目的 研究C1q/肿瘤坏死因子相关蛋白9(CTRP9)对哮喘幼鼠气道平滑肌细胞(airway smooth muscle cells,ASMCs)增殖和气道炎症的作用。方法 ELISA检测正常、哮喘儿童和小鼠血清中CTRP9含量;原代培养哮喘小鼠ASMCs,经pcDNA3.1-CTRP9转染后,MTT检测ASMCs增殖,ELISA测定TNF-α和IL-6含量,Western 印记检测TLR4和NF-κB p65蛋白表达以及NF-κB p65磷酸化水平;pcDNA3.1-CTRP9转染哮喘小鼠,HE染色观察肺组织炎性细胞的浸润程度;收集小鼠支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF),光镜下检测嗜酸性粒细胞、巨噬细胞和中性粒细胞数目;检测小鼠肺部TNF-α、IL-6、TLR4和NF-κB的表达。结果 哮喘儿童和小鼠血清中CTRP9含量低于正常组;CTRP9能抑制ASMCs增殖、炎症因子分泌和TLR4/NF-κB通路活化;CTRP9也能抑制哮喘小鼠肺部炎性细胞浸润、各炎性反应细胞数目、炎症因子分泌和TLR4/NF-κB通路活化。结论 CTRP9能抑制幼年哮喘小鼠ASMCs增殖和炎性反应。
Abstract
Objective To explore the effects of CTRP9 on proliferation of airway smooth muscle cells (ASMCs) and airway inflammation in infant asthmatic mice. Methods Elisa kits were used for detecting the contents of CTRP9 in serum from the normal and asthmatic children and mice respectively.ASMCs of asthmatic mice were isolated and cultured followed by transfection with pcDNA3.1-CTRP9,MTT were used to determine the level of cells proliferation and elisa kits were used again to measure the levels of TNF-α and IL-6.Besides,the levels of TLR4,NF-κB p65 and phosphorylation of NF-κB p65 were detected by Western blot analyses.Asthmatic mice were transfected with pcDNA3.1-CTRP9 and observed the infiltration of inflammatory cells in lung tissues by HE staining.The number of eosinophils,neutrophils and macrophages in BALF were counted under light microscope.Finally,the levels of TNF-α,IL-6,TLR4 and NF-κB in lung of mice were measured again as above methods. Results The asthmatic children and mice showed lower CTRP9 content and mRNA expression in serum compared to control group.After transfection ASMCs with pcDNA3.1-CTRP9,the cells proliferation,inflammatory cytokines secretion and TLR4/NF-κB pathway were all inhibited obviously.Moreover,overexpression of CTRP9 in asthmatic mice suppressed the infiltration of inflammatory cells in lung tissues,the number of inflammatory cells,as well as the inflammatory cytokines secreting and activation of TLR4/NF-κB. Conclusion CTRP9 inhibits the proliferation of airway smooth muscle cells and attenuates inflammation in asthmatic mice.
关键词
哮喘 /
C1q/肿瘤坏死因子相关蛋白9 /
气道平滑肌细胞 /
炎性反应 /
TLR4/NF-κB
Key words
airway smooth muscle cells /
inflammation /
TLR4/NF-κB /
asthma /
CTRP9
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