RESUMO
Objective@#To investigate the changes in coagulation of sepsis rats with protein-malnutrition or energy-malnutrition.@*Methods@#108 male Sprague-Dawley (SD) rats were divided into three groups by random number table, with 36 rats in each group. The rats in normal feeding group were given a free diet (27 g/d, containing 18% protein fodder), and the rats in protein-malnutrition group were given a low protein diet (27 g/d, containing 5% protein fodder). The rats in energy-malnutrition group were given a low energy diet (9 g/d, containing 18% protein fodder). After 4 weeks of continuous feeding, 8 rats from each group were sacrificed for malnutrition evaluation. The weights of body, thymus and spleen were measured. The percentages of spleen T lymphocyte subsets and M1 macrophage were determined by flow cytometry. Plasma interleukins (IL-6 and IL-10) levels were determined by enzyme-linked immunosorbent assay (ELISA). The remaining 28 rats in each group were collected for cecal ligation and puncture (CLP) to reproduce the sepsis model, 20 rats of which were used for Kaplan-Meier survival analysis, and the other 8 rats were sacrificed at 8 hours after CLP. The levels of plasma IL-6 and IL-10 were determined by ELISA, and the percentage of spleen M1 macrophages was determined by flow cytometry. The mRNA expressions of tissue factor (TF) and plasminogen activation inhibitor-1 (PAI-1) in liver tissue were determined by reverse transcription-polymerase chain reaction (RT-PCR). Pearson correlation method was used to analyze the correlation between the mRNA expressions of TF and PAI-1 and IL-6 in rats after CLP.@*Results@#① After 4 weeks of feeding, the rats in the normal feeding group and protein-malnutrition group gained weight, while those in the energy-malnutrition group lost 25% of their initial body weight. The weights of body, thymus and spleen in the protein-malnutrition group and the energy-malnutrition group were significantly lower than those in the normal feeding group. Compared with the normal feeding group and the protein-malnutrition group, the percentages of spleen CD3+ T lymphocytes, CD4+ T lymphocytes, M1 macrophages and plasma IL-6 levels were significantly increased in the energy-malnutrition group [CD3+ T lymphocytes percentage: (52.1±3.7)% vs. (46.9±3.9)%, (44.5±2.2)%; CD4+ T lymphocyte percentages: (35.0±3.6)% vs. (26.3±2.2)%, (25.1±2.3)%; M1 macrophage percentages: (8.7±2.0)% vs. (3.2±1.3)%, (4.2±1.1)%; IL-6 (ng/L): 129.4±16.2 vs. 48.1±10.0, 53.0±8.3, all P < 0.05]. ② Kaplan-Meier survival analysis at 7 days after CLP showed: all rats in the energy-malnutrition group died, and the 7-day cumulative survival rate was significantly lower than that in the normal feeding group and the protein-malnutrition group [0% (0/20) vs. 35% (7/20), 55% (11/20), both P < 0.05]. The mortality of the normal feeding group was 65%, which was consistent with moderate CLP mortality, indicating that the CLP model was successfully prepared. After CLP, the plasma IL-6 level in the protein-malnutrition group was significantly lower than that in the normal feeding group [IL-6 (ng/L): 154.6±34.7 vs. 233.4±41.2, P < 0.05]. Compared with the normal feeding group, the mRNA expressions of TF and PAI-1 in liver and plasma IL-6 levels in the energy-malnutrition group were significantly increased [TF mRNA (2-ΔΔCT): 4.5±2.2 vs. 1.1±0.7, PAI-1 mRNA (2-ΔΔCT): 3.3±1.8 vs. 1.3±0.9, IL-6 (ng/L): 382.7±118.2 vs. 233.4±41.2, all P < 0.05], the percentage of M1 macrophages in spleen was significantly lowered [(8.9±2.4)% vs. (15.2±5.4)%, P < 0.05]. There was no significant difference in plasma IL-10 level among all the groups. Correlation analysis showed that the mRNA expressions of TF and PAI-1 in the liver of rats after CLP were positively correlated with plasma IL-6 level (r1 = 0.644, r2 = 0.574, both P < 0.01).@*Conclusions@#Long-term sustained stress (starvation) leads to sustained chronic inflammatory state, and stimulated the release of related inflammatory factors and activation of the coagulation system after infection. And the inflammatory factors in sepsis rats without sustained stress protein malnutrition were significantly reduced.
