抗CD14单克隆抗体对脓毒症大鼠肺组织趋化因子和可溶性细胞间黏附因子-1表达的影响

Study of the Anti-CD14 monoclonal antibody on sI CAM-1 and KC expression of lung tissue with sepsis in rat

目的探讨抗CD14单克隆抗体(Anti-CD14Mc Ab)治疗大鼠脓毒症急性肺损伤的保护机制。方法在体部分:18只雄性Wistar大鼠(200~250 g)随机均分为3组,假手术组(Sham组)、脓毒症组(CLP组)和抗CD14治疗组(Anti-CD14组),CLP组和Anti-CD14组采用盲肠结扎穿孔术建立脓毒症急性肺损伤模型,Sham组作为对照组,开腹后只翻动盲肠,不进行结扎穿孔。Anti-CD14组术后经股静脉注射Anti-CD14Mc Ab 0.2 ml(1μg/ml),Sham组、CLP组术后经股静脉注射等量生理盐水0.2 ml,6 h后经腹主动脉抽血,测定3组大鼠血浆中趋化因子(KC)和可溶性细胞间黏附因子-1(s ICAM-1)表达水平,右肺下叶HE染色,观察肺组织病理结构的改变。离体部分:将培养的NR8383一部分接种于24孔培养板,分为3组,对照组(Ⅰ组)、脓毒症模型组(Ⅱ组)和Anti-CD14干预组(Ⅲ组)。Ⅰ组加入正常血浆,Ⅱ组加入脓毒症血浆,Ⅲ组加入脓毒症血浆和Anti-CD14Mc Ab共同干预,37℃、5%CO2培养箱孵育1 h,刮取NR8383,采用Western blot测定NR8383内NF-κB(p65)蛋白的表达水平;另一部分接种在预先放入适当大小灭菌消毒的盖玻片的24孔板上,分组及干预措施同上,采用激光扫描共聚焦显微镜(CLSM)检测NR8383内NF-κB(p65)蛋白入核情况。结果在体实验提示:(1)与Sham组[KC:(72.645±19.860)pg/ml;s ICAM-1:(252.766±19.921)pg/ml]相比,CLP组血浆KC、s ICAM-1水平[KC:(490.316±43.403)pg/ml;s ICAM-1:(686.952±36.411)pg/ml]和Anti-CD14组血浆KC、s ICAM-1水平[KC:(348.150±20.924)pg/ml;s ICAM-1:(411.050±47.170)pg/ml]明显升高,而Anti-CD14组KC、s ICAM-1水平明显低于CLP组,差异有统计学意义(P<0.05);肺组织病理结果提示CLP组肺间质明显增厚、肺间质及肺泡腔内有大量炎症细胞浸润,Anti-CD14组症状较CLP组明显减轻,但较Sham组有所增加;细胞实验Western blot检测显示:Ⅱ组(2.190 3±0.119 9)和Ⅲ组(1.365 8±0.101 8)NR8383的NF-κB(p65)入核量高于Ⅰ组(1.012 2±0.078 0),Ⅲ组的NF-κB(p65)入核量低于Ⅱ组,差别有统计学意义(P<0.05)。细胞实验CLSM检测结果提示:与Ⅰ组(0.131±0.009)相比,Ⅱ组(0.685±0.037)和Ⅲ组(0.210±0.008)NR8383 NF-κB(p65)蛋白入核明显升高,而Ⅲ组NR8383 NF-κB(p65)蛋白入核低于Ⅱ组(0.685±0.037),差异有统计学意义(P<0.05)。结论 Anti-CD14Mc Ab在脓毒症急性肺损伤时可能调控肺泡巨噬细胞NF-κB(p65)蛋白入核,使其KC表达减少,进而减少中性粒细胞浸润,起到肺保护作用;同时Anti-CD14Mc Ab能抑制内皮细胞表达s ICAM-1,减少中性粒细胞对内皮细胞的黏附作用,起到肺保护作用。

