巨噬细胞刺激蛋白及其受体酪氨酸激酶RON在慢性阻塞性肺疾病大鼠气道炎症中的作用

The Role of Macrophage-Stimulating Protein and Receptor Tyrosine Kinase RON in Airway Inflammation of COPD

目的研究巨噬细胞刺激蛋白(MSP)及其受体酪氨酸激酶RON在慢性阻塞性肺疾病(COPD)大鼠气道炎症中的作用及相关机制。方法单纯熏香烟法建立COPD大鼠模型;分离、培养正常和COPD大鼠肺泡巨噬细胞(AM),分别予不同剂量的MSP处理24 h。采用酶联免疫吸附法(ELISA)检测大鼠血清和支气管肺泡灌洗液(BALF)中MSP及AM上清液中IL-1β、TNF-α、IL-8和IL-10的浓度,逆转录-聚合酶链式反应(RT-PCR)法检测大鼠肺组织中酪氨酸受体激酶RON mRNA的表达情况,免疫组化法观察大鼠肺组织和离体培养的AM中RON蛋白的表达情况,比色法检测AM上清液中超氧化物歧化酶(SOD)的活性及丙二醛(MDA)的水平。结果与正常对照组比较,COPD组大鼠血清和BALF中MSP的浓度显著增高(P<0.01)。COPD组大鼠肺组织RON mRNA表达较正常对照组明显上调(P<0.01),COPD组大鼠支气管上皮及AM中RON蛋白水平也较正常对照组显著增加(P<0.01)。MSP呈剂量依赖性刺激正常和COPD大鼠AM细胞产生MDA增加并导致其SOD活性降低,亦能刺激两组大鼠AM释放炎症因子TNF-α、IL-8、IL-1β和IL-10呈剂量依赖性增加。在相同剂量MSP作用下,COPD组大鼠AM上清液中MDA及TNF-α、IL-8和IL-1β的浓度均高于正常对照组(P<0.01),而IL-10的浓度和SOD的活性则低于正常对照组(P<0.01);其中TNF-α、IL-8和IL-1β浓度的增加幅度及SOD活性降低的幅度大于正常对照组(P<0.01),而MDA和IL-10升高的幅度则低于正常对照组(P<0.01)。相关分析显示MSP和RON分别与BALF中细胞总数和AM计数呈正相关(P<0.01),与肺气肿指标呈一定的相关关系(P<0.01)。结论 MSP及其受体RON参与了COPD的气道炎症调节,其机制可能是MSP/RON促进了AM释放炎症因子和诱导氧自由基产生增多。

Objective To explore the role of macrophage-stimulating protein （MSP） and receptor tyrosine kinase RON in the airway inflammation of chronic obstructive pulmonary disease （ COPD）, and investigate its possible mechanism. Methods The rat COPD model was established by exposing the rats to cigarette smoke daily for three months. Rat alveolar macrophages （AMs） were isolated in vivo and cultured, and then challenged with different concentrations of MSP for 24 hours. The concentrations of MSP in broncho-alveolar lavage fluid （BALF） and serum, and the levels of IL-1β, TNF-α, IL-8, and IL-10 in the supernatants were measured by ELISA. The expression of RON mRNA in lung tissue was assessed by reverse transcription-polymerase chain reaction. The levels of RON protein in the lung tissue and AMs cultured in vitro were observed by immunohistochemistry. The activity of superoxide dismutase （SOD） and malondialdehyde （MDA） content in the culture solution were measured with chromatometry method. Results Compared with the control group, the concentrations of MSP in serum and BALF of the COPD rats were significantly higher （P 〈 0. 01 ）. The levels of RON mRNA and RON protein in the COPD rats were also up-regulated significantly （ P 〈 0.01 ）. MSP evoked the AMs isolated from the normal and COPD rats to generate more content of MDA and caused a reduction in activity of SOD. In addition, MSP stimulated TNF-α, IL-8, IL-1β and IL-10 release from AMs of the normal and COPD rats dose-dependently. The levels of TNF-α,IL-8,and IL-1β were higher,while the level of IL-10 and the SOl） activity were lower in AMs of the COPD group than those of the control group in the same dose of MSP （P 〈0. 01 ）. The more significant increase in the levels of TNF-ct, IL-8, IL-1β, and the more notable decrease in the activity of SOD was found in the COPD group compared with the control group. But the degree of increasing MDA and IL-IO in the AMs of the COPD group was lower than that in the control group. Linear correlation analysis showed that the MSP concentration and the RON protein level in the COPD rats were positively associated with the total cell counts and AM counts in BALF, and were related to the indexes for pulmonary emphysema. Conclusions There is a close correlation between the MSP and receptor tyrosine kinase RON with the airway inflammation of COPD. The mechanism might be that MSP promote the macrophages release inflammatory factors and increase the production of oxygen free radicals.