La prevenzione di stress termico effetti avversi nei ratti<em> Bacillus subtilis</em> Strain
Summary
We described a protocol for prevention of heat stress effects in rats by oral pre-treatment with beneficial bacteria. This protocol can be modified and used for various routes of administration and for analysis of different compounds.
Abstract
Questo studio è stato disegnato per valutare l'effetto protettivo del ceppo Bacillus subtilis contro complicazioni legate allo stress da calore. Trentadue ratti Sprague-Dawley sono stati utilizzati in questo studio. Gli animali sono stati trattati per via orale due volte al giorno per due giorni con B. subtilis ceppo BSB3 o PBS. Il giorno dopo l'ultimo trattamento, ciascun gruppo è stato diviso e due gruppi sperimentali (uno trattato con PBS e uno trattato con B. subtilis) sono stati posti a 45 ° C per 25 min. Due gruppi di controllo alloggiato per 25 min a temperatura ambiente. Tutti i ratti sono stati sacrificati e diversi parametri sono stati analizzati in tutti i gruppi. Gli effetti avversi di stress da calore sono registrati dalla diminuzione di altezza villi e lo spessore della mucosa totale a livello dell'epitelio intestinale; traslocazione di batteri dal lume; aumento vescicolazione degli eritrociti ed elevazione dei lipopolisaccaridi (LPS) concentrazione nel sangue. L'efficacia protettiva di trattamento viene valutata preintervento di questi effetti collaterali. Il protocollo è stato istituito per il trattamento orale di ratti con batteri per la prevenzione delle complicanze di stress termico, ma questo protocollo può essere modificato e utilizzato per altre vie di somministrazione e per l'analisi di composti diversi.
Introduction
Different stressors affect human and animal health. Temperature is one of the most stressful factors, causing chronic, acute and even lethal illnesses1. Changes in intestinal morphology and a loss of gut barrier integrity after heat stress were documented in many cases2,3. This protective barrier is responsible for defense against translocation of gut bacteria and their toxins, in particular lipopolysaccharides (LPS), from the lumen to the internal circulation4,5. Stability of the gut microbiota significantly influences intestinal barrier function, the immune response of the host, and tolerance to stress conditions6. Thus, modulation of the intestinal microbiota can provide a novel approach for prevention of stress-related adverse effects.
Beneficial bacteria have been used as a promising strategy to modify the gut microbiota for successful management of various clinical conditions, such as diarrhea, inflammatory bowel disease, atopic dermatitis, metabolic disorders7-10. Probiotic effects of beneficial bacteria include production of essential metabolites to support intestinal health, stimulation of the immune system, promotion of lactose tolerance, restoration of epithelial dysfunction. Probiotics were effective in prevention of the stress-related complications in vitro and in animal models11,12.
Researchers have given more attention to Bacillus bacteria as probiotics because these bacteria support intestinal homeostasis13 and have beneficial effects on the host14. Our previous data demonstrated high efficacy of Bacillus probiotics against pathogens in vitro15,16 and in clinical trials17,18. Here, we aim to evaluate the preventive effect of B. subtilis BSB3 strain against complications after heat stress.
Protocol
Representative Results
Discussion
L'esposizione a risultati di alta temperatura in gravi condizioni di salute 29. La prevenzione e la diagnosi precoce delle complicanze legate allo stress termico sono di vitale importanza 30. Il protocollo presentato può essere utilizzato per valutare l'efficacia di vari approcci per la prevenzione degli effetti avversi tensione termica.
Passaggi critici all'interno di questo protocollo sono: la procedura di trattamento termico (45 ?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
This work was supported by the Auburn University fund FOP 101002-139294-2050.