Objective To explore the protection mechanism of the Anti-CD14 Mc Ab on the treatment of sepsis with acute lung injury in rat. Methods 18 male Wistar rats(200-250 g) were randomly divided into 3 groups, group Sham, group CLP and group Anti-CD14. There were 6 rats in each group. The cecal ligation and puncture(CLP) method was used to make the animal model with acute lung injury, group Sham as control group, group Sham rats just flip the cecum without ligation and puncture, the group Anti-CD14 was injected 0.2 ml Anti-CD14 monoclonal antibody(1 μg/ml) by femoral vein after postoperative, group Sham, CLP were injected saline 0.2 ml by femoral vein after postoperative, after the success of modeling 6 h, we draw blood from abdominal aorta and detected plasma KC, s ICAM-1 of 3 group rats. Inferior lobe of right lung was taken for HE staining and observed the pathological changes in the structure of lung tissue. Sham group as control group. In vitro part: a part of cultivated NR8383 were seeded in 24-well culture plate, divided into three groups, control group(groupⅠ), sepsis model group(groupⅡ) and Anti-CD14 intervention group(groupⅢ). GroupⅠ was added to the normal plasma, groupⅡ was added to the sepsis plasma, groupⅢ was added to the sepsis plasma and Anti-CD14 monoclonal antibody, three group was incubated about 1 hour in incubator with 37 ℃ and 5% CO2, Western blot was used to detect the NF-kappa B(p65) protein expression levels of the NR8383 scraped from 24-well culture plate; another part of cultivated NR8383 were seeded in 24-well culture plate with appropriate size of the sterilized cover glass, grouping and intervention measures was the same as the above, the confocal laser scanning microscope(CLSM) was used to detect the NF-kappa B(p65) protein into the NR8383 nuclear situation. Results In vivo experiments: compared with the group Sham [KC:(72.645±19.860)pg/ml, s ICAM-1:(252.766±19.921)pg/ml], KC, s ICAM-1 expression level in plasma of the group CLP [KC:(490.316±43.403)pg/ml, s ICAM-1:(686.952±36.411)pg/ml] and the group Anti-CD14 [KC:(348.150±20.924)pg/ml, s ICAM-1:(411.050±47.170)pg/ml] significantly increased, but s ICAM-1, KC expression levels in plasma of the group Anti-CD14 below the group CLP, there were significant differences(P<0.05); lung tissue pathology results indicated pulmonary interstitial of the group CLP obvious thickening, a large number of inflammatory cells infiltrated the pulmonary interstitial and alveolar cavities, the group Anti-CD14 symptoms significantly lower than the group CLP, but higher than the group Sham; Cell experiment Western blot test showed: the group Ⅱ(2.190 3±0.119 9) and the group Ⅲ(1.365 8±0.101 8) NR8383 NF-kappa B(p65) into the NR8383 nucleus was higher than group Ⅰ(1.012 2±0.078 0), the group Ⅲ NF-kappa B(p65) into the NR8383 nucleus level was lower than the group Ⅱ, there were significant differences(P<0.05). Cell experimental CLSM test results indicated: compared with groupⅠ(0.131±0.009), the group Ⅱ(0.685±0.037), the group Ⅲ(0.210±0.008) NF-kappa B(p65) protein into the NR8383 nucleus increased significantly, while the group Ⅲ NF-kappa B(p65) protein into the NR8383 nucleus was lower than the group Ⅱ, there were significant differences(P<0.05). ConclusionsAnti-CD14 Mc Ab may control the NR8383 NF-kappa B(p65) protein into the nucleus and reduce its chemokines KC expression, thus reducing neutrophilic granulocyte infiltration, as a result, Anti-CD14 Mc Ab plays a protective role in sepsis with acute lung injury; with Anti-CD14 Mc Ab inhibit endothelial cells expressing s ICAM-1 and reduce neutrophil adhesion function of endothelial cells, therefore, Anti-CD14 Mc Ab plays a lung protective role.