. Stress provides opportunity for pathogens that increases inflammation. Previous studies have reported that L. fermentum could significantly decrease jejunal inflammation of the upper small intestine after 5-FU administration in rats [30]. Improved anti-inflammatory properties could be attributed to reduced pro-inflammatory cytokines, increased immature leukocyte production and interferon production [31, 32]. Probiotics also decrease pathogen-induced inflammation that is caused to improve the intestinal ecosystem. It could be stated that probiotics improve inflammation condition by mentioned mechanisms. Stress also decreased levels of antioxidant enzymes. Stress stimulates and increases metabolic rate that caused to produce high free radicals and also oxidative damage [6]. Stress decreases antioxidant enzymes but probiotic improved them. Antioxidant properties of probiotics have previously been reported [15] but mechanism is unknown.
In summary, the immobilization similar to other stresses increased inflammation and oxidation. Probiotic could improve inflammatory responses and antioxidant properties. It could be recommended to use the probiotics as daily supplement in patients related with stress such as depression and similar conditions. 

Ethical Considerations

Compliance with ethical guidelines

Approval for this study was obtained from Hormozgan University  of  Medical  Sciences  Research  Committee (HUMS-2017-122539).

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Authors' contributions
All authors contributed toward data analysis, drafting and revising the paper and agreed to be responsible for all the aspects of this work.
Conflict of interest
The authors declared no conflict of interest.


[1] Sheikh N, Ahmad A, Siripurapu KB, Kuchibhotla VK, Singh S, Palit G. Effect of Bacopa monniera onstress induced changes in plasma corticosterone and brain monoamines in rats. J Ethnopharmacol. 2007; 111: 671–676. [DOI:10.1016/j.jep.2007.01.025] [PMID]

[2] Ramadan KS, Alshamrani SA. Effects of Salvadora persica extract on the hematological and biochemicalalterations against immobilization-induced rats. Scientifica. 2015; 2015: 253195. [DOI:10.1155/2015/253195] [PMID] [PMCID]

[3] Patki, G, Solanki, N, Atrooz, F, Allam, F, Salim, S. Depression, anxiety-like behavior and memoryimpairment are associated with increased oxidative stress and inflammation in a rat model of social stress.Brain Res. 2013; 1539: 73–86. [DOI:10.1016/j.brain-res.2013.09.033] [PMID] [PMCID]

[4] Viswanathan K, Dhabhar FS. Stress-induced enhancement of leukocyte trafficking into sites of surgery orimmune activation. Proc Natl Acad Sci USA. 2005; 102: 5808–5813. [DOI:10.1073/pnas.0501650102] [PMID] [PMCID]

[5] Bailey MT, Dowd SE, Galley JD, Hufnagle AR, Allen RG, Lyte M. Exposure to a social stressoralters the structure of the intestinal microbiota: Implications for stressor-induced immunomodulation.Brain Behav Immun. 2011; 25: 397–407. [DOI:10.1016/j.bbi.2010.10.023] [PMID] [PMCID]

[6] Halliwell B. Free radicals, antioxidants, and human disease: Curiosity, cause, or consequence? Lancet. 1994; 344: 721–724. [DOI:10.1016/S0140-6736(94)92211-X]

[7] Madrigal JL, Moro MA, Lizasoain I, Lorenzo P, Castrillo A, Bosca L, Leza J.C. Inducible nitric oxidesynthase expression in brain cortex after acute restraint stress is regulated by nuclear factor κB-mediatedmechanisms. J Neurochem. 2001; 76: 532–538. [DOI:10.1046/j.1471-4159.2001.00108.x] [PMID]

[8] Sasaguri K, Kikuchi M, Hori N, Yuyama N, Onozuka M, Sato S. Suppression of stressimmobilization-induced phosphorylation of ERK 1/2 by biting in the rat hypothalamic paraventricularnucleus. Neurosci Lett. 2005; 383: 160–164. [DOI:10.1016/j.neu-let.2005.04.011] [PMID]

[9] Hebert MA, Serova LI, Sabban EL. Single and repeated immobilization stress differentially triggerinduction and phosphorylation of several transcription factors and mitogen-activated protein kinases in therat locus coeruleus. J Neurochem. 2005; 95: 484–498. [DOI:10.1111/j.1471-4159.2005.03386.x] [PMID]

[10] Ahmad A, Rasheed N, Chand K, Maurya R, Banu N, Palit G. Restraint stress-induced centralmonoaminergic & oxidative changes in rats & their prevention by novel Ocimum sanctum compounds.Indian J Med Res. 2012; 135: 548–554.

