miR506通过NF-κB/TGF-β通路对ARDS小鼠相关肺纤维化的影响及机制研究

Study on the role and mechanism of miR506 affecting ARDS-related pulmonary fibrosis through NF-κB/TGF-β pathway

目的 探究miR506通过核因子-κB(NF-κB)/转化生长因子-β(TGF-β)通路影响急性呼吸窘迫综合征(ARDS)小鼠相关肺纤维化的作用及机制,希望为ARDS相关肺纤维化患者的防治和预后改善提供新思路。方法39只小鼠随机分为对照组(n=10)、脂多糖(LPS)组(n=9)、LPS+NF-κB抑制剂组(n=10)和LPS+miR506抑制剂组(n=10)。除对照组外,其余3组小鼠均采用LPS诱导构建ARDS相关肺纤维化模型,LPS+NF-κB抑制剂组和LPS+miR506抑制剂组小鼠采用NF-κB抑制剂(BAY 11-7082)和miR506抑制剂组(miR506-antagomir)处理,对照组和LPS不做干预。28 d后,苏木精-伊红(HE)染色观察各组小鼠肺组织病理变化;比较各组小鼠体质量、肺湿干重比值(W/D)、肺系数、肿瘤坏死因子-α(TNF-α)、白细胞介素-4(IL-4)、IL-6、IL-1β、上皮细胞钙黏蛋白(E-cadherin)、α-平滑肌肌动蛋白(α-SMA)、波形纤维蛋白(Vimentin)、NF-κB、TGF-β1、SMAD同源物3(SMAD3)和SMAD7水平。结果 HE染色结果显示,对照组小鼠肺泡组织结构完整,未出现水肿及炎症细胞浸润;LPS组小鼠肺组织明显改变,肺泡间隔增厚,肺泡凹陷增多,有不同程度增生、融合出现,可见明显充血及炎症细胞浸润;LPS+NF-κB抑制剂组小鼠肺泡结构较LPS组相对完整,肺泡间隔增厚和肺泡凹陷情况明显改善,水肿及炎症细胞浸润明显缓解;LPS+miR506抑制剂组肺泡组织结构破坏严重,相较于LPS组肺泡间隔和凹陷加剧,增生及肺泡融合情况更明显,充血及炎症细胞浸润程度更重。与对照组比较,LPS组、LPS+NF-κB抑制剂组和LPS+miR506抑制剂组小鼠体质量均减轻,W/D和肺系数升高,差异均有统计学意义(P均<0.05);与LPS组比较,LPS+NF-κB抑制剂组体质量增加,W/D和肺系数下降,差异均有统计学意义(P均<0.05);与LPS+NF-κB抑制剂组比较,LPS+miR506抑制剂组小鼠体质量减轻,W/D和肺系数升高,差异均有统计学意义(P均<0.05)。与对照组比较,LPS组、LPS+NF-κB抑制剂组和LPS+miR506抑制剂组小鼠TNF-α、IL-6、IL-8和IL-1β含量均升高,小鼠肺组织中E-cadherin、SMAD7蛋白表达下调,α-SMA、Vimentin、NF-κB、TGF-β1和SMAD3蛋白表达上调,差异均有统计学意义(P均<0.05);与LPS组比较,LPS+NF-κB抑制剂组小鼠TNF-α、IL-6、IL-8和IL-1β含量均降低,肺组织中E-cadherin、SMAD7蛋白表达上调,α-SMA和Vimentin、NF-κB、TGF-β1和SMAD3蛋白表达均下调,差异均有统计学意义(P均<0.05);与LPS+NF-κB抑制剂组比较,LPS+miR506抑制剂组小鼠TNF-α、IL-6、IL-8和IL-1β含量均升高,肺组织中E-cadherin、SMAD7蛋白表达下调,α-SMA、Vimentin、NF-κB、TGF-β1和SMAD3蛋白表达上调,差异均有统计学意义(P均<0.05)。结论miR506通过抑制炎症反应、α-SMA、Vimentin、NF-κB、TGF-β1和SMAD3表达,上调SMAD7表达来抑制ARDS相关肺纤维化进展,其机制可能与调控NF-κB/TGF-β信号通路有关。

