Relationship Between Heart Damages and HSPs mrna in Persistent Heat Stressed Broilers

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1 2005,38(12): Scientia Agricultura Sinica mrna 1, ( ) PCR mrna HSC70 mrna HSP70 mrna HSC70 mrna HSC70 PCR Relationship Between Heart Damages and HSPs mrna in Persistent Heat Stressed Broilers SUN Pei-ming 1 2 LIU Yu-tian 2, ZHAO Yong-gang 2, BAO En-dong 1 WANG Zhi-liang 2 ( 1 College of Veterinary Medicine Nanjing Agricultural University Nanjing National Diagnostic Center for Exotic Animal Disease Ministry of Agriculture Qingdao ) Abstract: The relationship between heart damages and HSPs mrna of heat stressed broilers was studied using spectrophotometer, histopathological technique and fluorescence quantitative reverse transcription PCR (FQ RT-PCR). The results showed that the activity of creatine kinase (CK) and glutamic-pyruvic transaminase (GPT) was fluctuant during the persistent heat stress. The major lesions in the myocardial fibers were degeneration and necrosis. The transcription of constitutive or cognate heat shock protein 70 (HSC 70 ) mrna changed. The transcription of heat shock protein 70 (HSP 70 ) mrna significantly increased in the course of persistent heat stress. The results also suggested that the change of HSC 70 mrna was contrary to the activity of CK, and the level of HSC 70 mrna transcription could used as a symbol of myocardial cell damages in the course of persistent heat stress. Key words: Broiler; Heat shock protein (HSP); Fluorescence quantitative reverse transcription PCR (FQ RT-PCR); Heart; Heat stress stress response [3 4] [2 5] HSP [1] heat shock protein HSP [6] [2] HSP 70 HSP ( ) Tel sunpm2002@sohu.com Tel b_endong@njau.edu.cn

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3 12 mrna 2537 FQ RT-PCR HSC GAPDH 5 -TGAAA- constitutive or cognate heat shock protein 70 HSC 70 GTCGGAGTCAACGGAT-3 5 -ACGCTC- HSP 70 mrna HSC 70 HSP 70 CTGGAAGATAGTGAT-3 HSC ATTG- GCCTTTCACTGTGGT-3 5 -CCTGTCGCT- GGGAGTCGTT-3 HSP AGCGTAACA- 1 CCACCATTCC-3 5 -TGGCTCCCACCC- 1.1 TATCTC-3 TaKaRa AA GAPDH HSC 70 HSP bp 204 bp bp min 95 5 min 94 1 min s s min RT-PCR CK 1.5% GPT GAPDH HSC 70 HSP 70 Trizol pgem-t Easy 4 AMV RNA JM 109 SOC Taq DNA DNase Sca DNA 37 3 h LB Marker IPTG X-Gal JM109 IPTG X-Gal DNA SOC TaKaRa pgem-t Easy Vector PCR Promega SYBR Green Sca A B 6 h C Riboprobe in vitro Transcription Systems 12 h D 18 h E 24 h F 48 h DNase 15 RNA 25±1 20 min RT-PCR 10 40±1 FQ RT-PCR threshold cycle C t 10% HSC HSP 70 mrna CK GPT RT-PCR C t mrna % HSC 70 HSP 70 5 µm mrna H.E HSC 70 HSP 70 mrna 1 RT-PCR 2.1 Trizol RNA HSC 70 HSP glyceralde- hyde-3-phosphate dehydrogease GAPDH GAPDH [7] Primer Premier 3 h

4 h 48 h 2.2 CK GPT CK P< h GPT CK GPT 24 h CK GPT 1 CK 12 h 48 h P< h 24 h 2.3 P<0.05 P<0.01 GPT 1 CK GPT Table1 Activities of CK and GPT during heat stressing CK GPT Group Time h U ml -1 Katal A CK 75.2±21.5cdE 3.033±0.983bcdef B ±30.4cDf 6.374±2.471aCE C ±34.1abde 7.479±2.795aBeF D ±20.6aBcf 7.234±3.274eF E ±25.74Acf 5.179±1.758aBcd F ±35.7abde 6.004±2.342aCD P<0.05, P <0.01 The different capital and small letters in the same column stand for significance at 0.01 and 0.05 level, respectively P< h 18 h P< h 1-A 6 h 1-B 12 h 1-C 18 h 24 h 48 h 1-D 2.4 FQ RT-PCR GAPDH HSC 70 HSP 70 RT-PCR 25 µl 5 µl A B C D A B C D A. Normal myocardial fibers B. The myocardial fibers were not stained evenly C. The myocardial fibers were splited D. The myocardial fibers were splited and arose necrosis 1 H.E 40 Fig.1 The damage of myocardial fibers in heat stressed broilers (H.E staining 40)

