摘要
目的探讨二甲双胍(Met)调控微小RNA(miR)-34a对慢性阻塞性肺疾病(COPD)大鼠气道炎症的影响。方法每天烟熏30 min,持续30 d,并在第1、14、28天经气道滴注2 mL脂多糖(LPS)溶液制备COPD大鼠模型,实时荧光定量PCR(q RT-PCR)检测Met对肺组织中miR-34a水平的影响;支气管肺泡灌洗液(BALF)中白细胞计数和wright染色分类检测Met对其影响;苏木精-伊红(HE)检测Met对肺组织形态影响;酶联免疫吸附(ELISA)检测Met对血清中白介素IL-6、IL-8、IL-1β、肿瘤坏死因子-α(TNF-α)水平的影响;蛋白免疫印迹检测Met对肺组织中sirt1蛋白水平的影响;Target Scan查找sirt1 mRNA的3'UTR与miR-34a结合位点,并经双荧光素酶报告基因检测试剂盒鉴定。结果与对照组相比,模型组肺组织中sirt1蛋白水平,单核-巨噬细胞比例降低(P<0.05),miR-34a水平、BALF中白细胞数量,中性粒细胞、嗜酸性粒细胞、淋巴细胞比例,血清中IL-6、IL-8、IL-1β、TNF-α水平升高(P<0.05);添加Met后可缓解肺组织中sirt1蛋白水平,单核-巨噬细胞比例降低,miR-34a水平、BALF中白细胞数量,中性粒细胞、嗜酸性粒细胞、淋巴细胞比例,血清中IL-6、IL-8、IL-1β、TNF-α水平升高现象(P<0.05);升高miR-34a水平逆转Met对COPD大鼠的影响。并经双荧光素酶验证,miR-34a与sirt1存在靶向关系。结论Met可下调miR-34a从而上调sirt1实现对COPD气道炎症的缓解,为临床上Met治疗COPD提供一定的实验基础和依据。
Objective To investigate the effect of metformin(Met)on airway inflammation in rats with chronic obstructive pulmonary disease(COPD)by regulating microRNA(miR)-34a.Methods The COPD rat model was established by fumigation for 30 min daily for 30 d and instillation of 2 mL lipopolysaccharide(LPS)solution through the airway on days 1,14 and 28.Quantitative Real-time PCR(qRT-PCR)was performed to detect the effect of Met on the miR-34a level in lung tissue;white blood cell count in bronchoalveolar lavage fluid(BALF)and wright staining were used to detect the effect of Met;hematoxylin-eosin(HE)was used to detect the effect of Met on lung morphology;the serum interleukin IL-6,IL-8,IL-1βand tumor necrosis factor-α(TNF-α)levels were detected by enzyme-linked immunosorbent assay;Western blot was used to detect the effect of Met on the sirt1 protein level in lung tissue;TargetScan was applied to determine the 3′UTR and miR-34a binding sites of sirt1 mRNA,and was identified by the dual luciferase reporter gene detection kit.Results Compared with the control group,in the model group the sirt1 protein level and the proportion of monocytes to macrophages in lung tissue were decreased(P<0.05),whereas the miR-34a level,the number of white blood cells in BALF,the proportions of neutrophils,eosinophils and lymphocytes,and the serum IL-6,IL-8,IL-1βand TNF-αlevels were increased(P<0.05).Met in the COPD animals alleviated the reduced sirt1 protein level in lung tissues,the reduced proportion of monocytes to macrophages,and the increased miR-34a levels,number of white blood cells in BALF,proportions of neutrophils,eosinophils and lymphocytes,and the serum IL-6,IL-8,IL-1βand TNF-αlevels(P<0.05).An increase of the miR-34a level reversed the effect of Met on COPD rats.Dual luciferase verified that miR-34a and sirt1 had a targeting relationship.Conclusions Met down-regulated miR-34a and thereby up-regulated sirt1 to relieve airway inflammation in COPD,which provides an experimental basis for the potential clinical treatment of COP
作者
苏红见
乔亚红
安云霞
韩利
SU Hongjian;QIAO Yahong;AN Yunxia;HAN Li(Respiratory and Critical Care Unit 2,Henan Province Chest Hospital,Zhengzhou 450003,China;Department of Respiratory Medicine,Henan Provincial People’s Hospital,Zhengzhou 450003)
出处
《中国比较医学杂志》
CAS
北大核心
2021年第7期55-61,共7页
Chinese Journal of Comparative Medicine
基金
河南省医学科技攻关计划联合共建项目(LHGJ20190753)。
