3.1 WYHY and XQL decoction improves weight\anal temperature and pulmonary coefficient of asthmatic rats
It can be seen from Fig. 1-A that there is flow chart for establishing a cold asthma mouse model using OVA intraperitoneal injection\OVA atomization inhalation and ice water swim stress. It can be seen from Fig. 1-B that the body weight in control group is relatively stable, while that in model group, XQL group and WYHY group is decreased. At the end of the 22th day, compare with model group, the body weight in XQL group and WYHY group was significantly increased (P < 0.05).
It can be seen from Fig. 1-C.The anal temperature of model group was significantly lower than that of control group (P < 0.05), and the anal temperature of XQL group and WYHY group was significantly higher than that of model group (P < 0.05).Furthermore, through the results of pulmonary coefficient of rats in each group (Fig. 1-D), it can be found that the pulmonary coefficient of model group was significantly increased (P < 0.05), while that of XQL group and WYHY group was significantly decreased (P < 0.05).
3.2 WYHY and XQL decoction improves airway reactivity and pulmonary function in cold asthmatic rats
It can be seen from Fig. 2-A and Fig. 2-B, under the stimulation of acetylcholine (Different doses), the airway responsiveness of the model group was significantly increased compared with the control group (P < 0.01). The airway responsiveness of the XQL group and WYHY group was significantly decreased compared with the model group (P < 0.01).
It can be seen from Fig. 2-C, Fig. 2-D and Fig. 2-E, the inspiratory resistance (RI) and expiratory resistance (RE) of the model group was significantly increased compared with the control group (P < 0.01).The lung compliance (Cdyn) of the model group was significantly decreased compared with the control group (P < 0.01).The inspiratory resistance (RI) and expiratory resistance (RE) of the XQL group and WYHY group was significantly decreased compared with the model group (P < 0.01). The lung compliance (Cdyn) of the XQL group and WYHY group was significantly increased compared with the model group (P < 0.05). XQL decoction and WYHY decoction can significantly improve pulmonary function in cold asthmatic rats. The therapeutic effect of WYHY decoction is better.
3.3 XQL and WYHY decoction improves airway inflammation in cold asthmatic rats
As can be seen from Fig. 3A\3B\3C and 3D, compared with the control group, IL-10 in the serum of the model group were significantly decreased (P < 0.01), whereas IL-13\TNF-α\TGF-β1 were significantly increased (P < 0.01). After treatment with XQL decoction and WYHY decoction, the ratio of IL-10 was increased (P < 0.01), the expression of IL-13, TNF-α and TGF-β1was significantly decreased (P < 0.01).These results indicated that XQL and WYHY decoction can alleviate airway inflammation in cold asthmatic rats.
As shown in Fig. 3E\3F\3G\3H. Compared with the control group, the number of eosinophils, lymphocytes, macrophages and total cells in BALF in the model group was significantly increased; Compared with the model group, the number of eosinophils, macrophages, lymphocytes and total cells in XQL group and WYHY group was significantly decreased (P < 0.05). Compared with model group and XQL group, WYHY group had the lowest number of inflammatory cells. The results showed that the number of eosinophils, lymphocytes, macrophages and total cells in BALF could be reduced by both WYHY decoction and XQL decoction, and the effect of WYHY decoction was better.
Furthermore, the HE staining of model group is shown in Fig. 3-I (model): the epithelial cells of the bronchial mucosa appear serious hyperplasia and swelling, a large number of inflammatory cells infiltrate around the bronchus. The HE staining of XQL group is shown in Fig. 3-I (XQL): the epithelial cells of bronchial mucosa have hyperplasia and swelling, but the severity is lighter than that of the model group, there is slight inflammatory cell infiltration in the tissue, and the infiltration is improved. The HE staining of WYHY group is shown in figure Fig. 3-I (WYHY): the epithelial cells of bronchial mucosa can be seen to be slightly swollen and proliferated, the cilia have a small part of adhesion. The degree of infiltration is improved than that of model group and XQL group.
