基质金属蛋白酶9在Wistar大鼠与自发性高血压大鼠烟熏肺损伤中的变化

Comparison of matrix metalloproteinase-9 between Wistar rat and spontaneously hypertensive rat in pulmonary injury model

目的 比较基质金属蛋白酶9（MMP9）在Wistar大鼠和自发性高血压大鼠烟熏肺损伤中的变化.方法 雄性Wistar大鼠及雄性自发性高血压（SH）大鼠各10只,分别按随机数字表法随机分为Wistar对照组、Wistar烟熏组及SH对照组、SH烟熏组各5只.对照组常规饲养,烟熏组置于自制有机玻璃箱内,每天烟熏2次,每周烟熏6d.8周后观察大鼠一般情况,检测其肺功能及肺组织病理学;Western印迹法检测各组肺组织中核因子（NF）-κB、IκB-α、MMP9蛋白表达;实时定量PCR检测肿瘤坏死因子（TNF）-α和白细胞介素（IL）-6 mRNA表达的变化,免疫组化检测各组肺组织中MMP9蛋白的表达.结果 两烟熏组大鼠出现消瘦,间歇咳嗽及气促.Wistar对照组、Wistar烟熏组及SH对照组、SH烟熏组气道阻力分别为（0.23±0.04）、（0.33 ±0.05）、（0.33±0.02）、（0.46±0.08）cmH2O ·s· ml-1,Wistar烟熏组显著高于Wistar对照组,SH烟熏组显著高于SH对照组和Wistar烟熏组（均P＜0.05）.肺组织HE染显示两烟熏组大鼠均有慢性支气管炎和肺气肿的改变,SH烟熏组病变更为明显.两烟熏组平均肺泡数均显著低于其对照组（均P＜0.05）,Wistar烟熏组平均内衬间隔、肺泡破坏指数、支气管平滑肌指数和胶原指数均显著高于Wistar对照组,SH烟熏组均显著高于SH对照组和Wistar烟熏组（均P＜0.05）.Western印迹法检测Wistar对照组、Wistar烟熏组及SH对照组、SH烟熏组NF-κB蛋白表达分别为（0.322 ±0.014）、（0.558 ±0.044）、（0.373±0.029）、（1.156±0.197）,MMP9蛋白表达分别为（0.255±0.070）、（0.456±0.089）、（0.594±0.184）、（0.847 ±0.138）,SH烟熏组均显著高于SH对照组和Wistar烟熏组（均P＜0.05）;SH烟熏组IκB-α蛋白表达均显著低于SH对照组和Wistar烟熏组（均P＜0.05）.两烟熏组TNF-α mRNA表达水平均显著高于其对照组（均P＜0.05）,SH烟熏组IL-6 mRNA表达水平均显著高于SH对照组和Wistar烟熏组（均P＜0.05）.肺组织MMP9免疫组化结果显示烟熏组表达水平均明显高于对照组,SH烟熏组表达明显高于Wistar烟熏组（均P＜0.05）.结论 Wistar大鼠与SH大鼠烟熏后肺部均出现类似人类慢性阻塞性肺疾病的损伤,SH大鼠比Wistar大鼠肺损伤表现更为严重,MMP9表达水平明显增加。

Objective To compare the expression of matrix metalloproteinase-9 （MMP9） and observe the change of cigarette smoke-induced pulmonary injury in Wistar and spontaneously hypertensive （SH） rats.Methods A total of 10 male Wistar rats and 10 male SH rats were randomly divided into four groups of Wistar control,Wistar cigarette smoking,SH control and SH cigarette smoking （n =5 each）.The control groups were fed routinely while the cigarette smoking groups were placed into a homemade organic glass box and exposed to cigarette smoking for twice daily 6 days a week.After 8-week treatment,general condition,lung function and pathological changes of lung tissues were detected.The expressions of nuclear factor （NF）-κB,IκB-α and MMP9 were determined by Western blot.And real-time polymerase chain reaction （RT-PCR） was employed to detect the mRNA levels of tumor necrosis factor alpha （TNF-α） and interleukin-6 （IL-6）.Immunohistochemistry was used to detect the expression of MMP9 in lung tissue.Results Both cigarette smoking groups were marantic along with intermittent cough and polypnea.Resistance index （RI） in Wistar control,Wistar cigarette smoking,SH control and SH cigarette smoking groups was （0.23 ±.04）,（0.33 ±.05）,（0.33 ±.02） and （0.46 ±.08） cmH2O · s · ml-1 respectively.RI increased in Wistar cigarette smoking group versus Wistar control group.And SH cigarette smoking group was significantly higher than SH control and Wistar cigarette smoking groups （P 〈 0.05）.Hematoxylin ＆amp; eosin staining demonstrated that the lesion of chronic bronchitic inflammation and emphysema existed in two cigarette smoking groups while it was more severe in SH rats than Wistar rats.The mean alveolar number （MAN） was significantly reduced in two cigarette smoking groups compared with that in control groups （P 〈 0.05,respectively）.And mean lining interval （MLI）,damage index （DI）,bronchial smooth muscle index and collagen index significantly increased in Wistar cigarette smoking group compared with Wistar control group,SH cigarette smoking group significantly increased than that in SH control and Wistar cigarette smoking groups （P 〈 0.05,respectively）.On Western blot,the protein level of NF-κB in Wistar control,Wistar cigarette smoking,SH control and SH cigarette smoking groups was （0.322 ±0.014）,（0.558 ±0.044）,（0.373 ±0.029） and （1.156 ±0.197） respectively.The protein level of MMP9 in these groups was （0.255 ±.070）,（0.456 ±.089）,（0.594 ±.184） and （0.847 ±.138）respectively.The protein levels of NF-κB and MMP9 increased obviously in Wistar cigarette smoking group versus Wistar control group and those in SH cigarette smoking group were significantly higher than SH control and Wistar cigarette smoking groups （P 〈 0.05）.Additionally,IκB-α expression in SH cigarette smoking group was markedly decreased in comparison to that in SH control group and Wistar cigarette smoking group （P 〈 0.05）.The mRNA level of TNF-α increased obviously in two cigarette smoking groups in comparison to those in controls （P 〈 0.05） and the mRNA level of IL-6 significantly increased in the SH cigarette smoking group compared with SH control group and Wistar cigarette smoking group （P 〈 0.05）.As for immunohistochemistry,the MMP9 positive cells were prominently distributed in the lungs of cigarette smoking groups versus the stained cells in the control group.And SH cigarette smoking group was significantly higher than Wistar cigarette smoking group （P 〈 0.05）.Conclusion Both Wistar and SH rats induced by cigarette smoking may have an onset of lung injury symptom analogous to chronic obstructive pulmonary disease and SH rat are more severe.