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
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摘要
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 C57 BL/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.
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 C57 BL/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.
引文
[1] 钟南山.慢性阻塞性肺疾病在中国[J].中国实用内科杂志.2011,31(5):321-322.Zhong NS.Chronic obstructive pulmonary disease in China[J].Chin J Prac Intern Med,2011,31(5):321-322.
[2] Boers JE,Ambergen AW,Thunnissen FB.Number and proliferation of Clara cells in normal human airway epithelium[J].Am J Respir Crit Care Med,1999,159(5Pt1):1585-1591.
[3] Tsoumakidou M,Bouloukaki I,Thimaki K,et al.Innate immunity proteins in chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis[J].Exp Lung Res,2010,36(6):373-380.
[4] Plopper CG,Mango GW,Hatch GE,et al.Elevation of susceptibility toozone-induced acute tracheobronchial injury in transgenic mice deficient in Clara cell secretory protein[J].Toxicol Appl Pharmacol,2006,213(1):74-85.
[5] 庞敏,王冬,李婷,等.重组大鼠CC16蛋白质对LPS诱导大鼠气道上皮细胞表达TNF-α、IL-6和IL-8的影响[J].中国病理生理杂志,2016,32(10):1843-1847.Pang M,Wang D,Li T,et al.Effect of recombinant rat CC16 protein on LPS-induced expression of TNF-α,IL-6 and IL-8 in rat tracheal epithelial cells[J].Chin J Pathophys,2016,32(10):1843-1847.
[6] Pang M,Wang H,Bai JZ,et al.Recombinant rat CC16 protein inhibits LPS-induced MMP-9 expression via NF-kappaB pathway in rat tracheal epithelial cells[J].Exp Biol Med,2015,240(10):1266-1278.
[7] Liu MW,Liu R,Wu HY,et al.Atorvastatin has a protective effect in a mouse model of bronchial asthma through regulating tissue transglutaminase and triggering receptor expressed on myeloid cells-1 expression[J].Exp Ther Med,2017,14(2):917-930.
[8] Mannino DM.COPD:epidemiology,prevalence,morbidity and mortality,and disease heterogeneity[J].Chest,2002,121(5 Suppl):121S-126S.
[9] Lowrey GE,Henderson N,Blakey JD,et al.MMP-9 protein level does not reflect overall MMP activity in the airways of patients with COPD [J].Respir Med,2008,102(6):845-851.
[10] 朱平,严励,陈黎红,等.MMP-9/TIMP-1表达在糖尿病鼠皮肤伤口愈合过程中的变化及意义初探[J].中国病理生理杂志,2008,24(11):2204-2208.Zhu P,Yan L,Chen LH,et al.Imbalance between matrix metalloproteinases and tissue inhibitor of metalloproteinases during wound healing in diabetic rats [J].Chin J Pathophys,2008,24(11):2204-2208.
[11] Zhou XM,Hou G,Gu DX,et al.Peroxisome proliferator-activated receptor-γ in induced sputum is correlated with MMP-9/TIMP-1 imbalance and formation of emphysema in COPD patients[J].J Thorac Dis,2017,9(10):3703-3710.
[12] Pang M,Yuan Y,Wang D,et al.Recombinant CC16 protein inhibits the production of pro-inflammatory cytokines via NF-κB and p38 MAPK pathways in LPS-activated RAW264.7 macrophages[J].Acta Biochim Biophys Sin (Shanghai),2017,49(5):435-443.
[13] Pang M,Liu HY,Li T,et al.Recombinant club cell protein 16 (CC16) ameliorates cigarette smoke induced lung inflammation in a murine disease model of COPD[J].Mol Med Rep,2018,18(2):2198-2206.
[14] 庞敏,王海龙,张彰,等.不同药物对COPD大鼠Clara细胞和CC16表达的影响[J].中国比较医学杂志,2008,18(2):1-4.Pang M,Wang HL,Zhang Z,et al.Effects of different drugs on the number of Clara cells and CCl6 in rat COPD model[J].Chin J Comp Med,2008,18(2):1-4.
[15] Long XB,Hu S,Wang N,et al.Clara cell 10-kDa protein gene transfection inhibits NF-kappaB activity in airway epithelial cells[J].PLoS One,2012,7:e35960
[16] 郑鸿翱,何韶衡.建立慢性阻塞性肺疾病动物模型方法的研究进展[J].中国实验动物学报,2003,11(4):249-252.Zheng HA,He SH.Progress in methods to produce COPD animal model [J].Acta Lab Anim Sci Sin,2003,11(4):249-252.
