Based On Mucus Hypersecretion Mechanisms To Discuss The Regulation Of NRF2 On MiR-146, MUC5AC And Intervention Effect Of Shenqibufei Fang In Rats With COPD

Objective:To investigate the effect of Shenqibufei Fang on the expression of the airway epithelium cellular transcription factor-Nrf2,miR-146 and MUC5AC in rats with COPD and research the mechanism of Shenqibufei Fang on the regulation of airway mucus hypersecretion. To discuss the mechanism of Shenqibufei Fang treat with COPD in molecular biology level.Methods:1.The 60 healthy Wistar rats were randomly divided into control group,model group,acetylcysteine group, low dose group of traditional Chinese medicine,medicine dose group of traditional Chinese medicine, high dose group of traditional Chinese medicine. In addition to the control group,were treated with LPS after airway instillation and smoke modeling 2 weeks after the second instillation of LPS given the appropriate treatment total treatment for 30 days. Lung function in rats bronchoalveolar lavage fluid and lung tissue changes were observed. The mRNA and protein expression of miR-146,Nrf2 and MUC5AC in lung tissue were detected by REAL-TIME-PCR and Western blot.2.Were given airway epithelium smoke solution,Shenqibufei recipe on serum,Nrf2 siRNA,Myc3-Nrf2 four kinds of interference factors intervention culture,observe airway epithelial cell growth state intervention in different situations, using REAL-TIME-PCR and Western blot method to detect lung miR-146 and Nrf2 MUC5AC mRNA and protein expression, situation.Results:1. After modeling smoked, lung function in rats FEV0.3/FVC decreased lung expiratory resistance (Ri) and inspiratory resistance (Re) increased,decreased lung compliance. acetylcysteine and Chinese medicine can improve lung function in rats.2. After modeling,alveolar lavage fluid Leukocyte count and neutrophil count were significantly higher compared with the control group (p<0.05); leukocyte count in neutrophils based. Acetylcysteine and each dose of Chinese medicine can reduce the number of white blood cells BALF with the model group was statistically significant (p<0.05); p but no statistical significance between treatment groups comparison (p>0.05)3. Pathological specimens observation showed that after the treatment of Shenqibufei Fang relieve bronchial epithelial damage and inflammatory cell infiltration, decreased lung airway mucus plugs in rats. In acetylcysteine and high-dose groups, were very close to the normal group. Acetylcysteine and Chinese medicine are each dose can improve the situation of emphysema COPD rats, reduce pathological alveolar accounting field rate, each dose of Chinese medicine and acetylcysteine considerable effect (p>0.05), but the high dose significantly better than the Chinese low-dose (p<0.05)4. After modeling in each group of rats was significantly increased lung MUC5AC, acetylcysteine and Shenqibufei Fang can reduce the expression of MUC5AC expression in lung.And Shenqibufei Fang can reduce MUC5AC mRNA expression of Chinese low-dose, medium-dose and acetylcysteine quite effective (p>0.05). Chinese medicine dose group, high dose group and the acetylcysteine group reduced MUC5AC protein expression level was no significant difference (p>0.05)5. After modeling Nrf2 protein expression in rats was significantly higher (compared with the control group, p<0.05). Shenqibufei Fang and acetylcysteine can increase Nrf2 mRNA and protein expression levels (compared with the model group, p<0.05). The role of Shenqibufei Fang medium-dose and acetylcysteine was considerable (p> 0.05), while the protein expression of Nrf2 high dose group was significantly higher than acetylcysteine group (p<0.05)6. After the model increased expression of miR-146 in rats, and acetylcysteine and Fang Shenqibufei increase the expression of miR-146, compared with the control group and the model group (p<0.05). Chinese medicine dose, high dose is better than acetylcysteine (p<0.05)7. Smoke solution inhibited cell activity. Under 1%,5% concentration of smoke solution cell activity decreased relatively stable, and 25%,50%,100% smoke solution within 12 hours after cell activity decreased more rapidly, respectively cell activity 59%,36%,35%,10% solution of cells decreased more gentle, but after 24 hours decreased rapidly to 70%.8. Smoke solution may induce Nrf2 mRNA, MUC5AC mRNA, miR-146 expression. MUC5AC mRNA expression increased significantly,3h expression level that is significantly higher than Oh (p<0.05). Nrf2 mRNA, miR-146 slowly increase the degree of expression,but for 24h,the expression is significantly increased,a significant difference (p<0.05) compared with Oh.9. Smoke solution may induce