| Objective: Through the establishment of asthma model in mice and use TGF-beta 1 induced airway smooth muscle cells in mice,observed for particles to asthma airway remodeling in mice,airway smooth muscle cell proliferation,migration and TGF-? 1 / STAT3 signaling pathway and the effect of explore and asthma granule on airway remodeling of asthma and airway smooth muscle cell proliferation and migration ability to influence the mechanism of action.Methods:1.Experiment 1: 40 BALB/c mice were randomly divided into 4 groups with 10 mice in each group,namely,blank group,model group,pingchuan granule group and pulmicoreshu group.In addition to the blank group,the model group,the pingchuan granule group and the pulmicolinshu group were used to establish the asthma mouse model by the way of egg protein atomization and excitation.On the 21 st day,mice in the pingchuan granule group were given pingchuan granule(0.1ml/10g)by gavage,normal saline(0.1ml/10g)by gavage in the blank group and the model group,and pumicoreshu group were given pumicoreshu(1mg budesonide mixed with 2ml0.9% sodium chloride injection)by atomization for 30 minutes at a time for 4weeks,once a day.After 7 weeks,the general state of the mice in each group was observed,and the changes of pulmonary histopathological morphology in asthma mice in each group were observed by HE staining,PAS staining and Masson staining.Ig E level in BALF and TGF-? 1 level in serum were detected by ELISA.The expression level of alpha-SMA protein in lung tissue was detected by immunohistochemistry.Western blot analysis of protein expression levels of-sma,TGF-? 1,CTGF,STAT3 and p-stat3 in lung tissues.2.Experiment 2: airway smooth muscle cells of BALB/c mice were extracted and cultured in the primary generation,and the cultured airway smooth muscle cells were identified by ?-SMA in the cells detected by immunofluorescence.Airway smooth muscle cells were induced by TGF-? 1for modeling.The cultured airway smooth muscle cells were divided into four groups: blank group,TGF-? 1 group,pingchuan granule containing serum+TGF-?1 group,s3i-201(p-stat3 inhibitor)+TGF-? 1 group.The blank group was cultured normally,and the rest groups were induced with TGF-1.The drug serum containing pingchuan granules +TGF-? 1 group and s3i-201+TGF-?1 group were respectively cultured with the drug serum containing pingchuan granules and s3i-201 solution.MTT assay was used to detect the cellular activity of airway smooth muscle cells 48 hours after intervention.Transwell Chambers were used to detect the migration ratio of airway smooth muscle cells in each group.Experiment 3: airway smooth muscle cells were extracted and cultured,cells were grouped and the intervention methods were the same as experiment 2.After 48 hours of cell intervention,the expression levels of TGF-?1,CTGF,STAT3 and p-STAT3 protein in airway smooth muscle cells of each group were detected by Western blot.Results:1.HE staining of lung tissues of mice in the blank group showed no obvious pathological changes;Compared with the blank group,HE staining of the lung tissue of mice in the model group showed a large number of inflammatory cell infiltration,obvious proliferation of smooth muscle cells,obvious thickening of the airway wall and stenosis of the lumen.Compared with the model group,the above pathological features were alleviated in the pingchuan granule group and the pulmicorinshu group,while the pathological changes were significantly alleviated in the pulmicorinshu group.2.PAS staining of the lung tissue of mice in the blank group showed no obvious airway epithelial goblet cell hyperplasia and no mucinosis.Compared with the blank group,PAS staining in the lung tissue of the model group showed significantly increased airway mucus and goblet cells.Compared with the model group,the airway mucus and goblet cells were significantly reduced in the pingchuan granule group and the pulmicoreshu group,while the reduction was more obvious in the pulmicoreshu group.3.There was no significant abnormality under Masson staining in the lung tissue of mice in the blank group.Compared with the blank group,more collagen deposition can be seen in the lung tissue of the model group after