[2] Boers JE,Ambergen AW,Thunnissen FB.Number and proliferation of Clara cells in normal human airway epithelium[J].Am J Respir Crit Care Med,1999,159(5Pt1):1585-1591.
[3] Tsoumakidou M,Bouloukaki I,Thimaki K,et al.Innate immunity proteins in chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis[J].Exp Lung Res,2010,36(6):373-380.
[4] Plopper CG,Mango GW,Hatch GE,et al.Elevation of susceptibility toozone-induced acute tracheobronchial injury in transgenic mice deficient in Clara cell secretory protein[J].Toxicol Appl Pharmacol,2006,213(1):74-85.
[5] 庞敏,王冬,李婷,等.重组大鼠CC16蛋白质对LPS诱导大鼠气道上皮细胞表达TNF-α、IL-6和IL-8的影响[J].中国病理生理杂志,2016,32(10):1843-1847.Pang M,Wang D,Li T,et al.Effect of recombinant rat CC16 protein on LPS-induced expression of TNF-α,IL-6 and IL-8 in rat tracheal epithelial cells[J].Chin J Pathophys,2016,32(10):1843-1847.
[6] Pang M,Wang H,Bai JZ,et al.Recombinant rat CC16 protein inhibits LPS-induced MMP-9 expression via NF-kappaB pathway in rat tracheal epithelial cells[J].Exp Biol Med,2015,240(10):1266-1278.
[7] Liu MW,Liu R,Wu HY,et al.Atorvastatin has a protective effect in a mouse model of bronchial asthma through regulating tissue transglutaminase and triggering receptor expressed on myeloid cells-1 expression[J].Exp Ther Med,2017,14(2):917-930.
[8] Mannino DM.COPD:epidemiology,prevalence,morbidity and mortality,and disease heterogeneity[J].Chest,2002,121(5 Suppl):121S-126S.
[9] Lowrey GE,Henderson N,Blakey JD,et al.MMP-9 protein level does not reflect overall MMP activity in the airways of patients with COPD [J].Respir Med,2008,102(6):845-851.
[10] 朱平,严励,陈黎红,等.MMP-9/TIMP-1表达在糖尿病鼠皮肤伤口愈合过程中的变化及意义初探[J].中国病理生理杂志,2008,24(11):2204-2208.Zhu P,Yan L,Chen LH,et al.Imbalance between matrix metalloproteinases and tissue inhibitor of metalloproteinases during wound healing in diabetic rats [J].Chin J Pathophys,2008,24(11):2204-2208.
[11] Zhou XM,Hou G,Gu DX,et al.Peroxisome proliferator-activated receptor-γ in induced sputum is correlated with MMP-9/TIMP-1 imbalance and formation of emphysema in COPD patients[J].J Thorac Dis,2017,9(10):3703-3710.
[12] Pang M,Yuan Y,Wang D,et al.Recombinant CC16 protein inhibits the production of pro-inflammatory cytokines via NF-κB and p38 MAPK pathways in LPS-activated RAW264.7 macrophages[J].Acta Biochim Biophys Sin (Shanghai),2017,49(5):435-443.
[13] Pang M,Liu HY,Li T,et al.Recombinant club cell protein 16 (CC16) ameliorates cigarette smoke induced lung inflammation in a murine disease model of COPD[J].Mol Med Rep,2018,18(2):2198-2206.
[14] 庞敏,王海龙,张彰,等.不同药物对COPD大鼠Clara细胞和CC16表达的影响[J].中国比较医学杂志,2008,18(2):1-4.Pang M,Wang HL,Zhang Z,et al.Effects of different drugs on the number of Clara cells and CCl6 in rat COPD model[J].Chin J Comp Med,2008,18(2):1-4.
[15] Long XB,Hu S,Wang N,et al.Clara cell 10-kDa protein gene transfection inhibits NF-kappaB activity in airway epithelial cells[J].PLoS One,2012,7:e35960
[16] 郑鸿翱,何韶衡.建立慢性阻塞性肺疾病动物模型方法的研究进展[J].中国实验动物学报,2003,11(4):249-252.Zheng HA,He SH.Progress in methods to produce COPD animal model [J].Acta Lab Anim Sci Sin,2003,11(4):249-252.