姜黄素通过上调miR-124减轻脂多糖诱导的大鼠急性肺损伤

Curcumin alleviates lipopolysaccharide-induced acute lung injury in rats by up-regulating miR-124

目的探究姜黄素(Cur)对脂多糖(LPS)诱导的大鼠急性肺损伤(ALI)的保护作用及可能机制。方法体外培养大鼠肺泡巨噬细胞NR8383,采用双荧光素酶报告基因实验验证微小RNA-124(miR-124)与肿瘤坏死因子受体相关因子6(TRAF6)的靶向关系。SD大鼠采用随机数字表法分为对照组(NC组)、LPS组、LPS+Cur组、LPS+Cur+inhibitor-NC组、LPS+Cur+inhibitor组,每组10只。LPS组腹腔注射LPS 10 mg/kg制备ALI大鼠模型,LPS+Cur组在ALI模型制备30 min后尾静脉注射Cur 200 mg/kg,LPS+Cur+inhibitor-NC组、LPS+Cur+inhibitor组大鼠尾静脉分别注射miR-124 inhibitor-NC、miR-124 inhibitor 50 mg/kg 1周后腹腔注射LPS 10 mg/kg,30 min后尾静脉注射Cur 200 mg/kg,NC组注射等量生理盐水30 min后尾静脉注射等量生理盐水。干预24 h后,HE染色观察肺组织病理变化,酶联免疫吸附试验(ELISA)检测血清肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)水平,分别采用实时荧光定量PCR(RT-qPCR)、蛋白质印迹法(WB)检测肺组织miR-124、TRAF6 mRNA及蛋白表达。结果双荧光素酶报告基因实验结果显示,miR-124可直接靶向TRAF63'UTR区负调控其表达。动物实验结果表明,NC组大鼠肺组织结构清晰,肺泡结构完整,无明显异常改变;LPS组、LPS+Cur+inhibitor组大鼠肺组织结构破坏严重,肺间质增厚,大量炎性细胞浸润,弥漫性充血、渗出,肺泡萎缩;LPS+Cur组、LPS+Cur+inhibitor-NC组大鼠肺组织损伤减轻。与NC组比较,LPS组大鼠血清TNF-α、IL-6、肺组织TRAF6 mRNA及蛋白表达水平显著增加,miR-124表达水平显著降低(P<0.05);与LPS组比较,LPS+Cur组大鼠血清TNF-α、IL-6、、肺组织TRAF6 mRNA及蛋白表达水平显著降低,miR-124表达水平显著增加(P<0.05);与LPS+Cur+inhibitor-NC组比较,LPS+Cur+inhibitor组大鼠血清TNF-α、IL-6、肺组织TRAF6 mRNA及蛋白表达水平显著增加,miR-124表达水平显著降低(P<0.05)。结论Cur可通过上调miR-124靶向抑制TRAF6减轻LPS诱导的大鼠ALI及炎症反应,发挥治疗作用。

Objective To investigate the protective effect and its possible mechanism of Curcumin(Cur)on lipopolysaccharide(LPS)-induced acute lung injury(ALI)in rats.Methods Alveolar macrophages NR8383 of rats was cultured in vitro,double luciferase reporter gene assay was used to verify the targeting relationship between microRNA-124(miR-124)and tumor necrosis factor receptor associated factor 6(TRAF6).SD rats were randomly divided into control group(NC group),LPS group,LPS+Cur group,LPS+Cur+inhibitor NC group,LPS+Cur+inhibitor group,with 10 in each group.In LPS group,10 mg/kg LPS was injected intraperitoneally to prepare ALI rat model.In LPS+Cur group,after 30 minutes of ALI model preparation,Cur 200 mg/kg was injected into tail vein,while the rats in LPS+Cur+inhibitor NC group and LPS+Cur+inhibitor NC group were injected with miR-124 inhibitor NC and miR-124 inhibitor 50 mg/kg via caudal vein,respectively,and LPS 10 mg/kg was injected intraperitoneally 1 week later,and then Cur 200 mg/kg was injected into tail vein 30 minutes later.The rats in NC group were injected with equal amount of normal saline into tail vein 30 minutes after injection of the same amount of normal saline.After 24 hours of intervention,HE staining was used to observe the pathological changes of lung tissue,the levels of serum tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)were detected by enzyme-linked immunosorbent assay(ELISA),and the mRNA and protein expressions of miR-124 and TRAF6 were detected by real-time fluorescence quantitative PCR(RT-qPCR)and Western blot(WB)respectively.Results Double luciferase reporter gene showed that miR-124 could directly target TRAF63'UTR region and negatively regulate its expression.The results of animal experiments showed that the lung tissue structure of rats in NC group was clear,the alveolar structure was complete,and there was no obvious abnormal change;the lung tissue structure of rats in LPS group and LPS+Cur+inhibitor group was damaged seriously,the lung interstitium was thickened,a large number of inflammatory cells were infiltrated,diffuse hyperemia,exudation and alveolar atrophy were found;in addition,the lung injury in LPS+Cur group and LPS+Cur+inhibitor NC group was reduced.Compared with those in NC group,the expression levels of TNF-α,IL-6 in serum,TRAF6 mRNA and protein in lung tissue in LPS group were significantly higher,and the expression level of miR-124 was significantly lower(P<0.05);compared with those in LPS group,the expression levels of TNF-α,IL-6 in serum,TRAF6 mRNA and protein in lung tissue in LPS+Cur group were significantly lower,and the expression level of miR-124 was significantly higher(P<0.05);in addition,compared with those in LPS+Cur+inhibitor NC group,the expression levels of TNF-α,IL-6 in serum,TRAF6 mRNA and protein in lung tissue in LPS+Cur+inhibitor group were significantly higher,and the expression level of miR-124 was significantly lower(P<0.05).Conclusion Cur can reduce the ALI and inflammatory response induced by LPS by target inhibition of TRAF6 by up-regulating miR-124,and play a therapeutic role.