[11] Honad K, Littman DR: The microbiome in infectious disease and inflammation. Ann Rev Immunol.2012; 30:758-795. [DOI:10.1146/annurev-immunol-020711-074937] [PMID] [PMCID]

[12] Heyman M, Terpend K, Menard S. Effects of specific lactic acid bacteria on the intestinal permeability to macromolecules and the inflammatory condition. Acta Paediatr Suppl. 2005; 94(449):34-36. [DOI:10.1111/j.1651-2227.2005.tb02153.x] [PMID]

[13] Donato KA, Gareau M, Wang YJ, Sherman PM. Lactobacillus rhamnosus GG attenuates interferon-{gamma} and tumor necrosis factor-a-induced barrier dysfunction and pro-inflammatory signalling. Microbiol. 2010;156:3288-3297. [DOI:10.1099/mic.0.040139-0] [PMID]

[14] Okada Y, Tsuzuki Y, Hokari R, Komoto S, Kurihara C,Kawaguchi A., Nagao, S, MiuraS. Anti-inflammatoryeffects of the genus Bifidobacterium on macrophages by modificationof phospho-IkB and SOCS gene expression. Int J Exp Pathol. 2009; 90: 131–140. [DOI:10.1111/j.1365-2613.2008.00632.x] [PMID] [PMCID]

[15] Divyashri G, Krishna G, Muralidhara B, Prapulla SG. Probiotic attributes, antioxidant, anti-inflammatory and neuromodulatory effects of Enterococcus faecium CFR 3003: in vitro and in vivo evidence. J Med Microbiol. 2015; 64: 1527–1540. [DOI:10.1099/jmm.0.000184] [PMID]

[16] Yang HJ, Kim KY, Kang P, Lee HS, Seol GH. Effects of Salvia sclarea on chronic immobilization stress induced endothelial dysfunction in rats. BMC Complement Altern Med. 2014; 14: 396. [DOI:10.1186/1472-6882-14-396] [PMID] [PMCID]

[17] Rubisz-Brzezinska J, Jonderko G, Zebracka T, Dyczek-Parys E. Immunoglobulin E levels in selecteddermatoses. PrzDermatol. 1977; 64:17–22.

[18] Samarghandian S, Azimi-Nezhad M, Samini F.Preventive effect of safranal against oxidative damage in aged male rat brain.Exper Anim. 2015; 64: 65–71. [DOI:10.1538/expanim.14-0027] [PMID] [PMCID]

[19] Olfati A, Mojtahedin A, Sadeghi T, Akbari M, Martínez-Pastor F. Comparison of growth performance and immune responses of broiler chicks reared under heat stress, cold stress and thermoneutral conditions. Spanish J Agric Res. 2018; 16:1-7. [DOI:10.5424/sjar/2018162-12753]

[20] Quinteiro-Filho WM, Gomes AV, Pinheiro ML, Ribeiro A, Ferraz-de-Paula V, Astolfi-Ferreira CS, Ferreira AJ, Palermo-Neto J, 2012. Heat stress impairs performance and induces intestinal inflammation in broiler chickens infected with Salmonella enteritidis. Avi Pathol.2012; 41: 421- 427. [DOI:10.1080/03079457.2012.709315.] [PMID]

[21] Lin H, Decuypere E, Buyse J. Oxidative stress induced by corticosterone administration in broiler chickens (Gallus gallus domesticus) 1. Chronic exposure. Comp Biochem Physiol. 2004; 139: 737-744. [DOI:10.1016/j.cbpc.2004.09.013] [PMID]

[22] Fiore R, Miller, R, Coffman SM. Mycobacterium mucogenicum infection following a cosmeticprocedure with poly-L-lactic acid. J Drugs Dermatol. 2013; 12: 353–357.