Masson staining,which is widely distributed around the airway and infiltrates into the airway tissue.The pulmonary septum around the airway is thickened with fibrosis.The results of Masson staining on lung tissue of mice in the pulmicoreshu group and the pingchuan granules group showed that the collagen deposition was less than that of the model group,while the decrease was more obvious in the pulmicoreshu group.4.ELISA results showed that the level of Ig E in BALF and TGF-?1 in serum of mice in the model group was significantly higher than that in the blank group(P<0.05).The levels of Ig E in BALF and TGF-?1 in serum of the mice in the pingchuan granule group and the pulmicolinshu group were significantly lower than those in the model group(P<0.05).There was no significant difference between the pingchuan granules group and the pulmicoreshu group(P > 0.05).5.Immunohistochemical results showed that the protein expression level of ?-sma in the lung tissues of mice in the model group was significantly higher than that in the blank group(P<0.05).The protein expression level of-sma in lung tissues of pingchuan granule group and pulmicolinshu group was significantly lower than that of model group(P<0.05).The protein expression level of-sma in the pulmicorinshu group was lower than that in the pingchuan granules group,but there was no significant difference between the two groups(P > 0.05).6.Western blot results showed that the expression levels of ?-sma,TGF-?1,CTGF and p-stat3 in lung tissues of the model group were significantly higher than those of the blank group(P<0.05).The expression levels of ?-sma,TGF-?1,CTGF and p-stat3 in lung tissues of the pingchuan granule group and the pulmicolinshu group were lower than those of the model group(P<0.05).There was no significant difference between the pingchuan granules group and the pulmicoreshu group(P > 0.05).There was no significant difference in the expression level of STAT3 protein in the lung tissues of the blank group,model group,pingchuan granule group and pulmicoreshu group(P > 0.05).7.The cell activity and migration ratio of ASMCs in TGF-?1 group was significantly higher than that in the blank group(P<0.05),and the cell activity and migration ratio of pingchuan granules in drug-containing serum+TGF-?1 group was significantly lower than that in s3i-201 +TGF-?1 group(P<0.05).There was no significant difference between pingchuan granules in drug-containing serum +TGF-?1 group and s3i-201 +TGF-?1 group(P >0.05).8.Compared with the blank group,the expression levels of TGF-?1 and CTGF protein in ASMCs in the TGF-?1 group were significantly increased(P<0.05).The expression levels of TGF-?1 and CTGF in ASMCs of serum +TGF-?1 group and s3i-201 + TGF-?1 group were significantly lower than those in TGF-?1 group(P<0.05).There was no significant difference between serum + TGF-?1 group and S3I-201+ TGF-1 group(P > 0.05).9.Compared with the blank group,the expression level of p-stat3 protein in ASMCs in the TGF-?1 group was significantly increased(P<0.05),while the expression level of STAT3 protein was not significantly changed(P >0.05).The expression level of p-stat3 protein in ASMCs of serum +TGF-?1group and s3i-201 +TGF-?1 group was significantly lower than that of TGF-?1 group(P<0.05),and there was no significant change in the expression level of STAT3 protein(P > 0.05).There was no significant difference in the expression levels of p-stat3 and STAT3 in ASMCs between the serum group and s3i-201 group(P > 0.05).Conclusions:1.Pingchuan granules can improve the pathological changes of inflammatory cell infiltration,mucus secretion,goblet cell proliferation and collagen deposition in airway of asthmatic mice.2.Pingchuan granules can reduce Ig E level in BALF and TGF-?1 level in serum of asthmatic mice3.Pingchuan granules can reduce the protein expression levels of TGF-?1,-sma,CTGF and p-stat3 in the lung tissues of asthmatic mice.4.Pingchuan granules can reduce the expression levels of TGF-?1,CTGF and p-stat3 proteins in ASMCs.5.Pingchuan granules can inhibit the proliferation and migration of ASMCs by inhibiting the activation of TGF-?1/STAT3 signaling pathway. |
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