miR-22-3p靶向抑制SIRT1信号通路加重慢性阻塞性肺疾病

MiR-22-3p exacerbates chronic obstructive pulmonary disease by targetedly inhibiting SIRT1 signaling pathways

目的探讨miR-22-3p是否通过沉默信息调节因子1(SIRT1)信号通路影响慢性阻塞性肺疾病(COPD)的发生发展。方法将大鼠随机分为5组:对照组、模型组、antagomir-NC组、antagomir-miR-22-3p组和antagomir-miR-22-3p+EX527组,每组12只。对照组大鼠正常饲养,其他组大鼠建立香烟烟雾(CS)和脂多糖(LPS)诱导的COPD大鼠模型。CS暴露当天开始,模型组、antagomir-NC组、antagomir-miR-22-3p组和antagomir-miR-22-3p+EX527组大鼠分别尾静脉注射生理盐水、antagomir-NC、antagomir-miR-22-3p和SIRT1抑制剂EX527,每2周注射1次,CS暴露90 d内共注射6次。CS暴露90 d后,使用肺功能分析系统测定肺功能指标:最大自主分钟通气量(MVV)、0.3 s用力呼气量(FEV0.3)、用力肺活量(FVC)和最大呼气峰流速(PEF)。按照ELISA试剂盒测定支气管肺泡灌洗液(BALF)中白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)和巨噬细胞炎性蛋白-2(MIP-2)水平。按照试剂盒说明对肺组织进行苏木素伊红(HE)染色。按照试剂盒说明检测肺组织氧化应激指标[丙二醛(MDA)、超氧化物歧化酶(SOD)和过氧化氢酶(CAT)]。通过RT-PCR检测肺组织中miR-22-3p和SIRT1的mRNA水平。通过Western blot检测肺组织中SIRT1、核因子-κB(NF-κB)p65、p-NF-κB p65和叉头盒O3a(FoxO3a)蛋白表达。结果与模型组和antagomir-NC组比较,antagomir-miR-22-3p组大鼠中MVV、FEV0.3/FVC和PEF升高,BALF中IL-1β、TNF-α和MIP-2水平降低,肺组织病变减轻,肺组织中SOD和CAT水平升高,MDA水平降低,miR-22-3p水平降低,SIRT1 mRNA水平升高,SIRT1和FoxO3a蛋白水平升高,p-NF-κB p65蛋白水平降低(均P<0.05)。与antagomir-miR-22-3p组比较,antagomir-miR-22-3p+EX527组大鼠中MVV、FEV0.3/FVC和PEF降低,BALF中IL-1β、TNF-α和MIP-2水平升高,肺组织病变加重,肺组织中SOD和CAT水平降低,MDA水平升高,SIRT1 mRNA水平降低,SIRT1和FoxO3a蛋白水平降低,p-NF-κB p65蛋白水平升高(均P<0.05)。结论沉默miR-22-3p通过上调SIRT1的表达有效提高了COPD大鼠的肺功能并减轻了肺损伤。

Objective To investigate whether miR-22-3p affects the occurrence and development of chronic obstructive pulmonary disease(COPD)through silent information regulator 1(SIRT1)signaling pathway.Methods The rats were randomly divided into five groups(n=12 each group):control group,model group,antagomir-NC group,antagomir-miR-22-3p group and antagomir-miR-22-3p+EX527 group.The rats in control group were fed normally,and the rats in the other groups were given cigarette smoke(CS)and lipopolysaccharide(LPS)to establish the COPD model.From the day of CS exposure,the rats in model group,antagomir-NC group,antagomir-miR-22-3p group and antagomir-miR-22-3p+EX527 group were injected with normal saline,antagomir-NC,antagomir-miR-22-3p or SIRT1 inhibitor EX527 through tail vein once every two weeks,respectively.Injections were given six times within 90 d of CS exposure.After 90 d of CS exposure,the pulmonary function indicators,such as maximum voluntary minute ventilation volume(MVV),forced expiratory volume of 0.3 s(FEV0.3),forced vital capacity(FVC),and maximum peak expiratory flow rate(PEF)were measured using a pulmonary function analysis system.Levels of interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α)and macrophage inflammatory protein-2(MIP-2)in bronchoalveolar lavage fluid(BALF)were determined by ELISA kit.Lung tissue was stained with hematoxylin eosin(HE)according to kit instructions.The oxidative stress indexes,such as malondialdehyde(MDA),superoxide dismutase(SOD)and catalase(CAT),in lung tissue were detected according to the kit instructions.The miR-22-3p and SIRT1 mRNA levels in lung tissues were detected by RT-PCR.The protein expression levels of SIRT1,nuclear factor-κB(NF-κB)p65,p-NF-κB p65 and FoxO3a in lung tissues were detected by Western blot.Results Compared with model group and antagomir-NC group,the levels of MVV,FEV0.3/FVC and PEF were increased in antagomir-miR-22-3p group,the levels of IL-1β,TNF-αand MIP-2 in BALF were decreased,the pathological changes of lung tissue were alleviated,the levels of SOD and CAT in lung tissue were increased,the level of MDA was decreased,the level of miR-22-3p was decreased,the levels of SIRT1 mRNA,SIRT1 and FoxO3a were increased,and the level of p-NF-κB p65 protein was decreased(all P<0.05).Compared with antagomir-miR-22-3p group,the levels of MVV,FEV0.3/FVC and PEF were decreased in antagomir-miR-22-3p+EX527 group,the levels of IL-1β,TNF-αand MIP-2 in BALF were increased,the pathological changes of lung tissue were aggravated,the levels of SOD and CAT in lung tissue were decreased,the level of MDA was increased,the level of SIRT1 mRNA was decreased,the protein levels of SIRT1 and FoxO3a were decreased,and the level of p-NF-κB p65 protein was increased(all P<0.05).Conclusion Silencing miR-22-3p can effectively improve the lung function and reduce the lung injury in COPD rats by upregulating the expression of SIRT1.