【作者】 杨廷芳；

【导师】 王兴勇；

【作者基本信息】 重庆医科大学 ， 急诊医学， 2008， 硕士

【摘要】 目的:探讨小肠缺血再灌注(ischemia/reperfusion,I/R)所致的急性肺损伤(acute lung injury, ALI)信号转导机制和白藜芦醇苷(polydatin, PD)的保护机制。方法:224只健康雄性Sprague-Dawley(SD)大鼠,随机分为假手术对照组(C组)、肠缺血再灌注I/R肺损伤组(I/R组),I/R + NS组和I/R + PD治疗组。将大鼠腹腔打开后用暂时性夹持肠系膜上动脉(superior mesenteric artery, SMA)45分钟再松开的方法建立急性I/R肺损伤模型。在SMA松夹同时给予腹腔注射10 mg/kg PD为I/R + PD治疗组,注射生理盐水为I/R + NS组。假手术对照组的操作除不阻断SMA外其余操作与I/R组相同。分别于损伤后0h、0.5h、2h、6h、12h、24h、48h七个不同时间点处死实验大鼠留取大鼠肺标本保存待检。用苏木素-伊红(hematoxylin and eosin,HE)染色肺组织观察其在光镜下的病理形态学变化;用烘干前后肺组织的湿、干重(W/D)称量法测量肺组织水肿情况;比色法检测肺组织髓过氧化物酶(myeloperoxidase,MPO)活性变化;免疫组织化学染色法检测肺组织NF-κBp65和ICAM-1 (CD54)蛋白的表达;RT- PCR法检测肺组织HMGB1mRNA表达。结果:肺组织HE染色示假手术对照组仅有轻微的肺间质增宽改变,而I/R和I/R+NS组可见肺泡结构破坏,肺泡隔增宽且肺泡间隔炎性细胞浸润,肺泡腔内有渗出液、红细胞及渗出的炎性细胞,I/R+PD治疗组较I/R+NS组有明显改善。I/R组肺组织W/D值在0.5h、2h、6h、12h及24h升高与假手术对照组比较有显著统计学差异(P<0.01)。I/R组MPO活化表达升高,与假手术对照组在各时间点相比均有显著统计学差异(P<0.01)。I/R组ICAM-1(CD54)表达随肠I/R时间延长而增加,6h达峰值(160.67±21.62/HP),此后稍有下降,24h轻度升高(107.83±20.40/HP),与假手术对照组在各时间点相比均有明显升高(P<0.05)。I/R组MPO与ICAM-1之间存在较强的正相关(P<0.01)。I/R组NF-κBp65表达随肠I/R时间延长而增加,24h达峰值(169.00±22.65/HP),较假手术对照组在各时间点表达有统计学差异(P<0.05)。I/R组HMGB1mRNA随肠I/R时间延长表达升高,24h达峰值(1.81±0.22),较假手术对照组在2h、6h、12h、24h及48h表达有显著统计学差异(P<0.01)。I/R组NF-κBp6与HMGB1mRNA之间存在较强的正相关(P<0.01)。I/R+PD治疗组W/D值在0.5h、2h、6h及12h有显著降低,与I/R+NS组比较有显著统计学差异(P<0.01)。I/R+PD治疗组MPO活化表达较I/R+NS组在各时间点均有明显下降(P<0.05)。I/R+PD治疗组ICAM-1表达下降,在0.5h、2h、6h、12h及24h与I/R+NS组比较均有统计学差异(P<0.05)。I/R+PD治疗组NF-κBp65和HMGB1RNA表达均在2h后下降,与I/R+NS组比较有统计学差异(P<0.05)。结论:1.运用大鼠小肠缺血再灌注的方法成功建立了急性肺损伤动物模型。2. PMNs和ICAM-1表达有相互介导关系,而HMGB1肺内表达升高可能与NF-κB的转录调控有关。3. PD可能是通过减少PMNs和ICAM-1表达同时抑制NF-κB的激活,阻断其信号转导通路,下调HMGB1基因表达而实现对损伤肺组织的保护作用。更多还原

