Effects Of Metformin On AQP5 Expression And Mucus Secretion In Lung Tissue Of Asthmatic Mice

Objective: This study explored the interventional effects of metformin on ovalbumin(OVA)-induced aquaporin 5(AQP5)and mucin 5AC(MUC5AC)expression and mucus secretion in bronchial asthma mice,and discussed the possible mechanisms.Methods: BALB/c female mice were selected and divided into three groups according to the random number method: normal control group(NS group),asthma model group(AS group),and metformin intervention group(Met group),with 8 mice in each group.The first intraperitoneal injection of sensitization time was set on the first day of modeling,in which the AS and Met groups were sensitized by intraperitoneal injection of OVA sensitizing solution(including OVA 50 ug + aluminum hydroxide 4mg)on days 1,8 and 15,while the NS group was given the same amount of saline intraperitoneally;on days 22-28,the AS and Met groups were stimulated by nebulized inhalation of 1% OVA stimulating solution,while the NS group was stimulated by nebulized inhalation of the same amount of physiological saline.On day22-28,the AS and Met groups were stimulated by nebulized inhalation of 1% OVA excitation solution,while the NS group was given the same amount of saline;and half an hour before the nebulized excitation,the Met group was given metformin(250mg/kg)solution intraperitoneally,while the AS and NS groups were given the same amount of saline intraperitoneally for 1 week.Mice in each group were anesthetized and executed 12 h after the completion of the final excitation,and alveolar lavage fluid(BALF)and lung tissue specimens were collected.The lung tissues were paraffin sectioned,and the histopathological changes were observed by hematoxylin-eosin staining(HE)and periodic acid Schiff staining(PAS);the concentrations of TNF-α and MUC5 AC in BALF were measured by enzyme-linked immunosorbent assay(ELISA);the distribution and expression of MUC5 AC and AQP5 proteins in the lung tissues of mice were observed by immunohistochemistry;the immunoblotting method(Western Blot)to further determine the expression level of AQP5 in lung tissues.Results:1.Body weight of mice: The body weight of mice in NS group remained stable,while the body weight of mice in AS and MET groups showed a decreasing trend from the 22 nd to 28 th d.However,there was no statistical difference between the groups(P>0.05).2.Behavioral performance of mice:(1)sensitization stage: there was no significant change in the appearance,behavior,reaction and activity of mice in each group;(2)excitation stage: mice in the NS group showed increased activity within 3 min at the beginning of nebulization,but gradually adapted and soon returned to normal with the continuation of nebulization,and the spirit could be repeatedly nebulized,and the hair was still shiny and freely moving;compared with the NS group,mice in the AS group showed irritability,scratching and scratching during the nebulization process.In contrast to the NS group,the mice in the AS group showed irritability,scratching and nodding breathing during the nebulization process,and gradually increased,accompanied by open-mouthed panting and upright arching of the back,etc.The above discomfort was relieved after 1 h of rest at the end of nebulization,and the mice were seen to have rough and disorganized hair without luster after repeated nebulization excitation,and were mentally ill with reduced activity.Unlike the AS group,the symptoms of ear scratching and wheezing were reduced in the Met group,and the above symptoms could be relieved within 30 min after nebulization excitation.3.Lung histopathology:(1)HE staining: In the NS group,the airway structure was intact,the lumen was unobstructed,no secretion formation was seen in the airway,and no obvious inflammatory cell infiltration was seen in the lung tissue;in the AS group,the alveolar structure was disorganized and partially destroyed,the airway was seen to have cupular cell proliferation,the wall was thickened,the inner wall was rough,and some of them were seen to have a little secretion formation with lumen narrowing,and inflammatory cells,mainly eosinophils,were seen in the airway wall and lung tissue.Compared with the AS group,the airway structure of the Met group was relatively intact,the wall was slightly thickened,the lumen was not significantly narrowed and exudate was not seen,and the inflammatory cell infiltration in the lung tissue was reduced.(2)PAS staining: Compared with the NS group,the AS group showed significant proliferation of cupped cells in the airway mucosa with high mucus secretion,and the difference in the percentage of positive staining area between the two groups was statistically significant(P < 0.05);compared with the AS group,the Met group showed significantly less proliferation of cupped cells and mucus secretion in the airway mucosa,and the difference in the percentage of positive staining area between the groups was statistically significant(P < 0.05).4.The concentrations of TNF-α and MUC5 AC in BALF: The concentrations of TNF-α and MUC5 AC in BALF of mice in the AS group were significantly higher than those in the NS group,200.49±6.63 ng/L vs.111.09±8.25 ng/L and 6.31±0.06 μg/L vs.4.64±0.15 μg/L,respectively,and the differences were statistically significant(P<0.05).The concentrations of TNF-α and MUC5 AC in BALF of the Met group were141.61±3.83ng/L and 5.28±0.17μg/L,respectively,which were statistically significantly lower than those in the AS group(P<0.05).5.Immunohistochemical results of AQP5 and MUC5 AC in lung tissues: the expression of AQP5 in lung tissues was decreased in the AS group compared with the NS group,and the expression of MUC5 AC was increased,0.236±0.003 vs 0.327±0.017 and 0.264±0.012 vs 0.175±0.011,respectively.The differences were statistically significant(P < 0.05).6.Western blot results of AQP5: The expression of AQP5 in lung tissues was significantly decreased in the AS group compared with the NS group,and the expression of AQP5 was significantly increased in the Met group compared with the AS group,and the differences were statistically significant(P < 0.05).7.Correlation analysis: Pearson correlation analysis showed that TNF-αconcentration in BALF was significantly negatively correlated with the expression level of AQP5 in lung tissue(r=-0.9582,P<0.0001),and was significantly positively correlated with the secretion level of MUC5 AC in BALF(r=0.9681,P<0.0001),and was significantly positively correlated with the expression of MUC5 AC in lung tissue level in BALF(r=0.9568,P<0.0001);MUC5AC secretion level in BALF was significantly and positively correlated with its expression level in lung tissue(r=0.9439,P<0.0001);AQP5 expression in lung tissue was significantly and negatively correlated with MUC5 AC secretion level in BALF(r=-0.9328,P< 0.0001)and a significant negative correlation with the level of MUC5 AC expression in lung tissue(r=-0.9352,P<0.0001).Conclusion: Metformin ameliorated symptoms,airway inflammation and goblet cell proliferation in bronchial asthma mice,increased the protein expression level of AQP5,and reduced the expression of MUC5 AC in lung tissue and its secretion in BALF,thereby inhibiting mucus hypersecretion in the airways of asthmatic mice,and its reduction in the expression and secretion of MUC5 AC may be dependent on increasing the expression of AQP5 to achieve this.