In order to further explore the mechanism of autophagy affecting EMT, Western blot and Real-time PCR were used to investigate whether TGF-β/Smad pathway was involved in the influence of autophagy on EMT after referring to relevant literature and the previous experimental results of our research group. A549 cells were randomly divided into three experimental groups. The first group was added without any reagent, the second group was added with autophagy inhibitor 10mM 3-MA and pyocyanin 25ug/ml PCN, and the third group was added with 25ug/ml PCN. Figure (4A.B) Western blot assay was used to detect the expression level of downstream p-Smad2/3 protein of TGF-β/Smad pathway in each experimental group, the relative expression level of p-smad2/3 protein in PCN+3MA group was significantly increased compared with that in blank group (P<0.01), the expression of p-smad2/3 protein in PCN+3MA group was also increased compared with PCN group (P<0.01) (Figure 4B). Real-time PCR was used to detect changes in the nucleic acid level of TGF-β1 in cells, and the results (Figure 4C) indicated that the mRNA level of TGF-β in PCN+3MA group was significantly higher than that in PCN group and blank control group (p<0.01). It can be seen that TGF-β/smad expression changes significantly after the addition of 3MA to inhibit autophagy. These results suggest that autophagy may affect EMT levels through TGF-β/Smad signaling pathway.
Pulmonary fibrosis is a progressive disease that affects the respiratory function of patients, and can lead to respiratory failure or even death in severe cases [18,19, 20]. Mesenchymal cell transformation is caused by various factors such as infection, injury, stress, etc., causing epithelial phenotypic cells to transform into mesenchymal characteristic cells, which is involved in the occurrence and development of tissue and organ fibrosis [21]. Pulmonary fibrosis caused by mesenchymal transformation after alveolar epithelial cells are damaged is the main mechanism leading to lung function impairment [22, 23]. Clinically, when patients with compromised immunity are infected with pseudomonas aeruginosa, pyocyanin can stimulate cells to show oxidative stress, and then activate TGF-β1/smad signaling pathway to induce EMT in cells and aggravate pulmonary fibrosis [19, 20,24].
Autophagy can maintain cell balance and reduce cell damage caused by certain factors [25, 26], and uncontrolled autophagy and autophagy defects will aggravate cell damage [7, 25]. The occurrence of pulmonary fibrosis may be due to the imbalance between molecular mechanisms controlling oxidative stress and autophagy [27-29].Some researchers have replicated the mouse ARDS model with lipopolysaccharide (LPS) and observed that the alveolar epithelial cells are in a state of hypoxia and endoplasmic reticulum stress occurs. Endoplasmic reticulum stress activates autophagy by inhibiting the protein kinase B/ tuberculin/mammalian target of rapamycin (AKT/TSC/mTOR) pathway [30, 31].
Our study proves the fact that PCN can induce EMT phenomenon in alveolar epithelial cells. After PCN intervention in A549 cells, the expression of E-cadherin in epithelial cells decreased and α-SMA in fibroblasts increased compared with the control group by detecting the changes in protein and mRNA levels of EMT-related markers. In the experiment, we found that with the increase of PCN stimulation concentration, the changes of E-cadherin and α-SMA in protein and nucleic acid levels also gradually increased, indicating a concentration dependent relationship between EMT changes and PCN. This study also provided evidence that PCN can induce autophagy in alveolar epithelial cells. After stimulating A549 cells with PCN, the green fluorescence intensity representing autophagosomes was enhanced by immunofluorescence method compared with the control group, western-blot method also confirmed that the expression of autophago-related protein LC3 increased significantly after PCN stimulation.
At present, relevant studies have found that autophagy affects the progression of fibrosis by regulating the EMT degree of renal tubular epithelial cells, myocardial fibroblasts and tumor cells [32-36]. Some studies have found that autophagy can play a role in clearing ROS, and ROS accumulation increases the expression of HIF-1 factor through MAPK, Akt, NF-kB and other pathways, and then directly activates the transcription of downstream EMT-related target genes to promote the occurrence of EMT [11, 37, 38], autophagy can inhibit the development of EMT after clearing ROS. However, it was found that after autophagy was mediated by temozolomide and hypoglycemia, mesenchymal morphological changes and related protein changes occurred in cells, and EMT phenomenon was enhanced in glioma studies[39, 40], in the study of Hu et al., autophagy was induced after liver cancer cells were treated with balanced salt solution without nutrients and serum, epithelial markers of liver cancer cells decreased, interstitial markers increased, and morphological changes and aggressiveness increased, indicating that autophagy promoted EMT process [41].
Our study also proved that autophagy and EMT in A549 cells stimulated by PCN are not two independent processes, and the EMT phenomenon is enhanced after inhibition of autophagy. We selected a fixed concentration of PCN (25ug/ml) to act on A549 cells, meanwhile, different concentrations of autophagy inhibitor 3MA were added to each experimental group, and the results were detected by Western-blot and RT-PCR. After adding 3MA to inhibit autophagy, the protein and mRNA levels of autophagy marker LC3 and epithelial cell marker E-cadherin decreased significantly in all experimental groups, while the relative expression levels of protein and mRNA of mesenchymal cell marker α-SMA increased. With the increase of 3MA concentration, the changes of the above markers were more obvious. These results indicate that autophagy can inhibit EMT induced by PCN stimulation of A549 cells, and the EMT phenomenon will be enhanced after the degree of autophagy inhibition is aggravated.
Transforming growth factor TGF-β1 can induce autophagy by up-regulating the expression of autophagy related proteins such as ATG5 and ATG7 through Smad and JNK pathways [42]. Autophagy can also play a regulatory role in TGF-β, in which TGF-β receptor I (TβRI) protein is degraded through the autophagy pathway. In rat myocardial fibroblasts, TβRI was significantly increased after inhibition of autophagy by chloroquine, and significantly decreased after rapamycin mediated autophagy [43]. In renal tubular epithelial cells, autophagy blocks TGF-β1 signaling pathway by degrading TGF-β1 receptors, inhibits EMT mediated by TGF-β1/smad signaling pathway, and alleviates renal fibrosis [32, 33, 44].
This study confirmed that TGF-β1/smad pathway may be involved in the process of autophagy affecting EMT. It was found that the TGF-β mRNA level and p-smad2/3 protein relative expression level of the 3MA group were significantly increased compared with the blank group, and the TGF-β mRNA level and p-smad2/3 protein relative expression level of the PCN+3MA group were also significantly increased compared with the PCN group, indicating that after affecting the autophagy level, the mRNA level of the PCN+3MA group was significantly increased. TGF-β/smad also changes, suggesting that autophagy may affect EMT changes through TGF-β/smad pathway.
Our results show that pyocyanin can induce mesenchymal transformation and autophagy in alveolar type II epithelial cells at the same time, and the EMT phenomenon is enhanced after inhibition of autophagy, and there is a negative correlation between EMT changes and the degree of autophagy inhibition. The TGF-β/Smad signaling pathway changes after autophagy is inhibited, suggesting that autophagy may cause EMT changes through the TGF-β/Smad pathway. Therefore, our experimental conclusion is that in alveolar type II epithelial cells, PCN can cause EMT and autophagy, while autophagy inhibits EMT, and TGF-β/Smad pathway may be an important mechanism through which autophagy affects EMT.