let-7在小鼠哮喘模型气道炎性反应中的作用及机制

Role and mechanism of let-7 in airway inflammation in mice with asthma

目的观察let-7家族微RNA抑制剂（anti—let-7）对支气管哮喘（简称哮喘）小鼠气道炎性反应的影响，并探讨let-7参与哮喘形成的机制。方法32只小鼠按随机数字法分为4组（n=8），即正常对照组（A组）、哮喘组（B组）、干预对照组（c组）和干预组（D组）。其中B、C和D组用鸡卵蛋白（OVA）免疫，建立哮喘模型，A组以生理盐水替代OVA处理。D组小鼠激发前注射anti．1et-7以抑制内源性let-7表达，c组小鼠注射乱序siRNA对照。比较各组小鼠支气管肺泡灌洗液（BALF）的细胞计数，肺组织中let-7e以及BALF中白细胞介素-10（IL-10）含量；体外用anti．1et-7转染肺癌细胞A549，并检测细胞中let-7e表达和细胞培养上清中IL-10的含量；荧光素酶报告法检测let-7e是否直接靶向IL-10。结果与A组相比，B组和c组小鼠BALF中细胞总数和嗜酸粒细胞数显著增加[（20．32±5．33）×109／L和（24．74±6．69）×100／L比（7．12±1．88）×109／L，（6．45±2．5）×109／L和（7．12±2．66）×100／L比（0．04±0．01）×109／L，均P〈0．01]；肺组织中let-7e水平明显升高（分别为3．83倍和3．27倍，均P〈0．01）。与c组比较，D组BALF中细胞总数和嗜酸粒细胞数明显减少[（13．85±3．74）X109／L比（24．74±6．69）×109／L，（2．15±1．13）×109／L比（7．12±2．66）×109／L，均P〈0．05]；肺组织中let-7e显著降低[（0．45±0．22）比（3．28±0．45），P〈0．01]，同时BALF中IL-10水平明显升高[（4．68_＋0．85）比（1．70±0．29），P〈O．01]。此外，在肺癌细胞A549中转染anti—let-7，1et-7e表达显著下降[（0．22±0．03）比（1．00±0．11），P〈O．01]，同时培养上清中IL—10明显上升[（2．58±0．35）比（1．00±0．15），P〈0．01]。体外let．7e过表达显著降低IL-10报告载体的荧光素酶活性[（0．59±0．06）比（1．00±0．03），P〈0．01]，而对突变的IL．10报告载体没有抑制作用。结论anti—let-7对哮喘小鼠气道炎性反应具有明显的抑制作用，其作用机制可能与let-7直接靶向并抑制IL-10有关。

Objective To explore the effects of let-7 inhibitor（anti-let-7） on airway inflammation in rats with asthma, and to investigate the mechanisms of let-7 in the development of asthma. Methods A total of 32 mice were randomly assigned to control group （group A, n=8）, asthma group （group B, n=8）, treatment control group（group C, n=8） and treatment group （group D, n=8）, respectively. Groups B, C and D were sensitized with ovalbumin （OVA） for establishment of mouse asthma model. Group A was treated with normal saline orally. Group D was administered with anti- let- 7 prior to challenge, for suppression of endogenous let-7e expression, while scrambled-siRNA was injected in group C for treatment control. The cell count, let-Te in pulmonary tissues and interleukin-10 （IL-10） in brouchoalveolar lavage fluid （BALF） were detected. A549 cells, the lung cancer cells, were transfected with anti-let-7 in vitro followed by measurement of let-Te expression and IL-10 in cell culture supernatants. Luciferase report assay was employed to assess if let-Te targeted at IL-10. Results Compared with group A, groups B and C significantly increased in thetotal cell count [ （20.32±5.33） × 109/L and （24.74±6.69）×109/L vs （7.12±1.88） × 109/L, both/9〈0.011 and eosinophil count I （6.45_＋2.5） ×109/L and （7.12_＋2.66） × 109/L vs （0.04_＋0.01） × 109/L, both/9〈0.011 in BALF as well as let-7e of lung tissues （by 3.83-fold and 3.27-fold, both/9〈0.01）. When compared with group C, group D significantly reduced in total cell count I （13.85_＋3.74）× 109/L vs （24.74±6.69）×109/L, /9〈0.051 and eosinophil count I （2.15± 1.13 ） × 109/L vs （7.12± 2.66） ×109/L, /9〈0.051 in BALF, and in let-7e of lung tissues （0.45±0.22） vs （3.28±0.45）, P〈0.051, while markedly increased the level of IL- 10 in BALF [（4.68±0.85） vs （1.70±0.29）, P〈0.011. Additionally, transfection of A549 cells using anti-let-7 resulted in reduction of let-7e expression [ （0.22±0.03） vs （ 1.00±0.11 ）, P〈O.01 ] and elevation of IL- 10 in the supernatant [ （2.58± 0.35） vs （1.00± 0.15） , /9〈0.011. Moreover, over- expression of let- 7e led to significantly suppressed lueiferase activity of IL- 10 reporter vector I （0.59±0.06） vs （ 1.00± 0.03）, /9〈0.011, but did not affect the mutant IL-10 reporter vector. Conclusion The anti-let-7 can markedly inhibit airway inflammation in mice with asthma, possibly by targeting at let-7 and inhibition of IL-10.