穗花杉双黄酮对脂多糖致大鼠急性肺损伤的保护作用及机制研究

Protective Effect and Mechanism of Amentoflavone on Acute Lung Injury Induced by Lipopolysaccharide in Rats

目的探讨穗花杉双黄酮对脂多糖(LPS)致大鼠急性肺损伤(ALI)的保护作用及机制。方法将60只雄性SD大鼠按照随机数字表法分为对照组、模型组、阳性对照组和穗花杉双黄酮组(简称穗花杉组),每组15只。模型组、阳性对照组和穗花杉组大鼠均通过气道滴注3.0mg/kg LPS100μL,对照组大鼠气道滴注等体积的生理盐水,6h后阳性对照组大鼠腹腔注射2.5mg/kg地塞米松100μL,穗花杉组大鼠腹腔注射2.5mg/kg穗花杉双黄酮100μL,对照组和模型组注射等体积生理盐水。48h后称重测定大鼠肺组织湿/干重比;苏木精-伊红染色(HE)观察各组大鼠肺组织病理;ELISA法检测大鼠血清肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)和白细胞介素-1β(IL-1β)水平;RT-PCR检测肺组织miR-140-5p表达;蛋白印迹法(WB)检测肺组织Toll样受体4(TLR4)、核因子活化B细胞κ轻链增强子(NF-κB)蛋白表达水平。利用LPS刺激肺上皮细胞A549,加入穗花杉双黄酮RT-PCR检测miR-140-5P的表达,加入miR-140-5P抑制剂、mimic-NC干预,RTPCR检测TLR4、NF-κB表达。结果穗花杉组大鼠肺组织湿/干重比明显低于模型组[(4.77±0.19)比(5.84±0.27),P<0.05];HE染色结果显示,穗花杉组大鼠肺组织结构较ALI模型组大鼠炎症细胞明显减少,肺泡间隔明显变薄;ELISA结果显示,穗花杉组大鼠血清TNF-α、IL-6和IL-1β水平均明显低于模型组[TNF-α:(395.16±41.23)pg/mg比(846.23±29.26)pg/mg;IL-6:(315.26±30.25)pg/mg比(876.54±45.23)pg/mg;IL-1β:(650.16±29.46)pg/mg比(1056.26±54.16)pg/mg,P均<0.05];WB结果显示,穗花杉组大鼠肺组织TLR4、NF-κB表达量明显低于模型组;RT-PCR结果显示,穗花杉组大鼠肺组织和人肺上皮细胞miR-140-5P表达分别高于模型组和刺激组[(0.85±0.13)比(0.34±0.08);(0.81±0.15)比(0.27±0.14),P均<0.05];miR-140-5P mimic能明显抑制TLR4、NF-κB表达[TLR4:(0.95±0.12)比(2.31±0.08);NF-κB:(0.97±0.13)比(2.45±0.06),P均<0.05]。结论穗花杉双黄酮对LPS诱导的大鼠ALI具有保护作用,其机制可能与促进miR-140-5p mimic表达,抑制ALI炎症过程关键蛋白TLR4、NF-κB表达以及其下游炎症因子TNF-α、IL-6和IL-1β水平有关。

Objective To investigate the protective effect and mechanism of amentoflavone on acute lung injury(ALI)induced by lipopolysaccharide(LPS)in rat.Methods A total of 60 male SD rats were randomly divided into control group,model group,positive control group and amentoflavone group according to the random number table method,with 15 animals in each group.Rats in model group,positive control group and amentoflavone group were injected with 100μL LPS(3.00 mg/kg)by airway driping,rats in control group were treated with equal volume of normal saline.Rats in positive control group were injected with 100μL dexamethasone(2.5 mg/kg)intraperitoneally 6 hours later,amentoflavone group with 100μL amentoflavone(2.5 mg/kg),and control group and model group with equal volume of normal saline.The wet/dry weight ratio of lung tissue was measured by weighing 48 hours later.Hematoxylin-eosin staining(HE)was used to observe the lung tissue pathology in each group.ELISA was used to detect the levels of serum tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and interleukin-1β(IL-1β).RT-PCR was used to check miR-140-5 p expression in lung tissue.Western blot(WB)was used to test the expression levels of Toll-like receptor 4(TLR4)and nuclear factor activated B cell-derivedκlight chain enhancer(NF-κB)protein in lung tissue.RT-PCR was used to detect miR-140-5 p expression in lung tissue.After stimulating lung epithelial cell A549 by LPS,amentoflavone was added,and expression of miR-140-5 P was tested by RTPCR.In addition,expression of TLR4 and NF-κB was detected by RT-PCR with the addition of miR-140-5 P inhibitor and mimic-NC.Results The wet/dry weight ratio of rat lung tissue in the amentoflavone group was significantly lower than that in the model group[(4.77±0.19)vs(5.84±0.27),P<0.05].The results of HE staining showed that the lung tissue structure in the amentoflavone group reduced significantly compared with that of the ALI model group,and the alveolar septum was significantly thinner.ELISA results showed that the serum levels of TNF-α,IL-6 and IL-1βin amentoflavone group were significantly lower than those of model group[TNF-α:(395.16±41.23)vs(846.23±29.26)pg/mg;IL-6:(315.26±30.25)vs(876.54±45.23)pg/mg;IL-1β:(650.16±29.46)vs(1056.26±54.16)pg/mg,P<0.05,respectively].The results of WB indicated that the expressions of TLR4 and NF-κB in the lung tissue in amentoflavone group were significantly lower than those of model group.RT-PCR results showed that the expression of Mir-140-5 p in rat lung tissue and human lung epithelial cells in amentoflavone group was significantly higher than that in the model group and the stimulation group[(0.85±0.13)vs(0.34±0.08);(0.81±0.15)vs(0.27±0.14),P<0.05,respectively].Mir-140-5 p mimic inhibited the expression of TLR4 and NF-κB significantly[TLR4:(0.95±0.12)vs(2.31±0.08);NF-κB:(0.97±0.13)vs(2.45±0.06),P<0.05,respectively].Conclusion Amentoflavone has a protective effect on LPS-induced rat ALI,and its mechanism may be related to promoting the expression of Mir-140-5 p mimic,inhibiting the expression of key proteins TLR4 and NF-κB in the inflammatory process of ALI,and the maintenance of the levels of downstream inflammatory factors TNF-α,IL-6 and IL-1β.