RESUMO
Objective To investigate the changes in coagulation of sepsis rats with protein-malnutrition or energy-malnutrition. Methods 108 male Sprague-Dawley (SD) rats were divided into three groups by random number table, with 36 rats in each group. The rats in normal feeding group were given a free diet (27 g/d, containing 18% protein fodder), and the rats in protein-malnutrition group were given a low protein diet (27 g/d, containing 5% protein fodder). The rats in energy-malnutrition group were given a low energy diet (9 g/d, containing 18% protein fodder). After 4 weeks of continuous feeding, 8 rats from each group were sacrificed for malnutrition evaluation. The weights of body, thymus and spleen were measured. The percentages of spleen T lymphocyte subsets and M1 macrophage were determined by flow cytometry. Plasma interleukins (IL-6 and IL-10) levels were determined by enzyme-linked immunosorbent assay (ELISA). The remaining 28 rats in each group were collected for cecal ligation and puncture (CLP) to reproduce the sepsis model, 20 rats of which were used for Kaplan-Meier survival analysis, and the other 8 rats were sacrificed at 8 hours after CLP. The levels of plasma IL-6 and IL-10 were determined by ELISA, and the percentage of spleen M1 macrophages was determined by flow cytometry. The mRNA expressions of tissue factor (TF) and plasminogen activation inhibitor-1 (PAI-1) in liver tissue were determined by reverse transcription-polymerase chain reaction (RT-PCR). Pearson correlation method was used to analyze the correlation between the mRNA expressions of TF and PAI-1 and IL-6 in rats after CLP. Results ① After 4 weeks of feeding, the rats in the normal feeding group and protein-malnutrition group gained weight, while those in the energy-malnutrition group lost 25% of their initial body weight. The weights of body, thymus and spleen in the protein-malnutrition group and the energy-malnutrition group were significantly lower than those in the normal feeding group. Compared with the normal feeding group and the protein-malnutrition group, the percentages of spleen CD3+ T lymphocytes, CD4+ T lymphocytes, M1 macrophages and plasma IL-6 levels were significantly increased in the energy-malnutrition group [CD3+ T lymphocytes percentage: (52.1±3.7)% vs. (46.9±3.9)%, (44.5±2.2)%;CD4+ T lymphocyte percentages: (35.0±3.6)% vs. (26.3±2.2)%, (25.1±2.3)%; M1 macrophage percentages: (8.7±2.0)% vs. (3.2±1.3)%, (4.2±1.1)%; IL-6 (ng/L): 129.4±16.2 vs. 48.1±10.0, 53.0±8.3, all P < 0.05]. ② Kaplan-Meier survival analysis at 7 days after CLP showed: all rats in the energy-malnutrition group died, and the 7-day cumulative survival rate was significantly lower than that in the normal feeding group and the protein-malnutrition group [0% (0/20) vs. 35% (7/20), 55% (11/20), both P < 0.05]. The mortality of the normal feeding group was 65%, which was consistent with moderate CLP mortality, indicating that the CLP model was successfully prepared. After CLP, the plasma IL-6 level in the protein-malnutrition group was significantly lower than that in the normal feeding group [IL-6 (ng/L): 154.6±34.7 vs. 233.4±41.2, P < 0.05]. Compared with the normal feeding group, the mRNA expressions of TF and PAI-1 in liver and plasma IL-6 levels in the energy-malnutrition group were significantly increased [TF mRNA (2-ΔΔCT): 4.5±2.2 vs. 1.1±0.7, PAI-1 mRNA (2-ΔΔCT): 3.3±1.8 vs. 1.3±0.9, IL-6 (ng/L): 382.7±118.2 vs. 233.4±41.2, all P < 0.05], the percentage of M1 macrophages in spleen was significantly lowered [(8.9±2.4)% vs. (15.2±5.4)%, P < 0.05]. There was no significant difference in plasma IL-10 level among all the groups. Correlation analysis showed that the mRNA expressions of TF and PAI-1 in the liver of rats after CLP were positively correlated with plasma IL-6 level (r1 = 0.644, r2 = 0.574, both P < 0.01). Conclusions Long-term sustained stress (starvation) leads to sustained chronic inflammatory state, and stimulated the release of related inflammatory factors and activation of the coagulation system after infection. And the inflammatory factors in sepsis rats without sustained stress protein malnutrition were significantly reduced.