Objective To explore the effect and mechanism of mi R506 on acute respiratory distress syndrome(ARDS)-associated pulmonary fibrosis through nuclear factor-κB(NF-κB)/transforming growth factor-β(TGF-β) pathway and to provide new ideas for the prevention and treatment of ARDS-associated pulmonary fibrosis and the improvement of prognosis. Methods A total of 39 mice were randomly divided into control group(n=10), lipopolysaccharide(LPS) group(n=9),LPS+NF-κB inhibitor group(n=10) and LPS+mi R506 inhibitor group(n=10). Except for the control group, the other three groups of mice were induced by LPS to establish ARDS-associated pulmonary fibrosis models. LPS+NF-κB inhibitor group and LPS+mi R506 inhibitor group were treated with NF-κB inhibitor(BAY 11-7082) and mi R506 inhibitor(mi R506-antagomir), and control group and LPS group did not intervene. After 28 days, hematoxylin-eosin(HE) staining was used to observe the pathological changes in the lung tissues of mice in each group;the body mass, lung wet/dry weight ratio(W/D), lung coefficient, tumor necrosis factor-α(TNF-α), interleukin-4(IL-4), interleukin-6(IL-6), interleukin-1β(IL-1β), epithelial cadherin(E-cadherin), α-smooth muscle actin(α-SMA),Vimentin, NF-κB, TGF-β1, mothers against decapentaplegic homolog 3(SMAD3) and SMAD7 levels were compared among the groups. Results The results of HE staining showed that the structure of the alveoli in the control group was intact, no edema and inflammatory cell infiltration were observed, the lung tissues of the LPS group were obviously changed, the alveolar septum was thickened and the alveoli were depressed;in LPS +NF-κB inhibitor group, the alveolar structure of mice was relatively intact, the alveolar septum was thickened and the alveolar depression was obviously improved compared with that in LPS group;in the LPS+mi R506 inhibitor group, the structure of the alveoli was severely damaged, the alveolar septum and depression were more severe, the proliferation and alveolar fusion were more obvious than those in the LPS group, congestion and inflammatory cell infiltration were more severe. Compared with control group, the body mass of mice in LPS group, LPS+NF-κB inhibitor group and LPS+mi R506 inhibitor group was reduced while the W/D and lung coefficient were enhanced;the differences were statistically significant(all P<0.05). Compared with LPS group, the body mass was increased while the W/D and lung coefficient were decreased in LPS+NF-κB inhibitor group;the differences were statistically significant(all P<0.05). Compared with LPS+NF-κB inhibitor group, the body mass of mice was reduced while the W/D and lung coefficient were enhanced in LPS+mi R506 inhibitor group, with statistical differences(all P<0.05). Compared with the control group, the levels of TNF-α, IL-6, IL-8 and IL-1β were increased, and the expression levels of E-cadherin and SMAD7 protein in the lung tissue were down-regulated, the expression levels of α-SMA, Vimentin, NF-κB, TGF-β1 and SMAD3 protein were upregulated in in LPS group, LPS+NF-κB inhibitor group and LPS+mi R506 inhibitor group;the differences were statistically significant(all P<0.05). Compared with LPS group, the levels of TNF-α, IL-6, IL-8 and IL-1β decreased, and the expression levels of E-cadherin and SMAD7 protein in lung tissue were up-regulated;the expressions levels of α-SMA, Vimentin, NF-κB, TGF-β1 and SMAD3 protein were all down-regulated in LPS+NF-κB inhibitor group;the differences were statistically significant(all P<0.05). Compared with LPS+NF-κB inhibitor group, the levels of TNF-α, IL-6, IL-8 and IL-1β were increased, and the expression levels of E-cadherin and SMAD7 protein in lung tissue were down-regulated;the expression levels of α-SMA, Vimentin, NF-κB, TGF-β1 and SMAD3 protein were up-regulated in LPS+mi R506 inhibitor group;the differences were statistically significant(all P<0.05). Conclusion mi R506 could inhibit the progression of ARDS-associated pulmonary fibrosis by inhibiting the inflammatory response and expressions of α-SMA,Vimentin, NF-κB, TGF-β1 and SMAD3 and up-regulating the SMAD7 expression, and its mechanism might be related to the regulation of NF-κB/TGF-β signaling pathway.