[23] Zafir A, Banu, N. Induction of oxidative stress by restraint stress and corticosterone treatments in rats.Ind J Biochem Biophys. 2009; 46: 53–58.

[24] Marazza JA, Nazareno MA, de Giori GS, Garro MS. Enhancement of the antioxidant capacity ofsoymilk by fermentation with Lactobacillus rhamnosus. J Funct Food.2012; 4: 594–601. [DOI:10.1016/j.jff.2012.03.005]

[25] Divyashri G, Krishna G, Muralidhara Prapulla SG. Probiotic attributes, antioxidant, anti-inflammatoryand neuromodulatory effects of Enterococcus faecium CFR 3003: In vitro and in vivo evidence.J Med Microbiol. 2015; 64: 1527–1540. [DOI:10.1099/jmm.0.000184] [PMID]

[26] Mousavi ZE, Mousavi SM, Razavi SH, Hadinejad M, Emam-Djomeh Z, Mirzapour M. Effectof Fermentation of Pomegranate Juice by Lactobacillus plantarum and Lactobacillus acidophilus on theAntioxidant Activity and Metabolism of Sugars, Organic Acids and Phenolic Compounds. Food Biotechnol.2013; 27: 1–13. [DOI:10.1080/08905436.2012.724037] 

[27] Tamang JP, Shin DH, Jung SJ, Chae SW. Functional Properties of Microorganisms in Fermented Foods.Front Microbiol. 2016; 7: 578[DOI:10.3389/fmicb.2016.00578] 

[28] Quinteiro-Filho WM, Calefi AS, Cruz DSG, Aloia TPA, Zager A, Astolfi-Ferreira CS, Piantino Ferreira JA, Sharif S, Palermo-Neto J. Heat stress decreases expression of the cytokines, avian β-defensins 4 and 6 and Toll-like receptor 2 in broiler chickens infected with Salmonella enteritidis. Vet Immunol Immunopathol. 2017; 186: 19-28. [DOI:10.1016/j.vetimm.2017.02.006.] [PMID]

[29] de Waal Malefyt R, Abrams J, Bennett B, Figdor CG,de Vries JE. Interleukin 10 (IL-10) inhibits cytokine synthesisby human monocytes: an autoregulatory role of IL-10 roduced by monocytes. J Exp Med.1991;174 (5):1209-1220. [DOI:10.1084/jem.174.5.1209] [PMID]

[30] Smith CL, Geier MS, Yazbeck R, Torres DM,Butler RN, Howarth GS. Lactobacillus fermentumBR11 and fructo-oligosaccharide partially reducejejunal inflammation in a model of intestinalmucositis in rats. Nutr Cancer. 2008;60(6):757-67. [DOI:10.1080/01635580802192841.] [PMID]

[31] Park SY, Kim YH, Kim EK, Ryu EY, Lee SJ. Hemeoxygenase-1 signals are involved in preferentialinhibition of pro-inflammatory cytokine release bysurfactin in cells activated with Porphyromonas gingivalislipopolysaccharide. Chem Biol Interact. 2010;188(3):437-45. [DOI:10.1016/j.cbi.2010.09.007.] [PMID]

[32] Brunt J, Austin B. The development of probiotics for the controlof multiple bacterial diseases of rainbow trout , Oncorhynchusmykiss (Walbaum). J Fish Dis. 2007;30(10):573-9. [DOI:10.1111/j.1365-2761.2007.00836.x] [PMID]
Type of Study: Original Article | Subject: Immunology
Received: 2018/03/27 | Accepted: 2018/05/15 | Published: 2018/06/10

Add your comments about this article : Your username or Email:

Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

© 2021 CC BY-NC 4.0 | GMJ Medicine