miR-181a调控TLR4/MyD88/NF-κB通路抑制哮喘小鼠气道炎症反应的实验研究

MiR-181a regulated TLR4/MyD88/NF-κB pathway and inhibited airway inflammation in asthmatic mice

目的探讨miR-181a对哮喘小鼠气道炎症反应的影响及其可能作用机制。方法60只BALB/c小鼠随机分为正常对照组、模型组、miR-181a阴性对照组、miR-181a过表达组、脂多糖(LPS)[Toll样受体4(TLR4)特异性激活剂]组、miR-181a过表达+LPS组,除正常对照组外,其余各组均采用卵清蛋白(OVA)致敏及激发构建哮喘动物模型,在每次激发前30 min对各造模组给予相应药物干预。末次激发后24 h,测定各组小鼠气道阻力参数Penh值,苏木素-伊红(HE)染色观察小鼠肺组织病理学变化,分类并计数小鼠肺泡灌洗液中炎症细胞[嗜酸性粒细胞(EOS)、淋巴细胞、中性粒细胞]数量,ELISA法测定小鼠肺泡灌洗液中炎性因子[肿瘤坏死因子(TNF-α)、白细胞介素-6(IL-6)、IL-1β]水平,q RT-PCR法测定小鼠肺组织中miR-181a表达水平,WB测定小鼠肺组织中TLR4/髓样分化因子88/核因子κB(TLR4/MyD88/NF-κB)通路相关蛋白表达。结果与正常对照组比较,模型组小鼠Penh值,肺泡灌洗液中EOS数、淋巴细胞数、中性粒细胞数、TNF-α、IL-6、IL-1β水平,肺组织炎症细胞浸润、肺组织TLR4/MyD88/NF-κB通路相关蛋白TLR4、MyD88、NF-κB表达显著增加,肺组织miR-181a表达显著降低,差异均有统计学意义(P<0.05)。与模型组比较,miR-181a阴性对照组差异无统计学意义(P>0.05);LPS组小鼠Penh值、肺组织TLR4/MyD88/NF-κB通路相关蛋白TLR4、MyD88、NF-κB及miR-181a表达差异无统计学意义(P>0.05),肺泡灌洗液中EOS数、淋巴细胞数、中性粒细胞数、TNF-α、IL-6、IL-1β水平,肺组织炎症细胞浸润显著升高,差异均有统计学意义(P<0.05);miR-181a过表达组小鼠Penh值,肺泡灌洗液中EOS数、淋巴细胞数、中性粒细胞数、TNF-α、IL-6、IL-1β水平,肺组织炎症细胞浸润、肺组织TLR4/MyD88/NF-κB通路相关蛋白TLR4、MyD88、NF-κB表达显著降低,肺组织miR-181a表达显著升高,差异均有统计学意义(P<0.05)。与miR-181a过表达组比较,miR-181a过表达+LPS组小鼠肺组织炎症细胞浸润较重,小鼠Penh值,肺泡灌洗液中EOS数、淋巴细胞数、中性粒细胞数、TNF-α、IL-6、IL-1β水平,肺组织TLR4/MyD88/NF-κB通路相关蛋白TLR4、MyD88、NF-κB表达显著增加,差异均有统计学意义(P<0.05)。与LPS组比较,miR-181a过表达+LPS组小鼠Penh值,肺泡灌洗液中EOS数、淋巴细胞数、中性粒细胞数、TNF-α、IL-6、IL-1β水平,肺组织TLR4/MyD88/NF-κB通路相关蛋白TLR4、MyD88、NF-κB表达显著降低,肺组织miR-181a表达显著升高,差异均有统计学意义(P<0.05)。结论miR-181a抑制哮喘小鼠气道炎症反应可能是通过抑制TLR4/MyD88/NF-κB通路实现的。

Objective To investigate the effect of miR-181a on airway inflammation in asthmatic mice and its possible mechanism.Methods Sixty BALB/c mice were randomly divided into normal control group,model group,miR-181a negative control group,miR-181a overexpression group,lipopolysaccharide(LPS)[Toll like receptor 4(TLR4)specific activator]group,and miR-181a overexpression+LPS group.Except for the normal control group,the other groups were sensitized and challenged with ovalbumin(OVA)to construct the asthma animal model,and the corresponding drug intervention was given to each model group for 30 minutes before each challenge.24 hours after the last challenge,the airway resistance parameter Penh value was measured;the pathological changes of lung tissue were observed by hematoxylin-eosin(HE)staining;inflammatory cells[eosinophilia(EOS),lymphocyte,neutrophile]in bronchoalveolar lavage fluid were classified and counted;the levels of inflammatory factors[tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),IL-1β]in bronchoalveolar lavage fluid were determined by ELISA;the expression level of miR-181a in lung tissue was determined by qRT-PCR;the expression of TLR4/myeloid differentiation factor 88/nuclear factor kappa B(TLR4/MyD88/NF-κB)pathway related proteins in the lung tissue of mice was detected by WB.Results Compared with those in the normal control group,the Penh value,EOS count,lymphocyte count,neutrophil count,the levels of TNF-α,IL-6,IL-1βin bronchoalveolar lavage fluid,the infiltration of inflammatory cells in lung tissue,and the expression of TLR4/MyD88/NF-κB pathway related proteins in lung tissue of mice were significantly increased,the expression of miR-181a in lung tissue was significantly decreased in the model group,and the differences were statistically significant(P<0.05).Compared with those in the model group,there was no significant difference in the miR-181a negative control group(P>0.05);there were no significant differences in Penh value,the expression of TLR4/MyD88/NF-κB pathway related proteins and miR-181a in lung tissue of mice in LPS group(P>0.05),EOS count,lymphocyte count,neutrophil count,the levels of TNF-α,IL-6,IL-1βin bronchoalveolar lavage fluid,and the infiltration of inflammatory cells in lung tissue were significantly increased,and the differences were statistically significant(P<0.05);the Penh value,EOS count,lymphocyte count,neutrophil count,the levels of TNF-α,IL-6,IL-1βin bronchoalveolar lavage fluid,the infiltration of inflammatory cells in lung tissue,and the expression of TLR4/MyD88/NF-κB pathway related proteins in lung tissue of mice were significantly decreased,the expression of miR-181a in lung tissue was significantly increased in the miR-181a overexpression group,and the differences were statistically significant(P<0.05).Compared with those in the miR-181a overexpression group,the inflammatory cell infiltration was more severe,the Penh value,EOS count,lymphocyte count,neutrophil count,the levels of TNF-α,IL-6,IL-1βin bronchoalveolar lavage fluid,and the expression of TLR4/MyD88/NF-κB pathway related proteins in lung tissue of mice were significantly increased in the miR-181a overexpression+LPS group,and the differences were statistically significant(P<0.05).Compared with the LPS group,the Penh value,EOS count,lymphocyte count,neutrophil count,the levels of TNF-α,IL-6,IL-1βin bronchoalveolar lavage fluid,the expression of TLR4/MyD88/NF-κB pathway-related proteins TLR4,MyD88,and NF-κB in lung tissue were significantly decreased,and the expression of miR-181a in lung tissue was significantly increased in miR-181a overexpression+LPS group,and the differences were statistically significant(P<0.05).Conclusion MiR-181a could inhibit airway inflammation in asthmatic mice by inhibiting TLR4/MyD88/NF-κB pathway.