Materials
| Phosphate buffered saline (PBS) | Sigma-Aldrich, St. Louis, MO | P4417 | |
| Ethyl Alcohol | Pharmco products Inc. Brookfield, CT, USA | 64-17-5 | |
| Agar | VWR | 97064-334 | |
| MacConkey agar plates | VWR | 470180-742 | |
| 5% blood agar plates | VWR | 89405-024 | |
| Brucella blood with Hemin, and Vitamin K, plates |
VWR | 89405-032 | |
| Bouin’s Fixative | Electron Microscopy Sciences, Hatfield, PA, USA | 15990 | |
| IL-10 Rat ELISA kit | Invitrogen, Camarillo, CA, USA | KRC0101 | |
| IL-1beta Rat ELISA Kit | Invitrogen, Camarillo, CA, USA | KRC0011 | |
| IL-6 Rat ELISA Kit | Invitrogen, Camarillo, CA, USA | KRC0061 | |
| INF-gamma Rat ELISA Kit | Invitrogen, Camarillo, CA, USA | KRC4021 | |
| TNF-alpha Rat ELISA Kit | Invitrogen, Camarillo, CA, USA | KRC3011 | |
| Rat LPS ELISA Kit | NeoBioLab, MA, USA | RL0275 | |
| Environmental Chamber 6020-1 | Caron, Marietta, OH, USA | 6020-1 | |
| Centrifuge | Beckman Coulter, Indianapolis, IN, USA | Optima L-90K | |
| Ultra Centrifuge | |||
| Light microscope optical system | CitoViva Technology Inc., Auburn, AL | ||
| Colony counter | Fisher Scientific , Pittsburgh, PA, USA | RE-3325 | |
| Freezer | Haier, Brooklyn, NY, USA | HCMO50LA | |
| Ultra microplate reader | Bio-Tek Instrument, Winooski, VT, USA | ELx 808 | |
| Auto Strip Washer | Bio-Tek Instrument, Winooski, VT, USA | ELx50 | |
| Pipettes | Gilson, Pipetman, France | P100, P200, P1000 | |
| C24 Incubator Shaker | New Brunswick Scientific, Enfield, CT, USA | Classic C24 | |
| Rocking Shaker | Reliable Scientific, Inc., Nesbit, MS, USA | 55 | |
| Petri dishes | Fisher Scientific, Pittsburgh, PA, USA | 875713 | 100mmX15mm |
| SterilGard III Advance | The Baker Company, Sanford, ME, USA | SG403 | |
| Culture Growing Flasks | Corning Incorporated, Corning, NY, USA | 4995 | PYREX 250mL Erlenmeyer flasks |
| Optical Spectrometer Genesys 20 | Thermo Scientific, Waltham, MA, USA. | 4001 | |
| Sony DXC-33 Video camera | Sony | ||
| Richardson test slide | Electron Microscopy Science, Hatfield, PA, USA | 80303 | |
| Millipore water purification system | Millipore | Direct-Q | |
| Image-Pro Plus software | Media Cybernetics, MD, USA | ||
| Triple Beam Balance | OHAUS Corporation, Parsippany, NJ, USA | ||
| Tissue Homogenizer, Dounce | VWR | 71000-518 | |
Referencias
- Crandall, C. G., Gonzalez-Alonso, J. Cardiovascular function in the heat-stressed human. Acta Physiol. 199, 407-423 (2010).
- Lambert, G. P. Role of gastrointestinal permeability in exertional heatstroke. Exerc. Sport Sci. Rev. 32, 185-190 (2004).
- Yu, J., et al. Effect of heat stress on the porcine small intestine: A morphological and gene expression study. Comp. Biochem. Physiol. A-Mol. Integr. Physiol. 156, 119-128 (2010).
- Moseley, P. L., Gisolfi, C. V. New Frontiers in Thermoregulation and Exercise. Sports Med. 16, 163-167 (1993).
- Lambert, G. P. Stress-induced gastrointestinal barrier dysfunction and its inflammatory effects. J. Anim. Sci. 87, 101-108 (2009).
- Berg, A., Muller, H. M., Rathmann, S., Deibert, P. The gastrointestinal system – An essential target organ of the athlete’s health and physical performance. Exerc. Immunol. Rev. 5, 78-95 (1999).
- Khan, M. W., et al. Microbes, intestinal inflammation and probiotics. Expert Rev Gastroent. 6, 81-94 (2012).
- Quigley, E. M. M. Prebiotics and Probiotics: Their Role in the Management of Gastrointestinal Disorders in Adults. Nutr Clin Pract. 27, 195-200 (2012).
- Gore, C., et al. Treatment and secondary prevention effects of the probiotics Lactobacillus paracasei or Bifidobacterium lactis on early infant eczema: randomized controlled trial with follow-up until age 3 years. Clin Exp Allergy. 42, 112-122 (2012).
- Gomes, A. C., Bueno, A. A., de Souza, R. G. M., Mota, J. F. Gut microbiota, probiotics and diabetes. Nutr. J. 13, (2014).
