Expression Of MiRNA-19a/PTEN/AKT Pathway In Rat Asthma Model And Human Airway Smooth Muscle In Vitro

Part Ⅰ miRNA-19 a / PTEN / AKT pathway expression changes in chronic asthma rat modelsObjective: miRNA-19 a / PTEN / AKT pathway is related to the phenotype change of smooth muscle cells,and airway smooth muscle plays an important role in the course of asthma.Therefore,this study was to observe whether miRNA-19 a / PTEN / AKT pathway is involved in the process of asthma.Methods:(1)The rat model of chronic asthma was established,which was divided into asthma group and dexamethasone group.Another blank control group was set up.The methods like Hematoxylin-eosin staining(HE),Masson’s trichrom stain(MASSON),periodic acid-Schiff stain(PAS),α-smooth muscle actin(α-smooth muscle actin,α-SMA)immunohistochemistry have been used to verify whether the model is successfully established.(2)The expression of miRNA-19 a in the model lung was determined by real-time real-time PCR(RT-PCR).(3)The expression of PTEN,AKT and p-AKT in the model lung was determined by Western blot.(4)The statistical method was SPSS16.0 analysis.The quantitative data were showed by means of mean ± standard deviation(x±S).The analysis of one-way ANOVA was used when the variance was the same,and Welch correction test was used when the variance was not the same.P< 0.05 was statistically significant.Results:(1)In Hematoxylin-eosin staining,there were more inflammatory cells in the asthmatic group and dexamethasone group than in the blank control group.In Masson’s trichrom staining,there were more collagen fibers around the trachea in the asthma group and dexamethasone group than in the blank control group.In periodic acid-Schiff staining,glycogen protein secretion in asthmatic group and dexamethasone group was significantly higher than that in blank control group.In the immunohistochemistry of α-SMA,the smooth muscle layer in asthma group and dexamethasone group was significantly thicker than that in the blank control group.(2)The expression of miRNA-19 a in the asthmatic group and dexamethasone group was higher than that in the blank control group,and the expression of miRNA-19 a in dexamethasone group was lower than that in the asthmatic group.(3)The PTEN protein secretion of asthmatic group and dexamethasone group decreased significantly,while AKT and p-AKT protein secretion increased.Conclusions:(1)The successful establishment of rat model of chronic asthma was verified by HE staining,Masson staining,PAS staining and α-SMA immunohistochemistry.(2)In this study,we found that the expression of miRNA-19 a,AKT,p-AKT in the lung tissue of chronic asthma rats was higher than that in the blank group,and the expression of PTEN was lower than that in the blank group,suggesting that miRNA-19 a may participate in the disease process of asthma by regulating PTEN / AKT / p-AKT pathway.Part Ⅱ Expression of PTEN / AKT / p-AKT pathway in human airway smooth muscle cells in vitroObjective: In the previous study,we found that HMGB1 is involved in the process of asthma in the mouse model of chronic asthma,and it can regulate the inflammatory process of asthma through p38 MAPK and ERK1 / 2 pathway in human airway epithelial cells.However,the specific process of HMGB1 action on airway smooth muscle cells is not clear.PTEN / AKT / p-AKT pathway is related to the phenotype of smooth muscle cells.So we suppose that HMGB1 may participate in the biological process of smooth muscle cells through PTEN / AKT / p-AKT pathway.Methods:(1)The primary human airway smooth muscle cells were cultured and identified.Different concentrations(0ng / ml,500 ng / ml,5000 ng / ml,10000 ng / ml)of HMGB1 were used to stimulate human airway smooth muscle cells for 24 hours.(2)The expression of PTEN,AKT and p-AKT in the cells of each group was determined by Western blot.(3)The statistical method is the same as the first part.Results:(1)The cells grew as long fusiform adherent cells,and showed the "Valley peak" growth of typical smooth muscle cells.The positive rate of specific α-SMA immunofluorescence was 98%.Human airway smooth muscle cells were successfully cultured.(2)In HMGB1 stimulated human airway smooth muscle cells,PTEN secretion decreased significantly in 5000 ng / ml and 10000 ng / ml groups,and AKT and p-AKT secretion increased significantly.Conclusions:(1)Human airway smooth muscle cells were cultured successfully.(2)It was found that with the increase of HMGB1 concentration,the expression of PTEN decreased and the expression of AKT and p-AKT increased,suggesting that HMGB1 at high concentration may participate in the biological process of airway smooth muscle by activating PTEN / AKT / p-AKT pathway.