Nrf2, MUC5AC protein expression. After MUC5AC protein expression increased significantly, smoke-induced 3h, MUC5AC expressing cells is markedly increased (p<0.05). After 6h induction of protein expression was significantly increased Oh (p<0.05)10.After siRNA inhibits the expression of Nrf2, smoke-induced MU the expression of MUC5AC mRNA and miR-146 was increased in a time- dependent manner (p<0.05). Nrf2 and smoke suppression group 24h point solution, negative control 24h point comparison, a significant increase in expression level, was significant statistically (p<0.05).mRNA expression of MUC5AC decreased compared with Oh point, culture expression increased after 6h, but no significant difference,p>0.05;culture statistically significant (p<0.05); compared with Oh after 12h, but no difference in point 6h (p> 0.05). Myc3-Nrf2 group after 24h culture with smoke solution, negative control 24h point comparison, significantly decreased expression levels, statistically significant (p<0.05).After siRNA inhibits the expression of Nrf2 in smoke solution induced at different time points miR-146 expression is increased, but the increase rate of decline, but compared with Oh point, there are differences at all time points miR-146 expression level (p<0.05). siRNA group, negative control group and smoke solution group, cultured 24h miR-146 expression level decreased, but not statistically significant (p>0.05). Description suppressed Nrf2 expression affects miR-146 expression, but no significant effect relationship.11.After Myc3-Nrf2 overexpression cells Nrf2 gene, MUC5AC expression decreased and miR-146 expression increased in a time-dependent manner. Training 6h after MUC5AC protein expression statistically significantly increased (p<0.05). After 12h culture increased MUC5AC mRNA expression and Oh difference was statistically significant (p<0.05), after 24h culture, compared with the smoke solution group and negative control group, Myc3-Nrf2 group MUC5AC mRNA and protein expression was significantly decreased, miR-146 significantly increased expression (p<0.05).12.Chinese medicine serum can promote cell Nrf2, miR-146 expression and inhibit MUC5AC overexpression, medicine serum miR-146, Nrf2 mRNA and protein expression gradually increased, it showed a time-dependent manner. MUC5AC cultured cells in each group after 24h, miR-146 Nrf2 mRNA and protein expression were increased, compared with the smoke solution and blank serum group, chinese medicine serum MUC5AC increasing degree was low, miR-146, Nrf2 increased significantly (p<0.05), indicating that Shenqibufei Fang in order to improve the expression of miR-146, Nrf2 is.13.Chinese medicine serum can promote the expression of Nrf2 siRNA intervention cells, but Nrf2 mRNA expression level of the corresponding increase in expression level compared with the control group, but not statistically significant (p> 0.05); and no disincentive difference was statistically significant (p <0.05). Chinese medicine serum after 24h culture, Nrf2 protein expression with siRNA alone intervention and control serum+siRNA intervention compare expression increased (p <0.05), but compared with the case of the absence of siRNA, was statistically significant (p<0.05), the serum can be explained medicine promoting Nrf2 expression.14.After intervention cells at 24h, medicine can reduce serum MUC5AC expression of siRNA interference cell, but MUC5AC mRNA and protein expression level with the corresponding control group no difference (p>0.05); compared with no disincentives, MUC5AC mRNA expression level was significantly higher (p<0.05).15.Chinese medicine serum can promote cell miR-146 siRNA expression interventions, traditional Chinese medicine serum+ miR-146 expression levels of siRNA cells corresponding increase in expression level compared with the control group (p<0.05). In addition to the drug serum group,the remaining miR-146 expression levels betwe- en the groups was no significant difference. Description Shenqibufei Fang does not rely entirely on Nrf2 pathway improve miR-146 expression.Conclusions:1. Shenqibufei Fang can reduce the degree of BALF inflammation of in rats with COPD, improve lung function,inhibit pathological processes of lung tissue rats with COPD. Increase Nrf2, miR-146 expression in lung tissue of COPD rat lung tissue of rats reduced MUC5AC levels.2. Nrf2 involved in the regulation of smoke-induced chronic obstructive pulmonary disease pathogenesis solution MUC5AC expression.3. Nrf2 partially participation regulate smoke solution induced COPD pathogenesis of miR-146 expression.4 Shenqibufei Fang by Nrf2 regulating MUC5AC, miR-146 expression induced pathogenesis of COPD smoke solution.