CCR2b和CCR1拮抗剂RS504393对LPS诱导小鼠急性肺损伤的保护作用

Roles of CCR2b and CCR1 antagonist RS504393 in LPS-induced acute lung injury

目的探讨CC趋化因子受体（CCR）2b和CCR1的特异性拮抗剂RS504393在细菌脂多糖（LPS）诱导急性肺损伤中的作用及其机制。方法体外实验选用A549细胞系，应用LPS（10μg／mL）刺激合并RS504393（10μg／mL）治疗6h后，流式细胞仪技术检测CCR1和CCR2的表达，EHSA法检测细胞培养上清液内白介素（IL）-8的浓度；体内实验选用C57BL／6J小鼠，腹腔注射RS504393（5mg/kg）预处理30min后经鼻滴入LPS（5mg／kg），LPS刺激4h后收集BALF和肺脏标本，计数BALF内细胞总数，应用BCA法检测BALF内蛋白浓度，Realtime—RTPCR和EHSA法检测BALF和肺脏IL-1β凝血酶原激活物抑制物（PAI）-1和单核细胞趋化蛋白（MCP）-2的表达。多组间差别比较采用单因素方差分析（One—way ANOVA procedures），两组间差别比较采用成组t检验，以P〈0．05为差异具有统计学意义。结果A549细胞经LPS刺激后与对照组相比，CCRI、CCR2和108的表达明显升高，RS504393治疗可明显降低它们的表达。在LPS诱导肺损伤模型中，与对照组相比，BALF内细胞总数和蛋白浓度明显升高，BALF和肺脏内IL-1β PAl—1的mRNA和蛋白表达显著增加，经RS504393治疗后均显著下降；LPS刺激使BALF内MCP-2升高，RS504393治疗使其进一步增加。结论CCR2和CCR1在急性肺损伤的发病中充当着重要角色，应用CCR2b和CCR1的拮抗剂RS504393治疗急性肺损伤有一定的应用前景。

Objective To evaluate the potential effects of RS504393, CC chemokine receptor （CCR） 2b and CCR1 antagouist, on LPS-induced acute lung injury （ALI） and to investigate the underlying mechanisms. Method A549 cell line was stimulated with LPS （ 10μg/mL） and then treated with RS504393 （ 10μg/mL） for 6 hours. ALI model was established with intranasal administration of LPS （5 mg/kg） in C57BL/6J mice. RS504393 （5 mg/kg） was administered 30 rain before LPS dripped nasally. IL-8, IL-112, plasminogen activator inhibitor （PAI）-1, monoeyte chemoattractant protein （MCP）-2, and the expressions of CCR1 and CCR2b were studied by using Realtime-RT-PCR, ELISA and cyto-flowmetry. Results In A549 cell line treated with RS504393, the expressions of CCR1, CCR2b and IL-8 were significantly inhibited after LPS stimulation. In rats with LPS-induced ALI, treatment with RS504393 significantly protected mice against lung injury by attenuating influx of leukocytes and protein into bronehoalveolar space and by lessening pathological changes of lung. Treatment with RS504393 down-regulated IL-112 and PAI-1 expressions in bronehoal veolar lavage fluid （BALF） and lungs at mRNA and protein levels along with up-regulation MCP-2 expression compared to rats of vehicle-treated groups. Conclusions CCR2b and CCR1 play pivotal roles in the devalopment of ALI, and RS504393 as a antagonist can halt the development of ALI.