Summary

Preventie van hittestress schadelijke effecten bij ratten door<em> Bacillus subtilis</em> Strain

Published: July 11, 2016
doi:

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

Deze studie werd ontworpen om de beschermende werking van de Bacillus subtilis stam tegen complicaties bij hittestress evalueren. Tweeëndertig Sprague-Dawley ratten werden gebruikt in deze studie. De dieren werden oraal behandeld tweemaal daags gedurende twee dagen met B. subtilis BSB3 stam of PBS. De volgende dag na de laatste behandeling, elke groep werd gesplitst en twee experimentele groepen (één behandeld met PBS en behandeld met een B. subtilis) werden bij 45 ° C gedurende 25 min geplaatst. Twee controlegroepen bleven 25 minuten bij kamertemperatuur. Alle ratten werden gedood en verschillende parameters werden geanalyseerd in alle groepen. Nadelige effecten van hittestress zijn geregistreerd door de daling van de villi hoogte en totale mucosale dikte in het darmepitheel; translocatie van bacteriën uit het lumen; grotere blaasjes van erytrocyten en verhoging van de lipopolysacchariden (LPS) in het bloed. De beschermende werkzaamheid van de behandeling wordt geëvalueerd door preinterventie van deze bijwerkingen. Het protocol werd opgezet voor de orale behandeling van ratten met bacteriën voorkomen hittestress complicaties, maar dit protocol kan worden gemodificeerd en gebruikt voor andere toedieningswijzen en voor de analyse van verschillende verbindingen.

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

Alle experimentele procedures werden goedgekeurd door IACUC van Auburn University, Auburn, Alabama. 1. Voorbereiding van Cultuur Media, serviesgoed en bacteriecultuur Inoculeer 10 ml nutriënt bouillon in een kolf met een enkele kolonie van Bacillus subtilis BSB3 cultuur, overnacht gegroeid op een nutriënt agar plaat. Voeg 500 ml van sporulatie medium 19 (per liter: Bacto Nutrient Broth, 8 g, 10% (w / v) KCl, 10 ml; 1,2% (w / v) magnesiumsulfaat 4 x 7H 2<…

Representative Results

De gemiddelde lichaamstemperatuur van de dieren voor en na hittestress was 36,7 ± 0,07 o C en 40,3 ± 0,17 o C respectievelijk (p <0,05). Blootstelling van ratten warmte (groep 3) resulteerde in significante remming van villi hoogte en mucosale totale dikte (figuren 2, 3). Orale toediening van BSB3 stam voordat spanning beschermd darm tegen de schadelijke invloed van warmte. Transloca…

Discussion

Blootstelling aan hoge temperaturen leidt tot ernstige gezondheidsproblemen 29. Preventie en vroege opsporing van complicaties in verband met hittestress zijn van vitaal belang 30. Het gepresenteerde protocol kan worden gebruikt om de werkzaamheid van verschillende benaderingen voor het voorkomen van hittestress nadelige effecten ervan.

Kritische stappen in dit protocol zijn onder meer: ​​de warmtebehandeling procedure (45 o C, 25 min);…

Disclosures

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

References

  1. Crandall, C. G., Gonzalez-Alonso, J. Cardiovascular function in the heat-stressed human. Acta Physiol. 199, 407-423 (2010).
  2. Lambert, G. P. Role of gastrointestinal permeability in exertional heatstroke. Exerc. Sport Sci. Rev. 32, 185-190 (2004).
  3. 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).
  4. Moseley, P. L., Gisolfi, C. V. New Frontiers in Thermoregulation and Exercise. Sports Med. 16, 163-167 (1993).
  5. Lambert, G. P. Stress-induced gastrointestinal barrier dysfunction and its inflammatory effects. J. Anim. Sci. 87, 101-108 (2009).
  6. 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).
  7. Khan, M. W., et al. Microbes, intestinal inflammation and probiotics. Expert Rev Gastroent. 6, 81-94 (2012).
  8. Quigley, E. M. M. Prebiotics and Probiotics: Their Role in the Management of Gastrointestinal Disorders in Adults. Nutr Clin Pract. 27, 195-200 (2012).
  9. 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).
  10. Gomes, A. C., Bueno, A. A., de Souza, R. G. M., Mota, J. F. Gut microbiota, probiotics and diabetes. Nutr. J. 13, (2014).
  11. 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).
  12. 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).
  13. 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).
  14. Cutting, S. M. Bacillus probiotics. Food Microbiol. 28, 214-220 (2011).
  15. 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).
  16. Sorokulova, I. B., Kirik, D. L., Pinchuk, I. V. Probiotics against Campylobacter pathogens. J. Travel Med. 4, 167-170 (1997).
  17. 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).
  18. 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).
  19. 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).
  20. 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).
  21. 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).
  22. 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).
  23. 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).
  24. 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).
  25. 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).
  26. 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).
  27. Richardson, T. M. Test slides: Diatoms to divisions-What are you looking at. Proc Roy Microsc Soc. 22, 3-9 (1988).
  28. 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).
  29. Leon, L. R., Helwig, B. G. Heat stroke: Role of the systemic inflammatory response. J. Appl. Physiol. 109, 1980-1988 (2010).
  30. 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).

Play Video

Cite This Article
Sorokulova, I., Globa, L., Pustovyy, O., Vodyanoy, V. Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain. J. Vis. Exp. (113), e54122, doi:10.3791/54122 (2016).

View Video