PM2.5对慢性阻塞性肺疾病模型大鼠气道重塑的影响及补肺益肾组分方的保护作用

Effect of PM2.5 on the Airway Remodeling in Chronic Obstructive Pulmonary Disease Model Rats and the Protective Effect of the Effective-component Compatibility of Bufei Yishen Formula(补肺益肾组分方)

目的观察PM2.5对慢性阻塞性肺疾病(COPD)模型大鼠气道重塑的影响及补肺益肾组分方的保护作用与可能机制。方法72只SD大鼠随机分为空白组、PM2.5组、COPD组、PM2.5+COPD组、补肺益肾组分方组和氨茶碱组,每组12只。第1~8周,除空白组和PM2.5组外,其余各组给予香烟熏吸联合反复细菌感染制备COPD大鼠模型。第9~16周,除空白组和COPD组外,其余各组给予大气实时浓缩PM2.5全身暴露;同时,补肺益肾组分方组和氨茶碱组分别给予补肺益肾组分方6.48 mg/(kg·d)和氨茶碱混悬液54 mg/(kg·d)灌胃,其余各组给予生理盐水灌胃(雌鼠1.5 ml/只、雄鼠2 ml/只)。PM2.5末次暴露24 h内取材,观察肺组织病理形态和超微结构;采用免疫组化法检测肺组织碱性成纤维细胞生长因子(bFGF)、血管内皮生长因子(VEGF)、α平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原蛋白(COLⅠ),Ⅲ型胶原蛋白(COLⅢ)表达;采用ELISA法检测支气管-肺泡灌洗液中基质金属蛋白酶9(MMP-9)、基质金属蛋白酶12(MMP-12)和金属蛋白酶组织抑制因子1(TIMP-1)水平。结果与空白组比较,PM2.5组、COPD组和PM2.5+COPD组大鼠肺组织出现肺泡壁和支气管壁增厚、支气管狭窄、血管增生、炎症细胞浸润和Ⅱ型肺泡上皮结构破坏,且以PM2.5+COPD组尤为显著;与PM2.5+COPD组比较,补肺益肾组分方组和氨茶碱组以上肺组织病理损伤显著改善。与空白组比较,PM2.5组、COPD组和PM2.5+COPD组大鼠肺组织中bFGF、VEGF、α-SMA、COLⅠ、COLⅢ阳性表达,支气管-肺泡灌洗液中MMP-9和MMP-12均显著升高,TIMP-1显著降低(P<0.05或P<0.01);与PM2.5组或COPD组比较,PM2.5+COPD组bFGF、VEGF、α-SMA、COLⅠ、COLⅢ和MMP-9、MMP-12显著升高,TIMP-1显著降低(P<0.05或P<0.01);与PM2.5+COPD组比较,补肺益肾组分方组和氨茶碱组bFGF、VEGF、α-SMA、COLⅠ、MMP-9、MMP-12显著降低,TIMP-1显著升高,补肺益肾组分方组COLⅢ显著降低(P<0.05或P<0.01)。结论PM2.5暴露显著加重COPD模型大鼠气道重塑,补肺益肾组分方可以延缓PM2.5暴露环境下COPD气道重塑进程,其机制可能与抑制胶原沉积、改善蛋白酶/抗蛋白酶失衡等相关。

Objective To explore the effect of PM2.5 exposure on the airway remodeling of chronic obstructive pulmonary disease(COPD)model rats and the protective effect as well as the possible mechanism of the effective-component compatibility of Bufei Yishen Formula(补肺益肾组分方,ECC-BYF).Methods A total of 72 SD rats were randomized into control group,PM2.5 group,COPD group,PM2.5+COPD group,ECC-BYF group,and aminophylline group,with 12 rats in each group.The COPD rat model was prepared in all groups except for the control group and PM2.5 group by using repeated cigarette smoke inhalation and bacterial infection during week 1 to 8;then rats in all groups except for the control group and COPD group were exposed to real-time atmospheric concentrated PM2.5 during week 9 to 16.Meanwhile,rats in the ECC-BYF group and aminophylline group were given ECC-BYF(6.48 mg/kg·d)and aminophylline(54 mg/kg·d)by gavage,respectively,while rats in other groups were given normal saline(1.5 ml for female rat,2 ml for male rat)once daily from week 9 to 16.Animals were sacrificed within 24 hours of the last PM2.5 exposure.The histomorphological changes and ultrastructure of lung tissue were observed.The expression of basic fibroblast growth factor(bFGF),vascular endothelial growth factor(VEGF),α-smooth muscle actin(α-SMA),collagenⅠ(COLⅠ)and collagenⅢ(COLⅢ)in lung tissues were detected by immunohistochemistry,and the levels of matrix metalloproteinases(MMP)-9,MMP-12,and metalloproteinase-inhibitor(TIMP)-1 in bronchoalveolar lavage fluid(BALF)were determined by enzyme-linked immunosorbent assay(ELISA).Results Compared with the control group,lung tissues derived from rats in PM2.5 group,COPD group,and PM2.5+COPD group showed marked alveolar and bronchial wall thickness,airway narrowing,vascular hyperplasia,inflammatory cells infiltration,and disordered structure of typeⅡalveolar epithelium,and more severe changes were seen in PM2.5+COPD group;compared with the PM2.5+COPD group,more improvement on the above mentioned lung tissue outcomes were shown in the ECC-BYF group and aminophylline group.Compared with the control group,there were increased positive expressions of bFGF,VEGF,α-SMA and COLⅠand COLⅢ,elevated MMP-9 and MMP-12 in BALF,as well as decreased TIMP-1 in the PM2.5 group,COPD group,and PM2.5+COPD group(P<0.05 or P<0.01);compared with the PM2.5 group or the COPD group,there were increased bFGF,VEGF,α-SMA,COLⅠ,COLⅢand MMP-9,MMP-12,and decreased TIMP-1(P<0.05 or P<0.01)PM2.5+COPD group;compared with the PM2.5+COPD group,the ECC-BYF group and aminophylline group have significantly decreased bFGF,VEGF,α-SMA,COLⅠ,MMP-9,MMP-12,increased TIMP-1,and lowered COLⅢ(P<0.05 or P<0.01).Conclusion PM2.5 exposure markedly aggravates the airway remodeling in rats with preexisting COPD,while ECC-BYF could alleviate the airway remodeling progression of COPD rats caused by PM2.5 exposure;the mechanism may be related to the inhibition of collagen deposition,and the improvement of protease/anti-protease imbalance.