Sublethal hemorrhage induces tolerance in animals exposed to cecal ligation and puncture by altering p38, p44/42, and SAPK/JNK MAP kinase activation.
BACKGROUND
We have shown that SLH induces tolerance to endotoxin in vivo and in vitro, and is associated with alterations in MAP kinase (p38, p44/42, and SAPK/JNK) activation and TNF production. This study investigates the effect of sublethal hemorrhage (SLH) on cecal ligation and puncture (CLP) polymicrobial sepsis and examined the effect of the bioflavinoid, curcumin, a MAP kinase inhibitor, on this relationship.


METHODS
Sprague-Dawley rats underwent SLH (hemorrhage and MAP = 30 mm Hg for 15 min, with shed blood returned) or sham operation. After 24 h, rats had CLP (cecal base ligation with double puncture). Survival was determined +/- curcumin pretreatment (n = 10/group). Lung tissue, serum, and bronchoalveolar lavage (BAL) fluid were obtained 30 min after SLH and 4 and 12 h after CLP (n = 8/group). Lung tissue was analyzed for p38, p44/42 SAPK/JNK, and HSP-70 phosphorylation (Western). Lung myeloperoxidase (MPO) activity was measured as an index of neutrophil infiltration. TNF ELISA was performed on serum and BAL sample.


RESULTS
SLH significantly improved survival after CLP (21.5 vs. 7.5 h vs. sham, p = 0.008), and curcumin reversed this benefit (7.3 h, p = 0.0007 vs. SLH + CLP). MAP kinase activity was significantly greater in SLH rats 4 h post-CLP (p38: 720 vs. 331, p = 0.03, p44/42: 2759 vs. 1295, p = 0.006, SAPK: 413 vs. 254). Curcumin significantly inhibited MAPK activity both 30 min after SLH (p38: 297 vs. 3260, p44/42: 370 vs. 2628, SAPK: 748 vs. 1764, all p < 0.01 vs. SLH 30 min) and 4 h post CLP (p38: 146 vs. 720, p44/42: 616 vs. 2759, all p < 0.01 vs. SLH + CLP4 h). Four hours after CLP, SLH rats expressed more HSP72. Lung MPO activity was significantly lower in SLH + CLP rats at both 4 h (9.5 vs. 15.6, p = 0.02 vs. sham) and 12 h (18.1 vs. 37.5, p = 7 x 10(-5), vs. sham). Serum and BAL TNF levels were higher in SLH rats initially (serum: 145 vs. 28 pg/mL, p = 2 x 10(-5) BAL: 83 vs. 57 vs. sham + CLP4h); however, BAL TNF was significantly lower in SLH animals 12 h post-CLP (37 vs. 72.7 pg/mL, p = 0.003 vs. sham + CLP12h).


CONCLUSIONS
SLH induces tolerance to CLP. This tolerance is dependent on early MAP kinase activation, since the survival benefit is reversed by curcumin. Decreases in tissue cytokine levels and neutrophil infiltration are potential mechanisms by which SLH induces tolerance to CLP (polymicrobial sepsis), attenuates acute lung injury, and improves survival.
James A. Haley Veterans' Hospital and Department of Surgery, University of South Florida, Tampa, Florida 33612, USA.
Yvette
Y
Carter
Guoqing
G
Liu
Jun
J
Yang
Adam
A
Fier
Cynthia
C
Mendez
D000818:Animals; D015153:Blotting, Western; D001992:Bronchoalveolar Lavage Fluid / Q000166:cytology; D002432:Cecum / Q000601:surgery*; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D048031:JNK Mitogen-Activated Protein Kinases; D008026:Ligation / Q000379:methods; D048670:MAP Kinase Kinase 4; D008297:Male; D019950:Mitogen-Activated Protein Kinase 1 / Q000032:analysis / Q000378:metabolism*; D020929:Mitogen-Activated Protein Kinase Kinases / Q000032:analysis / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000032:analysis / Q000378:metabolism*; D009195:Peroxidase / Q000032:analysis / Q000378:metabolism*; D011336:Probability; D011677:Punctures; D011897:Random Allocation; D051381:Rats; D017207:Rats, Sprague-Dawley; D012680:Sensitivity and Specificity; D012720:Severity of Illness Index; D012771:Shock, Hemorrhagic / Q000201:enzymology*; D048051:p38 Mitogen-Activated Protein Kinases
D009195:Peroxidase; D048031:JNK Mitogen-Activated Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D020929:Mitogen-Activated Protein Kinase Kinases