5 12 mrna bp Y=-3.347X pgem-t Easy JM % HSC 70 Y=-3.380X % HSP 70 GenBank Y=-3.381X % RNA 260/ GAPDH HSC 70 HSP µg ml /ml FQ RT-PCR 2 4 FQ RT-PCR 5 7 FQ 4 HSP70 RT-PCR RT-PCR RT-PCR 1 µl Fig. 4 Standard curve on FQ RT-PCR of HSP SYBR Green 70 GAPDH ; 5. DNA Marker 1-4. Diluting of standard; 5. DNA Marker. The same as below 2 GAPDH RT-PCR Fig.2 Standard curve on FQ RT-PCR of GAPDH 5 GAPDH FQ RT-PCR Fig. 5 Agarose gel electrophoresis analysis of FQ RT-PCR productions of GAPDH standard 3 HSC70 RT-PCR Fig. 3 Standard curve on FQ RT-PCR of HSC HSC70 FQ RT-PCR Fig. 6 Agarose gel electrophoresis analysis of FQ RT-PCR productions of HSC 70 standard

6 HSP70 FQ RT-PCR Fig. 7 Agarose gel electrophoresis analysis of FQ RT-PCR productions of HSP 70 standard 2.5 HSC70 HSP70 mrna FQ RT-PCR C t HSC 70 mrna 18 h GAPDH HSC 70 HSP 70 mrna HSP 70 mrna HSC 70 HSP 70 mrna GAPDH mrna CK SPSS HSC 70 mrna 2 CK 2 HSC70 mrna HSP70 mrna 12 h Table 2 Change of HSC 70 mrna and HSP 70 mrna during heat stressing HSC 70 mrna Group Time h HSP 70 mrna A CK 0.053±0.019cdef 0.250±0.059def B ±0.012Cdef 0.322±0.114def C ±0.003aBdef 0.326±0.099def D ±0.076abcef 0.500±0.164abcF E ±0.044abcd 0.595±0.187abdF F ±0.037abcd 1.070±0.253abcde P <0.05, P <0.01 The different capital and small letters in the same column stand for significance at 0.01 and 0.05 level 5 h 10 h 6 h 12 h CK GPT CK GPT 12 h 48 h 18 h 24 h CK FQ RT-PCR HSC 70 mrna HSC 70 mrna 12 h 6 CK GPT HSC 70 mrna HSPs heat shock transcription factor HSF heat shock element HSE [8] HSC 70 HSP 70 mrna HSC 70 HSP 70 2 HSC 70 HSC 70 mrna HSP 70 mrna mrna 12 h HSP P<0.01 HSP HSF HSF 18 h P<0.01 HSF HSE HSP 70 mrna 48 [8] HSP mrna h P< [6] 3 h 2 3 h HSP HSC 70 mrna HSP 70 mrna

7 12 mrna 2541 HSC 70 mrna HSP 70 mrna Chemistry : HSP 18 h 48 h HSP 70 Research HSC 70 mrna CK HSC 70 mrna HSP HSP [6] HSC 70 mrna HSP 70 mrna 2004, 37: [2] Gabai V L Meriin A B Mosser D D Caron A W Rits S Shifrin V I Sherman M Y. HSP 70 prevents activation of stress kinase - A novel pathway of cellular thermotolerance. Journal of Biological [3] Benjamin I J McMilan D R. Stress (heat shock) proteins molecular chaperones in cardiovascular biology and disease. Circulation [4] Wang F, Zhao F J, Guo J S. The study on regulation of HSPs expression and hygiene significance. Foreign Medical Sciences (Section Hygiene) (in Chinese) [5] Nelson R J Ziegelhoffer T. The translation machinery and 70KD heat shock protein cooperate in protein synthesis. Cell 1992, 71: 97. Hartung J. HSP 70. Bao E D Gong Y Y Hartung J Fu X B Wang X J Zhang H T Wang Z L. Relation between pathologic damages and HSP 70 changes in acute heat stressed broilers. Scientia Agricultura Sinica : (in Chinese) [7] Bustin S A. Absolute quantification of mrna using real-time reverse transcription polymerase chain reaction assays. Journal of Molecular References Endocrinology [1],.. [8] Morimoto R I. Cell in stress transcriptional activation of heat shock genes. Science Xia L Z Liang S H. Advance of studies on reducing heat stress of chicken by nutritional factors. Ecology of Domestic Animal (in Chinese)