CAJ | 학술논문

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。目的：探討過氧化物酶體增殖物激活受體-γ/覈轉錄因子-κB（PPAR-γ/NF-κB）轉導通路是否參與膿毒癥所緻凝血功能障礙。方法健康雄性SD大鼠40隻，按隨機數字錶法分為對照組、脂多糖（LPS）刺激組、PPAR-γ選擇性激動劑囉格列酮（ROSI）預處理組、PPAR-γ選擇性拮抗劑2-氯-5-硝基苯胺（GW9662）預處理組4組，每組10隻。經舌下靜脈註射6 mg/kg LPS製備膿毒癥大鼠模型，對照組註射生理鹽水2 mL/kg；ROSI預處理組經舌下靜脈註射0.3 mg/kg ROSI後30 min註射LPS；GW9662預處理組經舌下靜脈註射0.3 mg/kg GW9662，15 min後給予0.3 mg/kg ROSI，30 min後再註射LPS。各組于製模後4 h取血，採用免疫細胞化學法結閤圖像分析法檢測外週血單箇覈細胞（PBMC）內PPAR-γ和NF-κBp65的錶達活性，併檢測血漿凝血酶原時間（PT）、活化部分凝血活酶時間（APTT）、纖維蛋白原（FIB）、D-二聚體水平。結果①PPAR-γ/NF-κB通路：對照組大鼠PBMC內PPAR-γ、NF-κBp65錶達均較少，主要位于細胞質。LPS刺激組PPAR-γ錶達（灰度值）稍有增高，但與對照組比較差異無統計學意義（111.01±4.06比98.46±5.99，P＞0.05）；ROSI預處理組PPAR-γ錶達（灰度值）較LPS刺激組明顯升高（214.38±5.79比111.01±4.06，P＜0.01），有覈移位；GW9662預處理組PPAR-γ錶達（灰度值）較少，與對照組比較差異無統計學意義（44.21±2.64比98.46±5.99，P＞0.05）。LPS刺激組PBMC內NF-κBp65錶達（灰度值）較對照組顯著升高（249.48±6.86比105.81±10.19，P＜0.01），齣現覈移位；ROSI預處理組NF-κBp65錶達（灰度值）較LPS刺激組顯著降低（102.47±8.05比249.48±6.86，P＜0.01），NF-κBp65從胞覈移位至胞質；GW9662預處理組NF-κBp65錶達（灰度值）與LPS刺激組比較差異無統計學意義（214.84±7.91比249.48±6.86，P＞0.05）。②凝血功能：與對照組比較，LPS刺激組PT、APTT明顯延長，FIB明顯降低，D-二聚體明顯升高〔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-二聚體（mg/L）：2.58±0.73比0.37±0.06，均P＜0.01〕；與LPS刺激組比較，ROSI預處理組PT、APTT明顯縮短，FIB 明顯升高，D-二聚體明顯降低〔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-二聚體（mg/L）：0.40±0.12比2.58±0.73，均P＜0.01〕；GW9662預處理組各指標與LPS刺激組比較差異均無統計學意義。結論 PPAR-γ選擇性激動劑ROSI能夠減輕膿毒癥大鼠凝血功能障礙；PPAR-γ/NF-κB轉導通路在膿毒癥引起的凝血功能障礙中有一定的作用。
목적：탐토과양화물매체증식물격활수체-γ/핵전록인자-κB（PPAR-γ/NF-κB）전도통로시부삼여농독증소치응혈공능장애。방법건강웅성SD대서40지，안수궤수자표법분위대조조、지다당（LPS）자격조、PPAR-γ선택성격동제라격렬동（ROSI）예처리조、PPAR-γ선택성길항제2-록-5-초기분알（GW9662）예처리조4조，매조10지。경설하정맥주사6 mg/kg LPS제비농독증대서모형，대조조주사생리염수2 mL/kg；ROSI예처리조경설하정맥주사0.3 mg/kg ROSI후30 min주사LPS；GW9662예처리조경설하정맥주사0.3 mg/kg GW9662，15 min후급여0.3 mg/kg ROSI，30 min후재주사LPS。각조우제모후4 h취혈，채용면역세포화학법결합도상분석법검측외주혈단개핵세포（PBMC）내PPAR-γ화NF-κBp65적표체활성，병검측혈장응혈매원시간（PT）、활화부분응혈활매시간（APTT）、섬유단백원（FIB）、D-이취체수평。결과①PPAR-γ/NF-κB통로：대조조대서PBMC내PPAR-γ、NF-κBp65표체균교소，주요위우세포질。LPS자격조PPAR-γ표체（회도치）초유증고，단여대조조비교차이무통계학의의（111.01±4.06비98.46±5.99，P＞0.05）；ROSI예처리조PPAR-γ표체（회도치）교LPS자격조명현승고（214.38±5.79비111.01±4.06，P＜0.01），유핵이위；GW9662예처리조PPAR-γ표체（회도치）교소，여대조조비교차이무통계학의의（44.21±2.64비98.46±5.99，P＞0.05）。LPS자격조PBMC내NF-κBp65표체（회도치）교대조조현저승고（249.48±6.86비105.81±10.19，P＜0.01），출현핵이위；ROSI예처리조NF-κBp65표체（회도치）교LPS자격조현저강저（102.47±8.05비249.48±6.86，P＜0.01），NF-κBp65종포핵이위지포질；GW9662예처리조NF-κBp65표체（회도치）여LPS자격조비교차이무통계학의의（214.84±7.91비249.48±6.86，P＞0.05）。②응혈공능：여대조조비교，LPS자격조PT、APTT명현연장，FIB명현강저，D-이취체명현승고〔PT（s）：18.32±2.03비12.22±1.38， APTT（s）：40.05±2.72비26.64±2.73，FIB（g/L）：1.65±0.51비3.60±0.37，D-이취체（mg/L）：2.58±0.73비0.37±0.06，균P＜0.01〕；여LPS자격조비교，ROSI예처리조PT、APTT명현축단，FIB 명현승고，D-이취체명현강저〔PT（s）：13.93±1.67비18.32±2.03，APTT（s）：30.29±0.86비40.05±2.72，FIB（g/L）：3.18±0.69비1.65±0.51，D-이취체（mg/L）：0.40±0.12비2.58±0.73，균P＜0.01〕；GW9662예처리조각지표여LPS자격조비교차이균무통계학의의。결론 PPAR-γ선택성격동제ROSI능구감경농독증대서응혈공능장애；PPAR-γ/NF-κB전도통로재농독증인기적응혈공능장애중유일정적작용。
Objective To determine the role of activated status of peroxisome proliferator-activated receptorγ/nuclear 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.