- Eutamene, H., et al. Synergy between Lactobacillus paracasei and its bacterial products to counteract stress-induced gut permeability and sensitivity increase in rats. J. Nutr. 137, 1901-1907 (2007).
- Ait-Belgnaoui, A., et al. Lactobacillus farciminis treatment attenuates stress-induced overexpression of Fos protein in spinal and supraspinal sites after colorectal distension in rats. Neurogastroenterol. Motil. 21, 585-593 (2009).
- Fujiya, M., et al. The Bacillus subtilis quorum-sensing molecule CSF contributes to intestinal Homeostasis via OCTN2, a host cell membrane transporter. Cell Host Microbe. 1, 299-308 (2007).
- Cutting, S. M. Bacillus probiotics. Food Microbiol. 28, 214-220 (2011).
- Pinchuk, I. V., et al. In vitro anti-Helicobacter pylori activity of the probiotic strain Bacillus subtilis 3 is due to secretion of antibiotics. Antimicrob Agents Ch. 45, 3156-3161 (2001).
- Sorokulova, I. B., Kirik, D. L., Pinchuk, I. V. Probiotics against Campylobacter pathogens. J. Travel Med. 4, 167-170 (1997).
- Gracheva, N. M., et al. The efficacy of the new bacterial preparation biosporin in treating acute intestinal infections. Zh Mikrobiol Epidemiol Immunobiol. , 75-77 (1996).
- Horosheva, T. V., Vodyanoy, V., Sorokulova, I. Efficacy of Bacillus probiotics in prevention of antibiotic-associated diarrhoea: a randomized, double-blind, placebo-controlled clinical trial. JMM Case Report. 1, (2014).
- Sorokulova, I. B., Krumnow, A. A., Pathirana, S., Mandell, A. J., Vodyanoy, V. Novel Methods for Storage Stability and Release of Bacillus Spores. Biotechnol. Prog. 24, 1147-1153 (2008).
- Vogel, H. G., Vogel, W. H., Vogel, H. G., Vogel, W. H. Drug Discovery and Evaluation: Pharmacological Assays. eds H.G. Vogel & W.H. Vogel. , 658-659 (1997).
- Bailey, M. T., Engler, H., Sheridan, J. F. Stress induces the translocation of cutaneous and gastrointestinal microflora to secondary lymphoid organs of C57BL/6 mice. J. Neuroimmunol. 171, 29-37 (2006).
- Rakoff-Nahoum, S., Paglino, J., Eslami-Varzaneh, F., Edberg, S., Medzhitov, R. Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis. Cell. 118, 229-241 (2004).
- Leon, L. R., Blaha, M. D., DuBose, D. A. Time course of cytokine, corticosterone, and tissue injury responses in mice during heat strain recovery. J. Appl. Physiol. 100, 1400-1409 (2006).
- Ribeiro, S. R., et al. Weight loss and morphometric study of intestinal mucosa in rats after massive intestinal resection. Influence of a glutamine-enriched diet. Rev. Hosp. Clìn. Fac. Med. S. Paulo. 59, 349-356 (2004).
- Vainrub, A., Pustovyy, O., Vodyanoy, V. Resolution of 90 nm (lambda/5) in an optical transmission microscope with an annular condenser. Opt Lett. 31, 2855-2857 (2006).
- Moore, T., Globa, L., Pustovyy, O., Vodyanoy, V., Sorokulova, I. Oral administration of Bacillus subtilis strain BSB3 can prevent heat stress-related adverse effects in rats. J Appl Microbiol. 117, 1463-1471 (2014).
- Richardson, T. M. Test slides: Diatoms to divisions-What are you looking at. Proc Roy Microsc Soc. 22, 3-9 (1988).
- Moore, T., Sorokulova, I., Pustovyy, O., Globa, L., Vodyanoy, V. Microscopic evaluation of vesicles shed by rat erythrocytes at elevated temperatures. J Therm Biol. 38, 487-492 (2013).
- Leon, L. R., Helwig, B. G. Heat stroke: Role of the systemic inflammatory response. J. Appl. Physiol. 109, 1980-1988 (2010).
- Kumar, Y., Chawla, A., Tatu, U. Heat Shock Protein 70 as a Biomarker of Heat Stress in a Simulated Hot Cockpit. Aviat Space Env Med. 74 (7), 711-716 (2007).
