支气管哮喘豚鼠肺组织中丝裂原活化蛋白激酶对γ-谷氨酰半胱氨酸合酶的作用

目的： 研究在支气管哮喘豚鼠肺组织中丝裂原活化蛋白激酶（MAPK）信号通路对γ-谷氨酰半胱氨酸合酶（γ-GCS）的影响。方法: 成年雄性豚鼠20只随机分成哮喘组和对照组，每组10只，以腹腔内注射联合雾化吸入卵蛋白复制哮喘模型。测支气管肺泡灌洗液（BALF）细胞总数并进行分类计数；原位杂交和RT-PCR测肺组织中γ-GCS-h mRNA表达；免疫组化测γ-GCS、磷酸化细胞外激活蛋白激酶（p-ERK）、磷酸化c-Jun氨基末端激酶(p-JNK)和磷酸化 p38（p-p38）在肺组织中的表达;Western blotting 测 p-ERK、p-JNK和p-p38在肺组织中的表达；双酶法测γ-GCS的活性。结果: （1）哮喘组BALF中细胞总数、嗜酸性粒细胞明显多于对照组 (P<0.01)。（2）免疫组化显示肺组织中p-ERK、p-p38、p-JNK和γ-GCS 蛋白质表达哮喘组明显高于对照组（P<0.01）；Western blotting 亦显示肺组织中p -ERK、p-JNK和p-p38 蛋白质表达哮喘组均明显高于对照组。（3)原位杂交和RT-PCR显示在肺组织中γ-GCS-h mRNA 表达哮喘组明显高于对照组(P<0.01)。（4) γ-GCS活性哮喘组明显高于对照组（P<0.01）。（5）直线相关性分析显示：在肺组织中p-ERK、p-p38与γ-GCS-h mRNA、γ-GCS蛋白质呈显著正相关，p-JNK与γ-GCS-h mRNA、γ-GCS蛋白质无明显相关性。结论: 支气管哮喘豚鼠肺中 p-ERK、p-p38、p-JNK和γ-GCS表达均增加；p-ERK和p-p38可能上调γ-GCS表达。

Effects of mitogen activated protein kinase on γ-glutamylcysteine synthase in lung of guinea pigs with bronchial asthma

AIM: To investigate the effects of mitogen activated protein kinase on γ-glutamylcysteine synthase (γ-GCS) in lung of guinea pigs with bronchial asthma.METHODS: Twenty adult male guinea pigs were divided into asthmatic group and control group (10 in each group).Asthmatic model was established by ovalbumin intraperitoneal injection combined with inhalation.The numbers of total and inflammation cells in bronchoalveolar lavage fluid (BALF) were measured.The γ-GCS-h mRNA in lung tissue was examined by in situ hybridization and RT-PCR.Immunohistochemistry was used to detecte the expression of γ-GCS,phosphorylated extracellular signal regulated kinase (p-ERK),phosphrylated c-Jun amino terminal kinase (p-JNK) and phosphorylated p38 (p-p38) in lung tissues.Western blotting was conducted to determine the expressions of p-ERK,p-JNK and p-p38 in lung tissue.The activity of γ-GCS was measured by coupled enzyme assay.RESULTS: (1) The total cell number and number of eosinophils in BALF of asthmatic group were significantly higher than those in control group (P<0.01).(2) Immunohistochemistry indicated that the p-ERK,p-p38,p-JNK and γ-GCS were stronger expressed in asthmatic group than those in control group (P<0.01).Western blotting also discovered that the expressions of p-ERK,p-JNK and p-p38 in lung tissue of asthmatic group were stronger than those in control group.(3) Both in situ hybridization and RT-PCR analysis showed that the expression of γ-GCS-h mRNA was more positive in asthmatic group compared with control group (P <0.01).(4) The activity of γ-GCS of asthmatic group was significantly higher than that in control group (P<0.01).(5) Linear correlation analysis indicated that in lung tissue of guinea pig with asthma,p-ERK and p-p38 markedly positive correlated with γ-GCS-h mRAN and γ-GCS protein.No relationship between p-JNK and γ-GCS-h mRAN,γ-GCS protein was observed.CONCLUSION: The expressions of p-ERK,p-p38,p-JNK and γ-GCS increase in lung of guinea pigs with bronchial asthma.p-ERK and p-p38 may positively regulate the expression of γ-GCS.