上调miR-24对急性肺损伤幼龄大鼠炎症反应的影响及作用机制

Effect and mechanism of upregulating miR-24 on the inflammation of acute lung injury in infant rats

目的 探究微小型RNA-24(microRNA-24,miR-24)对急性肺损伤(acute lung injury, ALI)幼龄大鼠炎症反应的影响及作用机制。方法 将幼龄大鼠分为Control、ALI、ALI+miR-24 mimic mock及ALI+miR-24 mimic组,利用脂多糖(LPS)建立幼龄大鼠ALI模型。miR-24 mimic mock及ALI+miR-24 mimic分别处理模型大鼠后,计算大鼠肺组织干湿重比,qRT-PCR检测miR-24的mRNA表达,HE染色观察大鼠肺组织病理变化,Tunel检测肺组织细胞凋亡情况,Western blot检测肺组织中磷酸化核因子κB p65(nuclear factor κB p65, NF-κB p65)、磷酸化核因子κB抑制蛋白(phosphorylation inhibitor of NF-κB, p-IκBα)及IκBα激酶(IκBα kinase, p-IKK)的表达。将大鼠肺泡巨噬细胞分为Control、LPS、LPS+miR-24 mimic、LPS+pcDNA-IL-1α(pc-IL-1α)及LPS+miR-24 mimic+pc-IL-1α组,利用LPS刺激细胞。miR-24 mimic及pc-IL-1α分别或同时转染细胞后,qRT-PCR检测miR-24的mRNA水平,生物信息预测miR-24与IL-1α的靶向关系,并利用荧光素酶报告试验检测。ELISA检测肿瘤坏死因子α(tumor necrosis factor, TNF-α)、白介素-6(interleukin-6, IL-6)、IL-1α和IL-1β在各组幼龄大鼠血清中及各组大鼠肺泡巨噬细胞培养上清中的浓度。结果 miR-24在ALI幼龄大鼠肺组织的mRNA水平降低,miR-24 mimic可上调miR-24 mRNA表达。与Control组比较,ALI组幼龄大鼠肺泡内充满炎性细胞,肺干湿重比值增大,肺组织凋亡细胞百分比升高。与ALI组比较,ALI+miR-24 mimic mock组无统计学差异,ALI+miR-24 mimic组肺泡内炎性细胞明显减少,肺干湿重比值减小,凋亡细胞百分比降低。miR-24 mimic可降低ALI幼龄大鼠血清中TNF-α、IL-6、IL-1α和IL-1β的浓度,减少肺组织中 p-IKK 、p-IκBα及NF-κB p65的表达。miR-24在经LPS处理大鼠肺泡巨噬细胞中的mRNA水平降低。miR-24 mimic减弱野生型IL-1α质粒(IL-1α wt)的荧光素酶活性,并可降低经LPS处理大鼠肺泡巨噬细胞上清中炎症因子 TNF-α、IL-6、IL-1α和IL-1β的浓度,而pc-IL-1α可提高这些炎症因子,并减弱miR-24 mimic对炎症反应的抑制作用。结论 miR-24 mimic可通过靶向IL-1α及抑制NF-κB激活,减轻ALI幼龄大鼠肺组织的炎症反应。

Objective To investigate the effect and mechanism of microRNA-24 (miR-24) on the inflammation of acute lung injury (ALI) in infant rats.Methods The infant rats were divided into control, ALI, ALI+miR-24 mimic mock and ALI+miR-24 mimic groups.Then ALI model was established by LPS.After treatment with miR-24 mimic mock or ALI+miR-24 mimic respectively, the lung W/D ratio was calculated, qRT-PCR was performed to evaluate the mRNA level of miR-24, HE stain was performed to evaluate the pathologic change of the lung, Tunnel was performed to evaluate the apoptosis level, Western blot was performed to evaluate the expressions of nuclear factor κB p65 (NF-κB p65), phosphorylation inhibitor of NF-κB (p-IκBα), and IκBα kinase (p-IKK).Rat alveolar macrophages were divided into control, LPS, LPS +miR-24 mimic, LPS+pc-IL-1α and LPS+miR-24 mimic+ pc-IL-1α groups;then these cells were stimulated by LPS.After transfection with miR-24 mimic and pc-IL-1α respectively or meanwhile, qRT-PCR was performed to evaluate the mRNA level of miR-24, the targeted-relationship between miR-24 and IL-1α was predicted by bioinformatics and conformed by luciferase reporter assay.ELISA was performed to evaluate the concentrations of tumor necrosis factor (TNF-α), interleukin-6 (IL-6), IL-1α and IL-1β in the infant rats serum and rat alveolar macrophages of each group.Results The mRNA level of miR-24 declined in the lung tissue of ALI infant rats, and miR-24 mimic could upregulate miR-24 significantly.Compared with those in control group, the lung W/D ratio and the apoptosis rate increased in ALI group, and the alveoli in infant rats with inflammatory cells.Compared with that in ALI group, ALI+miR-24 mimic mock did not differ significantly, but the lung W/D ratio and the apoptosis rate were lowered in ALI+miR-24 mimic group, and inflammatory cells decreased remarkably.MiR-24 mimic could reduce the concentrations of TNF-α, IL-6, IL-1α and IL-1β in the serum, decrease the expressions of p-IKK, p-IκBα and NF-κB p65 in the lung tissue of ALI infant rats.The mRNA level of miR-24 declined in the rat alveolar macrophages treated with LPS.miR-24 mimic inhibited the luciferase activity of IL-1α wt, and it could reduce the concentrations of TNF-α, IL-6, IL-1α and IL-1β in the rat alveolar macrophages supernatant treated with LPS, but pc-IL-1α could enhance these inflammatory cytokines and weaken the inhibitory effect of miR-24 mimic on inflammation.Conclusion MiR-24 mimic can weaken the inflammation of ALI in infant rats via targeting IL-1α and inhibiting NF-κB activation.