Study On The Mechanism Of Pingchuan Granule Intervention On Airway Epithelial Cell Autophagy And Apoptosis Based On PI3K/Akt Signaling Pathway

Aim:This study is to clarify the regulation and mechanism of autophagy and apoptosis on epithelial cells of airway,and to reveal the intervention effect of Pingchuan granule on autophagy and apoptosis on epithelial cells from airway,which provide some scientific evidence for the treatment of bronchial asthma by Traditional Chinese Medicine.Methods:1.Forty-eight healthy clean Wistar rats(male and female)were randomly divided into 3 groups with 16 animals in each group,namely group A(blank group),group B(model group)and group C(Pingchuan granule group).The model of asthma was prepared by inhalation of ovalbumin,HE staining was used to observe the morphological changes of the lung,and immunohistochemistry was used to detect the expression of Beclin-1,LC3B and caspase-3 in lung tissue.RT-PCR and Western Blot were used to detect the expression of Beclin-1,LC3B and caspase-3 in lung tissue.The mechanism of pingchuan granules on epithelial cell apoptosis and autophagy in asthmatic rats was observed.2.16HBE cells was induced by rapamycin to establish the model and the MTT assay was used to determine the proliferation inhibition rate of 16HBE cells with different concentrations of rapamycin and serum containing Pingchuan Granules.The cells were treated with the best concentration of rapamycin and the drug-containing serum,including normal control group,rapamycin group,rapamycin treated with chloroquine group,and rapamycin treated with serum which contain Traditional Chinese Medicine group.Electron microscopy was used to observe the autophagic and apoptotic cells in each group.Cell apoptosis was detected by flow cytometry.The expression of LC3B,Beclin-1,and Caspase-3 was detected by RT-PCR and Western Blot.The mechanism of pingchuan granules on epithelial cell apoptosis and autophagy in asthmatic rats was observed.3.The model of 16HBE cells induced by rapamycin was treated with PI3K/Akt pathway blocker LY294002 and drug-containing serum.The cells were divided into normal control group,rapamycin group,rapamycin treated with serum containing Traditional Chinese Medicine group,andrapamycin treated with LY294002 group.The fluorescence intensity of autophagic vesicles and the autophagic intensity of cells were detected by fluorescence microscope and fluorescence microscope.The expression of p-Akt?p-mTOR in PI3K/Akt signaling pathway was detected by RT-PCR and Western Blot.ignal pathways associated with autophagy and apoptosis were determined.Result:1.After ovalbumin stimulation,the infiltration of inflammatory cells in the trachea,lung tissue,and alveolar walls of rats could be observed by HE staining.The structure of epithelial cells is disordered and destroyed.2.The results of immunohistochemistry showed that the expression of Beclin-1,LC3B and caspase-3 protein in the model group was higher than that in the control group(P<0.01).The expression of Beclin-1 and LC3B protein in Pingchuan Granule group was lower than that in the model group(P<0.01).3.The results of Western Blot and PCR showed that:Pingchuan Granule can reduce the expression and content of beclin-1,LC3B and caspase-3 in asthmatic rats,compared with the model group,there is statistical significance(P<0.05).4.0.1 ?M rapamycin is the best concentration to induce 16HBE cell proliferation inhibition.5.The best serum concentration of Pingchuan Granule was 20%.To compare with other concentrations,there was a statistically significant difference(P<0.05).6.After 16hBE cells were induced by rapamycin,the early apoptosis rate and late apoptosis rate of 16HBE cells increased(P<0.01).However,after chloroquine blocking or Pingchuan Granule intervention,rapamycin-induced late cell apoptosis rate significantly decreased(P<0.01),and the total apoptotic rate also showed a decreasing trend(P<0.01).7.After 16HBE cells were induced by rapamycin,the expression of Beclin-1,LC3-II/LC3-I,caspase-3 and PI3K were significantly higher than those in control group(P<0.05).After chloroquine blocking or Pingchuan Granule intervention,the expression of Beclin-1,LC3-II/LC3-I,caspase-3 and PI3K were significantly decreased to compare with the model group(P<0.01).8.The results of electron microscopy showed that autophagy and apoptosis increased significantly in rapamycin-induced 16HBE cells,which was inhibited after intervention with chloroquine or Pingchuan granules.9.Compared with normal control cells,the fluorescence intensity of MDC induced by rapamycin was significantly increased(P<0.05).Compared with rapamycin group,LY294002 treatment significantly inhibited the fluorescence intensity of MDC(P<0.05).Pingchuan Granules could also inhibit the increase of fluorescence intensity of MDC(P<0.05),which suggested that both LY294002 and Pingchuan Granules could inhibit autophagy.10.Compared with the normal control group,the expression of p-Akt and p-mTOR protein in rapamycin group was significantly decreased(P<0.01),and the expression levels of-Akt and mTOR protein had no significant changes.Compared with rapamycin group,after LY294002 blocking or Pingchuan Granule intervention,the expressions of p-Akt and p-mTOR protein were significantly decreased(P<0.01).There was no significant change in the expression of Akt and mTOR protein.Conclusion:1.Pingchuan Granules could inhibit autophagy and apoptosis in asthmatic rats induced by ovalbumin.2.After rapamycin-induced 16HBE cells,both autophagy and apoptosis increased.3.Pingchuan Granule could inhibit the expression of Beclin-1,LC3-?/LC3-?,caspase-3 and PI3K induced by rapamycin in 16HBE cells.4.Pingchuan Granule could inhibit the phosphorylation of Akt and mTOR and the occurrence of autophagy.5.The PI3K/Akt signaling pathway is one of the autophagy pathways in 16HBE cells.