急性肺栓塞后肺泡巨噬细胞Latexin表达上调及其对炎症的影响

目的:研究大鼠急性肺栓塞模型肺组织中羧肽酶抑制剂Latexin的表达变化及其对肺部炎症的影响。方法:建立大鼠急性肺栓塞模型,分别在急性肺栓塞后1、8、24和48 h开胸取出肺组织。常规提取肺组织的总RNA和总蛋白,以正常组为对照,采取半定量RT-PCR的方法研究Latexin在mRNA水平表达的变化;采用Western-blot方法进一步验证Latexin在蛋白水平表达的变化;采用免疫组织化学的方法检测大鼠肺组织中Latexin在肺栓塞前后表达的变化及其组织分布情况。分别在大鼠急性肺栓塞后1、8、24和48 h暴露气管,进行支气管肺泡灌洗,支气管肺泡灌洗液(BALF)上清采用放射免疫法(RIA)检测内皮素-1(ET-1)的浓度;采用ELISA法检测白三烯C4(LTC4)的浓度;细胞沉淀用于分离正常大鼠肺泡巨噬细胞(AMs),并进一步用免疫磁珠法纯化AMs。采用半定量RT-PCR和Western blot法检测AMs在急性肺栓塞后不同时间点Latexin及其作用底物羧肽酶A3(CPA3)的表达。结果:在大鼠急性肺栓塞后8h,肺组织内Latexin的mRNA水平和蛋白水平均逐渐升高。免疫组化研究表明急性肺栓塞后AMs在肺泡腔内的浸润增加,Latexin主要表达于AMs。急性肺栓塞大鼠BALF中分离的AMs无论Latexin和CPA3的表达都明显升高。随着Latexin表达的升高,ET-1和LTC4的浓度也随之升高。结论:急性肺栓塞后AMs中Latexin及其作用靶蛋白CPA3的表达均升高,二者处于一种动态的平衡。大鼠急性肺栓塞后AMs中Latexin的表达显著升高,抑制了CPA3的酶解作用,使得ET-1和LTC4的浓度增加,从而促进了肺部炎症的发展和肺血管收缩。

Up-regulation of Latexin in the alveolar macrophages and its effects on the inflammation of lung tissues after acute PE

Objective: To study the expression changes of carboxypeptidase inhibitor Latexin in the alveolar macrophages(AMs) of a rat acute pulmonary embolism(PE) model and its effects on the AMs-mediated inflammation.Methods: We constructed a rat acute PE model.The samples of lung tissues were collected at different time points 1h,8h,24h and 48h.The normal rats were used as control.The mRNA level changes of Latexin were identified by semi-quantitative RT-PCR,and the protein level changes of it were validated by western blot methods.The immunohistochemical method was employed to study the distribution and expression changes of Latexin in the lung tissues.Bronchoalveolar lavage fluid(BALF) of rats with acute PE was collected at different time points 1h,8h,24h and 48h.Radioimmunoassay(RIA) was employed to determine the concentration of endothelin-1(ET-1) in the supernatant of BALF and ELISA to determine the concentration of leukotriene C4(LTC4).AMs were collected from the cell precipitate of BALF and then separated by anti-CD68 mAbs using immunomagnetic beads(d≤50 am).The expression changes of latexin and its target protein carboxypeptidase A3(CPA3) in AMs were validated by semi-quantitative RT-PCR and western blot methods.Results: The mRNA levels and the protein levels of latexin were up-regulated obviously in the lung tissues after acute PE.The immunohistochemical study indicated more infiltration of AMs in the alveolar space than normal,and that latexin was mainly expressed in AMs. The expression of both latexin and CPA3 in AMs from BALF increased obviously after acute PE.The concentration of ET-1 and LTC4 in BALF also increased accompanying with the up-regulation of latexin in AMs.Conclusion: The expression of both latexin and its target protein CPA3 in AMs from BALF increased obviously after acute PE,and their expression was in a state of dynamic balance.The up-regulation of latexin suppressed the activity of CPA3,which resulted in the increasing of ET-1 and LTC4 in the lung tissues.These molecules would promote the progress of inflammation and pulmonary vasoconstriction in the lung tissues after acute PE.