| [Objective] To investigate the expression and relationship levels of v-glutamylcysteine synthetase (γ -GCS) and p-ERK, p-JNK, p-p38, p-PKB in lung of rat and human with chronic obstructive pulmonary disease, to elucidate the possible important role of Y -GCS, MAPK, PKB in pathogenesis of COPD, and provide theoretical basis and new remedy for COPD.[Methods] The study was composed of two parts: animal experiment and clinical trial.Part1: twenty-eight male Wistar rats were randomly divided into COPD modeland control groups. The rat COPD model was established by intratracheal instillation of lipopolysaccharide (LPS200μg/200μL) twice and exposed to cigarette smoke daily. The level of the activity of Y -GCS in lung tissue was measured, the levels of Y -GCS mRNA expression in lung tissue was measured by in site hybridization (ISH). The protein expression of Y -GCS and p-ERK, p-JNK, p-p38, p-PKB were observed by imrnunohistochemistry and western blot.Part2: Surgically resected lung carcinoma and para-carcinoma tissues in 18 patients were collected, half of tissues were acquired from lung cancer patients with COPD. The level of the activity of Y -GCS in lung tissue was measured, the levels of Y -GCS mRNA expression in lung tissue was measured by in site hybridization (ISH). The protein expression of Y -GCS and p-ERK, p-JNK, p-p38, p-PKB were observed by immunohistochemistry.[Results]1. PEE, FEV0.3, were decreasing significantly in COPD group compared with control group( all P<0.05), which suggested that there be airflow obstruction, anincrease in residual volume and pulmonary disfunction. The pathological findings were consistent with chronic bronchitis and obstructive emphysema.2. The levels of the activity of Y -GCS were higher in COPD rat group (3.242 +0.526U) than that in control group (2.269+0.431U, P<0.01).3. The ISH showed that the levels of Y -GCS mRNA expression of the lung tissue in COPD group (0.446+0.056) were significantly higher than that in the control group (0.322+0.049, P<0.01).4. In immunohistochemical (EH), the granules of Y -GCS, p-ERK, p-JNK and p-p38,, p-PKB positively stained, were observed in the cytoplasm of pulmonary alveolar wall, intrapulmonary vascular and bronchial wall in COPD group; p-ERK, p-JNK and p-p38 could be observed in nucleus too. The protein expression of Y -GCS, p-ERK, p-p38 and p-PKB was significantly higher in the COPD group (0.358+0.024, 0.373+0.023,0.339+0.020,0.317+0.042) than the control group (0.253 + 0.014, 0.256+0.023,0.232+0.014,0.221 ±0.038, respectively, all P<0.01); while the expression of p-JNK (0.291 ±0.034) was lower than the other and had no significant difference with the control group (0.269 ±0.031, P>0.05).5. The western blot assay demonstrated that the protein expression of Y -GCS, p-ERK, p-p38 and p-PKB(0.218± 0.011,0.223 ±0.014,0.205 ±0.013,0.204 ±0.033) was significantly higher than the control group (0.133±0.014,0.119±0.010,0.073± 0.008,, 0.074 ±0.014, respectively, all P<0.01); while p-JNK (0.081 ±0.013) showed no significant difference with the control group (0.069±0.011, P>0.05).6. The levels of the activity of Y -GCS were higher in COPD group patient (23.868 ±2.646U) than that in control group (15.784 ±2.406U, P<0.01).7. The levels of Y -GCS mRNA expression in COPD group (0.376 ±0.045) were significantly higher than that in the control group (0.257 ±0.038, P<0.01). The expression of Y -GCS, p-ERK, p-p38 and p-PKB protein were significantly higher in COPD group (0.360+0.051,0.368±0.054, 0.342±0.043,0.376±0.065) as compared to control group (0.251 ±0.048,0.257+0.037,0.256 ±0.034,0.283 ± 0.051 all P<0.05); while the expression of p-JNK (0.269 ± 0.043) had no significant... |
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