重组CC16蛋白对慢性阻塞性肺疾病小鼠肺组织结构及MMP-9和TIMP-1表达的影响

Effect of rCC16 protein on the lung histology and expressions of MMP-9 and TIMP-1 in mice with chronic obstructive pulmonary disease

目的研究重组大鼠CC16(rCC16)蛋白质对减轻慢性阻塞性肺疾病(chronic obstructive pulmonary disease,COPD)小鼠模型肺组织损伤的作用,及对肺组织中基质金属蛋白酶(MMP)-9和基质金属蛋白酶组织抑制因子(TIMP)-1表达的调节作用。方法将40只清洁级C57BL/6小鼠随机分为4组,分别为空白组、COPD模型组、rCC16剂量组1和剂量组2。除空白组外,其余小鼠均采用吸烟3月法制备COPD模型,从第3月开始空白组和模型组给予PBS滴鼻,rCC16剂量组1和剂量组2分别给予1μg/g体重和2.5μg/g体重的rCC16滴鼻干预。观察各组小鼠精神状态、饮食情况、体重变化及大小便情况等,H&E染色观察各组小鼠肺组织形态结构变化,荧光定量聚合酶链式反应、免疫组织化学法检测MMP-9和TIMP-1信使RMA(mRNA)和蛋白质的表达变化。结果空白组小鼠体重随饲养周期增大,COPD组小鼠体重较空白组明显减轻,rCC16干预组后,干预组2小鼠体重逐周增大,但干预组1小鼠体重增加较缓慢,差异均有统计学意义(P<0.05);COPD模型组小鼠肺组织结构明显破坏,肺泡间隔增宽,部分形成肺气肿,rCC16干预组后,小鼠肺部的肺泡结构趋于完整,肺大泡的形成也减少;COPD模型组小鼠肺组织MMP-9和TIMP-1的表达均明显高于空白组(P<0.05);rCC16干预后,均可降低MMP-9和TIMP-1的表达,差异均有统计学意义(P<0.05),rCC16对MMP-9的降低作用具有剂量依赖性,而对TIMP-1的调节不呈剂量依赖性。结论 rCC16滴鼻干预可以减轻COPD小鼠肺组织的损伤,降低肺组织中MMP-9和TIMP-1的表达,对COPD的治疗具有积极意义。

Objective To study the protective effect of recombinant rat Club cell 16 (rCC16) protein on lung injury and expressions of matrix metalloproteinase (MMP)-9 and tissue inhibitor of metalloproteinase (TIMP)-1 in mice suffering from chronic obstructive pulmonary disease (COPD). Methods C57BL/6 mice ( n =40) were randomly divided into four groups: blank, COPD model, rCC16 treatment 1, and rCC16 treatment 2 groups. The mice were exposed to tobacco smoke for 3 months to create a COPD model. The blank-group mice were exposed to room air. When the model was stable, mice in the blank and model groups were administered phosphate-buffered saline (PBS;i.n.), and mice in the other two groups were treated with rCC16 (1.0 or 2.5 μg/g body weight, i.n., respectively). The mental state, diet, body-weight changes and urine of mice were observed. Histological changes in the lung tissues in different groups were observed with hematoxylin and eosin staining. mRNA and protein levels of MMP-9 and TIMP-1 were analyzed by quantitative reverse transcription-polymerase chain reaction and immunohistochemistry. Results Body weights in the COPD group were decreased compared with those in the blank group, which were increased with the feeding period. Body weights in the rCC16 treatment 2 group were increased obviously, whereas the body weights in the rCC16 treatment 1 group increased more slowly, and the differences were significant ( P < 0.05). Lung structure in the COPD group was changed with widened interalveolar septa and development of emphysema. rCC16 treatment alleviated those pulmonary alterations and reduced the formation of pulmonary bullae. The expressions of MMP-9 and TIMP-1 were significantly higher than those in the blank group ( P < 0.05). rCC16 treatment inhibited the expressions of MMP-9 and TIMP-1, which were overexpressed in the COPD group, and the differences were significant ( P < 0.05). Moreover, the rCC16’s regulation on MMP-9 expression was dose-dependent. Conclusions Intranasal administration of rCC16 reduces the pulmonary injury and expressions of MMP-9 and TIMP-1 in lung tissues of COPD mice. Our results demonstrate that rCC16 has a promising therapeutic effect against COPD.