中医分期序贯治疗对哮喘大鼠气道重构基质金属蛋白酶-9及其抑制剂表达影响的研究

目的：利用免疫组化、Western Blot和实时荧光定量PCR方法检测哮喘分期序贯治疗对哮喘大鼠气道重构时肺组织中基质金属蛋白酶MMP-9及其抑制剂TIMP-1表达的影响。方法：SD大鼠随机分成7组，即正常组（Z组）、哮喘组（X组）、序贯治疗1组（A1组）、序贯治疗2组（A2组）、序贯治疗3组（A3组）、孟鲁司特钠组（M组）、布地奈德组（B组），除Z组外，其余各组均以卵蛋白（OVA）辅以氢氧化铝为佐剂，与第1、8、15天注射致敏，22天后隔天雾化吸入卵蛋白8周激发哮喘，建立哮喘模型。从实验第8天开始，A1组急性期麻杏二陈汤灌胃，缓解期和稳定期胃饲生理盐水；A2组急性期麻杏二陈汤灌胃，缓解期胃饲金水六君煎加减，稳定期胃饲生理盐水；A3组急性期麻杏二陈汤灌胃，缓解期胃饲金水六君煎加减方，稳定期胃饲六味地黄颗粒；M组激发后雾吸沙丁胺醇，缓解期和稳定期胃饲孟鲁司特钠；B组激发后雾吸沙丁胺醇，缓解期和稳定期雾吸布地奈德，X组以生理盐水胃饲代替，干预7周后分别运用免疫组化、Western Blot和实时荧光定量PCR方法对SD大鼠进行MMP-9、TIMP-1定位、定量表达的分析, 拟从分子水平、基因水平上探讨哮喘大鼠肺组织MMP-9、TIMP-1表达影响的生物学机制。结果：免疫组化法、Western Blot法和实时荧光定量PCR法结果均显示：①与正常组比较，哮喘组大鼠肺组织中MMP-9及TIMP-1表达含量明显增高；②与哮喘组比较，各治疗组对哮喘大鼠肺组织中MMP-9及TIMP-1的表达均有所降低。结论：①哮喘时，MMP-9及TIMP-1表达升高；②中药分期序贯治疗具有通过降低哮喘大鼠肺组织中MMP-9及其抑制剂的表达，调节两者比值，达到阻止气道重构的作用，这可能为其重要机制之一。

Effects of TCM Staged Sequential Therapy on Expressions of Matrix Metalloproteinase-9 and Its Inhibitor in Asthmatic Rats with Airway Remodeling

This study was aimed to verify the effects of staged sequential therapy on expressions of matrix metalloproteinase-9 (MMP-9) and its inhibitor TIMP-1 within lung tissues in asthmatic rats with the airway remodeling, by applying a series of tests such as the immunohistochemistry, western blot and real-time fluorescent quantitative PCR. SD rats were randomly divided into 7 groups, which were the asthmatic group (Group X), the normal group (Group Z), the No. 1 sequential therapy group (Group A1), the No. 2 sequential therapy group (Group A2), the No. 3 sequential therapy group (Group A3), the montelukast group (Group M), and the budesonide group (Group B). The asthmatic model was established in each group except Group Z, by sensitization with both ovalbumin (OVA) and aluminium hydroxide via injection at the 1st, 8th and 15th day in a 22-day duration, followed by OVA aerosol inhalation every other day for 8 weeks for asthma activation. At the 8th day after the asthmatic model was established, Group A1 was orally given Ma-Xing Er-Chen Tang (MXECT) during acute phase while given normal saline (NS) during catabasis and stable phase; Group A2 was given MXECT during acute phase, and given modified Jin-Shui Liu-Jun Jian (JSLJJ) during catabasis as well as given NS during stable phase; Group A3 was given MXECT during acute phase, and given modified JSLJJ during catabasis as well as given Liu-Wei Di-Huang (LWDH) Powders during stable phase; Group M was given salbutamol via aerosol inhalation after stimulation, while orally given montelukast during catabasis and stable phase; Group B was given salbutamol via aerosol inhalation after stimulation, while given inhaled budesonide during catabasis and stable phase; Group X was given NS. After the 7-week intervention, the immunohistochemistry, western blot and real-time quantitative PCR were applied to analyze the location and quantitative expression of MMP-9 and TIMP-1, so as to find out the biological mechanism on expressions of MMP-9 and TIMP-1 in lung tissues of asthmatic rats from molecular levels to gene levels. The results of immunohistochemistry, western blot and real-time fluorescent quantitative PCR showed as follows. Compared with Group Z, the contents of MMP-9 and TIMP-1 increased significantly within lung tissues in Group X. Compared with Group X, the contents of MMP-9 and TIMP-1 decreased within lung tissues of asthmatic rats in each treatment group. It was concluded that the expression of MMP-9 and TIMP-1 elevated during asthma. TCM staged sequential therapy can regulate the ratio between MMP-9 and its inhibitor so as to block the airway remodeling, by decreasing the expression of MMP-9 and its inhibitor within lung tissues in asthmatic rats. This is one of the important action mechanisms.