Effects Of Mitogen Activated Protein Kinase And AP-1 On γ-glutamylcysteine Synthase In Bronchial Asthma

Objective: To investigate the effects of mitogen activated protein kinase (MAPK) and AP-1 (active protein-1)onγ–glutamylcysteine in lung of guinea pigs with bronchial asthma synthase and the inhibitory role of glucocorticosteroid and the inhibitor of protein tyrosine kinase genistein on the expression ofγ–glutamylcysteine. In addition, inflammatory cell in sputum from bronchial asthma rats were analyzed.Methods: Forty adult male guinea pigs were randomly divided into control group (group A), asthmatic group (group B), dexamethasone group (group C) and genistein group (group D) (10 in each group). Asthmatic model was established by ovalbumin intraperitoneal injection combined with inhalation. The rats in group C and D were injected with 1.5mg/kg dexamethasone or genitein 15 mg/kg through intraperitoneal injection daily for 14 days, respectively. In the control group, ovalbumin was injected by saline. The total cell and the proportion of inflammation cell in bronchoalveolar lavage fluid (BALF) were measured. The reduced glutathione (GSH) and total GSH in BALF and lung tissue were examined, respectively. Inflammatory cell infiltration and index of bronchiole remodeling were measured; The expression of phosphorylated extracellular signal regulated kinase (p-ERK), phosphrylated c-Jun amino terminal kinase (p-JNK), phosphorylated P38(p-P38), AP-1 andγ-GCS were tested using immunohistochemistry in lung tissue. The expression of p-ERK, p-JNK, p-P38 , total ERK, total JNK, total P38 and AP-1 were detected using western blot in lung tissue. The expression ofγ-GCS-h mRNA and AP-1 mRNA were measured by RT-PCR in lung tissue. The activity ofγ-GCS was measured by coupled enzyme assay.Results:1. The total cell and proportion of eosinophils in BALF of group B were significantly higher than that in group A compared with group B. The total cell and proportion of eosinophils in BALF of group C and group D were decreased. However, there is no significantly difference between group C and D.2. The infiltration of eosinophils and lymphocytes, as well as the remodeling of bronchiole was demonstrated in group B in HE staining. The above findings were less apparent in group C and group D.3. The total GSH and GSSG in BALF and lung tissue of group B, C, D were significantly higher than those of group A; but GSH/GSSG is lower. Compared with group B, the total GSH and GSSG in BALF and lung tissue of group C and group D was decreased, but GSH/GSSG is significantly higher. The GSH in BALF and lung tissue was not statistically significant among all groups(P>0.05.4. The expression of p-ERK,p-P38,p-JNK , AP-1 andγ-GCS in group B was increased in group B than that in group A indicated by immunohistochemistry. Compared with group B, the expression of p-ERK, p-P38, p-JNK, AP-1 andγ-GCS in C group were decreased; the expression of p-ERK, AP-1 andγ-GCS in D group were decreased, The expression of p-P38 and p-JNK was not statistically significant(P>0.05)among group B, D. The expression of p-ERK, p-JNK,p-P38 and AP-1 in lung tissue of B group was more positive than that of group A demonstrated by Western blot. Compared with B, the expression of p-ERK, p-P38, p-JNK and AP-1 in C group was decreased; the expression of p-ERK and AP-1 in D group was decreased, However, the expression of p-P38 and p-JNK had no statistical difference(P>0.05), the expression of total ERK, total JNK and total P38 in group A, B, C and D was not statistically significant (p>0.05).5. The expression ofγ-GCS-h mRNA and AP-1 mRNA in lung tissue of the 4 animal groups was correlated with the expression ofγ-GCS protein.6. Linear Correlation analysis: there was significantly positive correlation between total GSH, GSSG and total cell, E%; there was strong negative correlation between GSH/GSSG and total cell, E%. There was strongly positive correlation between total GSH,GSSG and AP-1 protein,γ-GCS mRNA,γ-GCS protein. There was strong negative relationship between GSH / GSSG and AP-1 protein,γ-GCS mRNA,γ-GCS protein. In the lung tissue of guinea pig with asthma, the expression of p-ERK, p-JNK and p-P38 was strongly positively correlated with AP-1 protein,γ-GCS-h mRNA andγ-GCS protein; the expression of AP-1 protein was also strongly positively correlated withγ-GCS mRNA,γ-GCS protein in lung tissue.Conclusions:1. The oxidative stress likely up-regulatedγ-GCS gene through AP-1 in the lung of guinea pig with asthma and the activation of AP-1 is likely the combinational effect of p-ERK, p-JNK and p-P38.2. The glucocorticoid down-regulated the expression of AP-1 possibly via decreasing the oxidative stress and suppressing the activity of ERK, p-JNK and P38 in the lung of guinea pig with asthma. Genistein down-regulated the expression of AP-1 possibly via decreasing the oxidative stress and inhibiting the activity of ERK.