IL-9及其受体通过STAT3通路激活巨噬细胞促进慢性阻塞性肺疾病进展

IL-9 and its receptors promote progression of chronic obstructive pulmonary disease via activation of macrophages through STAT3 pathway

目的探讨IL-9及其受体(IL-9R)在慢性阻塞肺疾病进展中的作用机制。方法24只健康清洁雄性C57BL/6小鼠随机均分为对照组、吸烟组和戒烟组。构建香烟烟雾诱导的慢性阻塞性肺疾病小鼠模型(吸烟组)和戒烟模型(戒烟组)。HE染色比较3组肺气肿和气道重塑程度;免疫组化检测3组肺组织中IL-9及IL-9R表达情况;实时定量PCR、Western blotting检测3组肺组织中IL-9、IL-9R及其下游通路MMP-12、STAT3的表达。ELISA法检测不同浓度IL-9刺激小鼠肺泡巨噬细胞(MH-S)分泌IL-6、TNF-α和MMP-12的水平。将小鼠肺泡巨噬细胞MH-S分为对照组、干预组(加入30 ng/mL IL-9)、AG490+IL-9干预组(加入AG490和30 ng/mL IL-9),PCR、Western blotting检测各组小鼠肺泡巨噬细胞MMP12的表达。结果HE染色结果显示,与对照组比较,吸烟组小鼠肺泡腔变大,肺泡间隔破裂,炎症细胞浸润明显;戒烟组小鼠肺泡腔扩大,肺泡间隔破裂,炎症细胞浸润程度较吸烟组严重。免疫组化结果显示,与对照组比较,吸烟组及戒烟组小鼠IL-9、IL-9R阳性细胞显著增加(P<0.05)。实时定量PCR、Western blotting检测结果显示,与对照组比较,吸烟组小鼠肺组织IL-9、IL-9R、p-STAT3和MMP-12表达水平显著升高(均P<0.05);与吸烟组比较,戒烟组IL-9、IL-9R、p-STAT3及MMP12表达显著降低(均P<0.05)。ELISA检测结果显示,与0 ng/mL IL-9比较,10、30 ng/mL IL-9刺激肺泡巨噬细胞分泌IL-6、TNF-α和MMP-12水平显著提高(均P<0.05);体外实验表明IL-9通过JAK/STAT3通路促进肺泡巨噬细胞分泌MMP-12。结论IL-9及IL-9R通过STAT3通路激活巨噬细胞,促进慢性阻塞性肺疾病进展。

Objective To investigate action mechanism of IL-9 and IL-9 receptor(IL-9R)in the progression of chronic obstructive pulmonary disease.Methods Twenty-four healthy clean male C57BL/6 mice were randomly divided into control,smoking and smoking-cessation groups.A mouse model of chronic obstructive pulmonary disease induced from exposure to cigarette smoke(smoking group)and a smoking-cessation model(smoking-cessation group)were constructed.Hematoxylin and eosin(HE)staining was used to compare the degree of emphyzema and airway remodeling among the three groups.The expressions of IL-9 and IL-9R in the lung tissues of the mice in the three groups were detected using immunohistochemistry.The expression levels of IL-9,IL-9R,and its downstream MMP-12 and STAT3 in the lung tissues of the three groups were detected via PCR and Western blotting.The levels of IL-6,TNF-α,and MMP-12 in mouse alveolar macrophages(MH-S)stimulated by IL-9 at different concentrations were detected using ELISA.Mouse alveolar macrophages(MH-S)were further divided into normal control,IL-9(30 ng/mL)intervention,and AG490+IL-9(30 ng/mL)intervention groups.PCR and Western blotting were used to detect whether IL-9 can promote the expression of MMP-12 in mouse alveolar macrophages through the JAK/STAT3 pathway.Results The results of HE staining showed that compared with the control group,the alveolar cavity of the smoking group was enlarged,the alveolar interval was broken,and the inflammatory cells were infiltrated.In the smoking-cessation group,the alveolar cavity was enlarged,the alveolar septum was ruptured,and the degree of inflammatory cell infiltration was more serious than that in the smoking group.The immunohistochemical results showed that,compared with control group,the numbers of IL-9-and IL-9R-positive cells were significantly higher in the smoking and smoking-cessation groups(all P<0.05).The results of PCR and Western blotting showed that,compared with the control group,the expression levels of IL-9,IL-9R,p-STAT3,and MMP-12 in the lung tissue of the mice in the smoking group were significantly higher(all P<0.05).Compared with the smoking group,the expressions of IL-9,IL-9R,p-STAT3,and MMP-12 in the smoking-cessation group were significantly lower(all P<0.05).The ELISA results showed that compared with 0 ng/mL IL-9,10 and 30 ng/mL IL-9 stimulated significantly higher levels of IL-6,TNF-α,and MMP-12 in the alveolar macrophages(all P<0.05).The results of in vitro experiments showed that IL-9 promoted the secretion of MMP-12 via alveolar macrophages through the JAK/STAT3 pathway.Conclusion IL-9 and IL-9R may promote the progression of chronic obstructive pulmonary disease by activating macrophages through the STAT3 pathway.