The administration of XQL decoction and WYHY decoction can significantly reduce the infiltration of airway inflammatory cells and significantly improving asthma lesions. WYHY decoction has better therapeutic effect.
3.4 XQL and WYHY decoction regulates autophagy in lung tissue of cold asthmatic rats
As shown in Fig. 4A\4B\4C\4D, compared with the control group, the mRNA level of autophagy genes (ATG3\ATG5\ATG7\ATG12) in the model group was significantly decreased (P < 0.01).Compared with the model group, the mRNA level of ATG3\ATG5\ATG7\ATG12 in XQL group and WYHY group was significantly increased (P < 0.01).
Compared with the XQL group, the mRNA level of ATG3\ATG5\ATG12 mRNA in the lung tissue of rats was significantly increased in the WYHY group. These results indicated that XQL decoction and WYHY decoction can regulating autophagy gene (ATG3\ATG5\ATG7\ATG12) in cold asthmatic rats. WYHY decoction has better regulation ability to ATG3\ATG5 and ATG12.
Furthermore, the results of transmission electron microscopy of control group is shown in Fig. 4E (control): that the structure and tissue of type-II alveolar cells in control group were normal, organelles were relatively rich, with normal overall morphology and normal endoplasmic reticulum structure. The model group is shown in Fig. 4E (model): Type-II alveolar cells have obvious variability characteristics compared with the control group, which is mainly manifested as severe deformation, nuclear membrane depression. XQL group and WYHY group is shown in Fig. 4E (XQL\WYHY): Type-II alveolar cells deformation is improved compared with that in the model group, and the cell nuclear membrane surface is normal without depression. The matrix surface structure of mitochondrial crista is relatively complete.
The result proves that the autophagy level in lung tissue of cold asthma rats is decreased. After treatment with XQL decoction and WYHY decoction, the level of autophagy is increased, indicating that XQL decoction and WYHY decoction has the effect of upregulating the level of autophagy in lung tissue of asthma rats. WYHY decoction has better effect of upregulating.
3.5 Expression of PI3K-p110 α, p-mTOR, LC3 and ATG5 in rats
It can be seen from Fig. <link rid="fig14">5</link>-A and 5-C.Compared with the control group, the levels of PI3K-p110α in the lung tissue of the model group was significantly increased (P < 0.01).Compared with the model group, the levels of PI3K-p110α in the XQL group and WYHY group was significantly decreased (P < 0.05). Compared with the XQL group, the levels of PI3K-p110α in the lung tissue of the WYHY group was significantly decreased (P < 0.05).It can be seen from Fig. <link rid="fig14">5</link>-B and 5-C.Compared with the control group, the levels of p-mTOR in the lung tissue of the model group was significantly increased (P < 0.01).Compared with the model group, the levels of p-mTOR in the XQL group and WYHY group was significantly decreased (P < 0.05). Compared with the XQL group, the levels of p-mTOR in the lung tissue of the WYHY group was significantly decreased (P < 0.05).
Therefore, we conclude that XQL decoction and WYHY decoction can inhibit the activation of PI3K-mTOR signal pathway, WYHY decoction has better regulation ability.
It can be seen from Fig. <link rid="fig15">6</link>-A and 6-C.Compared with the control group, the levels of ATG5 in the lung tissue of the model group was significantly decreased (P < 0.01).Compared with the model group, the levels of ATG5 in the XQL group and WYHY group was significantly increased (P < 0.05). Compared with the XQL group, the levels of ATG5 in the WYHY group was significantly increased (P < 0.05). It can be seen from Fig. <link rid="fig15">6</link>-B and 6-C.Compared with the control group, the levels of LC3-II in the lung tissue of the model group was significantly decreased (P < 0.01).Compared with the model group, the levels of LC3-II in the XQL group and WYHY group was significantly increased (P < 0.05). Compared with the XQL group, the levels of LC3-II in the WYHY group was significantly increased (P < 0.05).
Therefore, we conclude that XQL decoction and WYHY decoction can upregulation of ATG and LC3-II to improve autophagy, WYHY decoction has better regulation ability.