蛋白营养不良与能量营养不良状态下脓毒症大鼠凝血的变化

Changes in coagulation of sepsis rats with protein-malnutrition or energy-malnutrition

目的探讨蛋白营养不良和能量营养不良状态下脓毒症大鼠凝血的变化。方法将108只雄性SD大鼠按随机数字表法分为3组,每组36只。正常喂养组给予含18%蛋白饲料27 g/d自由饮食;蛋白营养不良组给予含5%蛋白饲料27 g/d低蛋白自由饮食;能量营养不良组给予含18%蛋白饲料9 g/d低能量饮食。连续喂养4周后,从各组分别选取8只大鼠进行营养不良评价,称量体重以及胸腺、脾脏重量,应用流式细胞仪检测脾脏T淋巴细胞亚群比例和M1型巨噬细胞比例,采用酶联免疫吸附试验(ELISA)检测血浆白细胞介素(IL-6、IL-10)水平。各组剩余28只大鼠用盲肠结扎穿孔术(CLP)制备脓毒症模型,其中20只用于Kaplan-Meier生存分析;其余8只于术后8 h处死,采用ELISA法检测血浆IL-6、IL-10水平,应用流式细胞仪检测脾脏M1型巨噬细胞比例,采用反转录-聚合酶链反应(RT-PCR)检测肝脏组织因子(TF)及凝血酶原激活物抑制因子-1(PAI-1)的mRNA表达。采用Pearson相关法对CLP术后大鼠TF、PAI-1的mRNA表达与IL-6进行相关性分析。结果①4周喂养结束后,正常喂养组和蛋白营养不良组大鼠体重增加,而能量营养不良组丢失初始体重的25%;蛋白营养不良组及能量营养不良组大鼠体重、胸腺和脾脏重量均明显低于正常喂养组。与正常喂养组和蛋白营养不良组比较,能量营养不良组大鼠脾脏CD3+T淋巴细胞、CD4+T淋巴细胞、M1型巨噬细胞比例以及血浆IL-6水平均明显升高〔CD3+T淋巴细胞比例:(52.1±3.7)%比(46.9±3.9)%、(44.5±2.2)%,CD4+T淋巴细胞比例:(35.0±3.6)%比(26.3±2.2)%、(25.1±2.3)%,M1型巨噬细胞比例:(8.7±2.0)%比(3.2±1.3)%、(4.2±1.1)%,IL-6(ng/L):129.4±16.2比48.1±10.0、53.0±8.3,均P<0.05〕。②CLP术后7 d Kaplan-Meier生存分析显示,能量营养不良组大鼠全部死亡,7 d累积生存率明显低于正常喂养组和蛋白营养不良组〔0%(0/20)比35%(7/20)、55%(11/20),均P<0.05〕。正常喂养组大鼠死亡率为65%,符合中度脓毒症死亡率,说明CLP模型制备成功。CLP术后,蛋白营养不良组大鼠血浆IL-6水平明显低于正常喂养组〔IL-6(ng/L):154.6±34.7比233.4±41.2,P<0.05〕;能量营养不良组大鼠肝脏TF、PAI-1的mRNA表达和血浆IL-6水平均明显高于正常喂养组〔TF mRNA(2-ΔΔCT):4.5±2.2比1.1±0.7,PAI-1 mRNA(2-ΔΔCT):3.3±1.8比1.3±0.9,IL-6(ng/L):382.7±118.2比233.4±41.2,均P<0.05〕,脾脏M1型巨噬细胞比例明显低于正常喂养组〔(8.9±2.4)%比(15.2±5.4)%,P<0.05〕。各组大鼠血浆IL-10水平比较差异均无统计学意义。相关分析显示,CLP术后大鼠肝脏TF、PAI-1 mRNA表达与血浆IL-6水平均呈显著正相关(r1=0.644、r2=0.574,均P<0.01)。结论长期持续应激(饥饿)导致大鼠持续慢性炎症状态,并在感染后刺激相关炎性因子释放及凝血系统激活,无持续应激的蛋白营养不良脓毒症大鼠炎性因子释放明显减少。

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.Results①After 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].②Kaplan-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.