前B细胞克隆增强因子在肥胖大鼠 急性呼吸窘迫综合征中的表达研究

Expression of pre-B-cell colony enhancing factor in obesity rats with acute respiratory distress syndrome

目的：探讨前B细胞克隆增强因子（PBEF）在肥胖大鼠急性呼吸窘迫综合征（ARDS）中的变化及其可能作用。方法将40只健康雄性SD大鼠按随机数字表法分为对照组、肥胖组、ARDS组和肥胖ARDS组，每组10只。肥胖两组大鼠采用高脂饲料喂养，另两组喂养普通饲料；待高脂饲养大鼠体重超过正常饲养大鼠20%时为肥胖模型建立成功。此时ARDS两组大鼠经尾静脉注射脂多糖（LPS）构建ARDS模型，另两组则注射等量生理盐水。于ARDS制模后8h开胸取肺组织，测定肺干/湿重（D/W）比值；采用实时荧光定量反转录-聚合酶链反应（RT-PCR）检测PBEFmRNA表达；用蛋白质免疫印迹试验（WesternBlot）检测PBEF、核转录因子-κBp50（NF-κBp50）蛋白表达；采用免疫组化法检测PBEF阳性表达；苏木素-伊红（HE）染色后光镜下观察肺组织病理学改变。结果高脂饲料喂养第6周成功建立肥胖大鼠模型。肥胖组、ARDS组、肥胖ARDS组肺D/W比值明显低于对照组（0.195±0.005、0.179±0.003、0.153±0.011比0.224±0.007，均P＜0.05），且肥胖ARDS组显著低于ARDS组，说明肥胖对非ARDS及ARDS大鼠的肺含水量均有影响。肥胖组、ARDS组、肥胖ARDS组肺组织PBEFmRNA表达量依次升高〔分别为对照组的（1.77±0.22）、（3.29±0.14）、（5.52±0.14）倍，均P＜0.01〕，肺组织PBEF、NF-κBp50蛋白表达量也依次升高〔PBEF分别为对照组的（1.75±0.16）、（2.71±0.19）、（3.83±0.18）倍，NF-κBp50分别为对照组的（1.56±0.12）、（1.95±0.12）、（2.48±0.24）倍，均P＜0.01〕，且各组间两两比较差异均有统计学意义。免疫组化显示，对照组肺组织中几乎无PBEF阳性表达；肥胖组有少量PBEF阳性表达；ARDS组PBEF阳性表达明显增多；肥胖ARDS组肺泡壁、肺泡腔内的渗出液中有强阳性反应。HE染色显示，对照组肺组织结构正常；肥胖组有少许炎性细胞浸润；ARDS组肺泡间隔增厚，肺泡腔内有大量红细胞浸润；肥胖ARDS组肺部炎症程度最为明显，肺泡腔内可见大量渗出液、红细胞及炎性细胞。结论 PBEF可能通过调控NF-κB的活性参与肥胖大鼠ARDS的发生发展。

Objective To investigate the variation and potential function of pre-B cell colony enhancing factor （PBEF） in obesity rats with acute respiratory distress syndrome （ARDS）. Methods Forty healthy male Sprague-Dawley （SD） rats were randomly divided into control group, obesity group, ARDS group, and obesity ARDS group, with 10 rats in each group. Rats in the two obesity groups were fed with high-fat diet, and the rats in other groups were fed with normal fodder. After successful obesity models were reproduced as the mean weight of rats in obesity model groups was up over 20% compared to other groups, the rats in two ARDS groups were injected with lipopolysaccharide （LPS） through tail vein to reproduce ARDS models, and the rats in other groups were injected with the same volume of saline. The lung tissues were harvested at 8 hours after ARDS model reproduction to determine lung dry/wet weight （D/W） ratio; real-time fluorescence quantitative reverse transcription-polymerase chain reaction （RT-PCR） was used to detect the expression of PBEF mRNA, and the proteins expressions of PBEF and nuclear factor-κB p50 （NF-κB p50） were detected by Western Blot; the distribution and expression of PBEF was also examined by immunohistochemical staining. Pathological changes of lung tissues were observed after hcmatoxylin-eosin （HE） staining. Results Obesity rat models were set up successfully with 6 weeks of high fat diet. The D/W ratio of rats in obesity, ARDS, and obesity ARDS groups was significantly lower than that of control group （0.195±0.005, 0.179±0.003, 0.153±0.011 vs. 0.224±0.007, all P 〈 0.05）, and D/W ratio in obesity ARDS group was significantly lower than that of ARDS group, indicating that the lung water content of ARDS rats and non ARDS rats could be influenced by obesity. In obesity group, ARDS group and obesity ARDS group, the expression of PBEF mRNA in lung tissues was increased in turn [it was （1.77±0.22）, （3.29±0.14）, （5.52±0.14） folds of that in control group respectively, all P 〈 0.01], and the proteins expression of PBEF and NF-κB p50 in lung tissues were also increased in turn [PBEF protein was （1.75±0.16）, （2.71±0.19）, （3.83±0.18） folds of that of control group, and NF-κB p50 protein was （1.56±0.12）, （1.95±0.12）, （2.48±0.24） folds of that of control group respectively, all P 〈 0.01], and there were significant differences between any two groups. There was almost no PBEF expression in lung tissues of control group detected by immunohistochemical staining, and a little PBEF expression in obesity group, the obvious increased PBEF expression was found in ARDS group, as well as PBEF expressed strikingly in alveolar walls exudates and alveolar spaces exudates of obesity ARDS group. It was shown by HE staining there were no significant pathological changes in lung tissue of rats in the control group; a few inflammatory cells infiltrated in lung tissue of obesity group, thicken alveoli septum and a number of the red cells infiltrated in alveolar spaces were found in ARDS group; the inflammation degree in lung tissue is most serious, a huge amount of exudates, red cells and inflammatory cells in alveolar spaces were found in the obesity ARDS group. Conclusions PBEF might be involved in the process of the development of ARDS in obese rats through the regulation of activity of NF-κB.