过氧化物酶体增殖物激活受体-γ/核转录因子-κB转导通路在脓毒症所致凝血功能障碍中的作用

摘要

目的：探讨过氧化物酶体增殖物激活受体-γ/核转录因子-κB（PPAR-γ/NF-κB）转导通路是否参与脓毒症所致凝血功能障碍。方法健康雄性SD大鼠40只，按随机数字表法分为对照组、脂多糖（LPS）刺激组、PPAR-γ选择性激动剂罗格列酮（ROSI）预处理组、PPAR-γ选择性拮抗剂2-氯-5-硝基苯胺（GW9662）预处理组4组，每组10只。经舌下静脉注射6 mg/kg LPS制备脓毒症大鼠模型，对照组注射生理盐水2 mL/kg；ROSI预处理组经舌下静脉注射0.3 mg/kg ROSI后30 min注射LPS；GW9662预处理组经舌下静脉注射0.3 mg/kg GW9662，15 min后给予0.3 mg/kg ROSI，30 min后再注射LPS。各组于制模后4 h取血，采用免疫细胞化学法结合图像分析法检测外周血单个核细胞（PBMC）内PPAR-γ和NF-κBp65的表达活性，并检测血浆凝血酶原时间（PT）、活化部分凝血活酶时间（APTT）、纤维蛋白原（FIB）、D-二聚体水平。结果①PPAR-γ/NF-κB通路：对照组大鼠PBMC内PPAR-γ、NF-κBp65表达均较少，主要位于细胞质。LPS刺激组PPAR-γ表达（灰度值）稍有增高，但与对照组比较差异无统计学意义（111.01±4.06比98.46±5.99，P＞0.05）；ROSI预处理组PPAR-γ表达（灰度值）较LPS刺激组明显升高（214.38±5.79比111.01±4.06，P＜0.01），有核移位；GW9662预处理组PPAR-γ表达（灰度值）较少，与对照组比较差异无统计学意义（44.21±2.64比98.46±5.99，P＞0.05）。LPS刺激组PBMC内NF-κBp65表达（灰度值）较对照组显著升高（249.48±6.86比105.81±10.19，P＜0.01），出现核移位；ROSI预处理组NF-κBp65表达（灰度值）较LPS刺激组显著降低（102.47±8.05比249.48±6.86，P＜0.01），NF-κBp65从胞核移位至胞质；GW9662预处理组NF-κBp65表达（灰度值）与LPS刺激组比较差异无统计学意义（214.84±7.91比249.48±6.86，P＞0.05）。②凝血功能：与对照组比较，LPS刺激组PT、APTT明显延长，FIB明显降低，D-二聚体明显升高〔PT（s）：18.32±2.03比12.22±1.38， APTT（s）：40.05±2.72比26.64±2.73，FIB（g/L）：1.65±0.51比3.60±0.37，D-二聚体（mg/L）：2.58±0.73比0.37±0.06，均P＜0.01〕；与LPS刺激组比较，ROSI预处理组PT、APTT明显缩短，FIB 明显升高，D-二聚体明显降低〔PT（s）：13.93±1.67比18.32±2.03，APTT（s）：30.29±0.86比40.05±2.72，FIB（g/L）：3.18±0.69比1.65±0.51，D-二聚体（mg/L）：0.40±0.12比2.58±0.73，均P＜0.01〕；GW9662预处理组各指标与LPS刺激组比较差异均无统计学意义。结论 PPAR-γ选择性激动剂ROSI能够减轻脓毒症大鼠凝血功能障碍；PPAR-γ/NF-κB转导通路在脓毒症引起的凝血功能障碍中有一定的作用。%Objective To determine the role of activated status of peroxisome proliferator-activated receptorγuclear factor-κB ( PPAR-γ/NF-κB ) in coagulation disorders induced by sepsis. Methods Forty male Sprague-Dawley ( SD ) rats were randomly divided into four groups, n = 10 in each group: control group, lipopolysaccharide ( LPS ) challenged group, rosiglitazone ( ROSI, selective agonist of PPAR-γ) pretreatment group, and GW9662 ( PPAR-γ antagonist ) pretreatment group. The sepsis model was reproduced by injection of 6 mg/kg LPS via sublingual vein, and the rats in control group were injected with 2 mL/kg normal saline. The rats in ROSI pretreatment group were given 0.3 mg/kg ROSI by sublingual venous injection followed by injection of LPS 30 minutes later;and in GW9662 pretreatment group rats were given 0.3 mg/kg GW9662 by sublingual venous injection followed by 0.3 mg/kg ROSI 15 minutes later, followed by injection of LPS 30 minutes later. Blood was collected at 4 hours after LPS administration, and the expressions of PPAR-γ and NF-κBp65 in peripheral blood mononuclear cell ( PBMC ) were determined with immunocytocheminal technique and graph analysis. Plasma prothrombin time ( PT ), activated partial thromboplastin time ( APTT ), fibrinogen ( FIB ), and D-dimer were determined simultaneously. Results① PPAR-γ/NF-κB pathway: the expressions of PPAR-γ and NF-κBp65 were lowered in control group, and they were expressed in cytoplasm. In LPS challenged group the expression of PPAR-γ ( gray value ) was slightly increased but with no significant difference as compared with control group ( 111.01±4.06 vs. 98.46±5.99, P >0.05 ). In ROSI pretreatment group the expression of PPAR-γ( gray value ) was significantly higher than that in LPS challenged group ( 214.38±5.79 vs. 111.01±4.06, P<0.01 ), with dislocation into nuclei. In GW9662 pretreatment group the expression of PPAR-γ ( gray value ) was lowered but without significant difference compared with that of control group ( 44.21±2.64 vs. 98.46±5.99, P>0.05 ). In LPS challenged group the expression of NF-κBp65 ( gray value ) was significantly higher than that in control group ( 249.48±6.86 vs. 105.81±10.19, P < 0.01 ), and it was translocated into the nuclei. In ROSI pretreatment group the expression of NF-κBp65 ( gray value ) was significantly lower than that in LPS challenged group ( 102.47±8.05 vs. 249.48±6.86, P < 0.01 ), and it lied in cytoplasm. In GW9662 pretreatment group the expression of NF-κBp65 ( gray value ) showed no significant difference as compared with that of LPS challenged group ( 214.84±7.91 vs. 249.48±6.86, P>0.05 ).②Coagulation:compared with control group, PT and APTT were significantly prolonged, FIB was significantly decreased, and D-dimer was significantly increased in LPS challenged group [ PT ( s ):18.32±2.03 vs. 12.22±1.38, APTT ( s ):40.05±2.72 vs. 26.64±2.73, FIB ( g/L ): 1.65±0.51 vs. 3.60±0.37, D-dimer ( mg/L ): 2.58±0.73 vs. 0.37±0.06, all P < 0.01 ]. Compared with LPS challenged group, APTT and PT were significantly shortened, FIB was significantly increased, and D-dimer was significantly lowered in ROSI pretreatment group [ PT ( s ):13.93±1.67 vs. 18.32±2.03, APTT ( s ):30.29±0.86 vs. 40.05±2.72, FIB ( g/L ):3.18±0.69 vs 1.65±0.51, D-dimer ( mg/L ):0.40±0.12 vs. 2.58±0.73, all P<0.01 ]. All parameters in GW9662 pretreatment group showed no significant difference as compared with those of LPS challenged group. Conclusions PPAR-γagonist ROSI may ameliorate coagulation disorders in septic rats. PPAR-γ/NF-κB transduction pathway plays an important role in septic coagulopathy.