热火预防应激在大鼠不利影响<em>枯草芽孢杆菌</em>应变
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
此研究的目的是评估对热应力有关的并发症的枯草芽孢杆菌菌株的保护作用。三十二个的Sprague-Dawley大鼠用于该研究。动物口服治疗，一天两次两天与B枯草芽孢杆菌菌株BSB3或PBS。第二天在最后一次治疗后，各组被划分和两个实验组（一个用PBS处理和一个与枯草芽孢杆菌处理的）被放置在45℃下25分钟。两个对照组留在室温下25分钟。所有大鼠安乐死，并在各组不同的参数进行了分析。热应力的不良影响是由绒毛高度，并在肠上皮细胞总粘膜厚度的减小登记;从管腔细菌易位;增加红细胞的囊泡和仰角的脂多糖（LPS）在血液中的水平。治疗的保护效力是通过预评估这些副作用公约。该协议被设定为大鼠细菌预防热应力并发症的口服治疗，但该协议可以被修改，并用于其它给药途径和不同的化合物的分析。
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
暴露在高温下会导致严重的健康状况29。预防和早期发现与热应激的并发症是至关重要的30。 所提出的协议可以用来评估用于预防热应力的不良影响的各种方法的功效。
这个协议中的关键步骤包括：在热处理过程（45℃，25分钟）;无热原的材料的收集和血液以防止肠毒素污染处理期间的使用;保持血清等分在-20℃至避免反复冻融;和…
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 | |
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