【Abstract】 【Objective】To investigate the mechanism of signal transduction and the protective effect of polydatin (PD) in intestinal ischemia- reperfusion (I/R) induced acute lung injury in rats.【Methods】Two hundred and twenty-four Male Sprague-Dawley (SD) rats were randomly divided into four groups, the sham-operation control group (group C), the intestinal I/R induced lung injury group (I/R group), I/R + NS group and I/R + PD treatment group. The I/R induced ALI model was established by temporally clamping the superior mesenteric artery (SMA) for 45 minutes. Either 10 mg/kg PD (I/R + PD treatment group) or normal saline (I/R + NS group) was intraperitoneally administered immediately before the clamp released. The operative procedures of the sham-operation control group were the same as those of I/R group except not clamping the SMA. The rates were sacrificed and the lung tissues were separated at different time points of 0h, 0.5h, 2h, 6h, 12h, 24h or 48h after I/R injury. The pathological changes of lung tissues were examined under the light microscope with hematoxylin and eosin (HE) stain. The lung tissue Wet/dry weight ratio (W/D) were examined. The levels of lung tissue myeloperoxidase (MPO), the neutrophil sequestration indicator, were detected by colorimetric method. Formalin-fixed and paraffin embedded lung tissues were stained with immunohistochemistry technique for nuclear factor-κB(NF-κB) and intercellular adhesion molecule-1 (ICAM-1) detection. The lung tissue High mobility group box 1(HMGB1mRNA) expressions were determined by RT-PCR.【Results】Histological changes of the lung tissues showed slight thickening of the alveolar walls in sham-operation control group. There were massive inflammatory cell infiltrations, byperemia and of interstitial lung tissue edema, diffusate, akaryocyte, inflame-cellule in alveolar intracavitary, alveolar wall thickening in I/R group and I/R + NS group. The above lung pathological changes were not so severe in I/R + PD treatment group. The lung tissue wet-to-dry weight ratios markedly rose in I/R group compared with those in sham-operation control group at any corresponding time point of 0.5h, 2h, 6h, 12h and 24h, with statistical significance(P<0.01). The expressions of lung tissue MPO markedly rose in I/R group compared with those in sham-operation control group at any corresponding time point, with statistical significance(P < 0.01). The expressions of lung tissue ICAM-1 increased with time after intestinal I/R, peaked at 6h (160.67±21.62/HP), then decreased, and slightly increase at 24h(170.83±20.40/HP)in I/R group. The expressions of lung tissue ICAM-1 were higher than those in sham-operation control group at any corresponding time point (P < 0.05). There exists a strong positive correlation between MPO and ICAM-1, with statistical significance (P<0.01). The expressions of lung tissue NF-κBp65 in I/R group increased with time after intestinal I/R, peaked at 24h (169.00±22.65/HP). The expressions of lung tissue NF-κBp65 in I/R group was lower than those in sham-operation control group at any corresponding time point (P<0.05). The expressions of lung tissue HMGB1mRNA in I/R group increased with time after intestinal I/R, peaked at 24h (1.81±0.22), The expressions of lung tissue HMGB1mRNA markedly rose in I/R group compared with those in sham-operation control group at any corresponding time point of 2h, 6h, 12h, 24h and 48h, with statistical significance(P<0.01). There exists a strong positive correlation between NF-κBp65 and HMGB1 mRNA, with statistical significance (P<0.01). Lung tissue wet-to-dry weight ratios markedly decreased in I/R + PD treatment group compared with those in I/R + NS group at any corresponding time point of 0.5h, 2h, 6h and 12h,with statistical significance(P<0.01). The expressions of lung tissue MPO in I/R + PD treatment group was lower than those in the I/R + NS group at any corresponding time point (P<0.05). The expressions of lung tissue ICAM-1in I/R + PD treatment group was lower than those in the I/R + NS group at any corresponding time point of 0.5h, 2h, 6h, 12h and 24h (P < 0.05). The expressions of lung tissue NF-κBp65 and HMGB1mRNA in I/R + PD treatment group was lower than those in the I/R + NS group at any corresponding time point of 2h, 6h, 12h, 24h and 48h (P<0.05).【Conclusion】1. Acute lung injury model was successfully induced by intestinal ischemia/reperfusion injury in rats.2. The expressions of PMNs and ICAM-1 inter-mediated each other. The increased lung tissue HMGB1 expressions may be regulated by NF-κB through transcriptional control in intestinal I/R induced ALI.3. The protective effect of polydatin may be through decreasing the lung tissue PMNs and ICAM-1 expressions, inhibiting the NF-κB activiation, blocking the signal transduction and down-regulating the expressions of HMGB1 during intestinal I/R induced lung injury.更多还原