Chlorogenic Acid Inhibited Epithelial–Mesenchymal Transition to Treat Pulmonary Fibrosis through Modulating Autophagy

Abstract

Chlorogenic acid (CGA), derived from dicotyledons and ferns, has been demonstrated with anti-inflammatory, anti-bacterial, and free radical-scavenging effects and can be used to treat pulmonary fibrosis (PF). However, the specific mechanism by which CGA treats PF needs to be further investigated. In this study, in vivo experiment was firstly performed to evaluate the effects of CGA on epithelial–mesenchymal transition (EMT) and autophagy in bleomycin (BLM)-induced PF mice. Then, the effects of CGA on EMT and autophagy was assessed using transforming growth factor beta (TGF-β) 1-induced EMT model in vitro. Furthermore, autophagy inhibitor (3-methyladenine) was used to verify that the inhibitory mechanism of CGA on EMT was associated with activating autophagy. Our results found that 60 mg/kg of CGA treatment significantly ameliorated lung inflammation and fibrosis in mice with BLM-induced PF. Besides, CGA inhibited EMT and promoted autophagy in mice with PF. In vitro studies also demonstrated that 50 µM of CGA treatment inhibited EMT and induced autophagy related factors in TGF-β1-induced EMT cell model. Moreover, the inhibitory effect of CGA on autophagy and EMT in vitro was abolished after using autophagy inhibitor. In conclusion, CGA could inhibit EMT to treat BLM-induced PF in mice through, activating autophagy.

INTRODUCTION

Pulmonary fibrosis (PF) is the end result of many interstitial lung diseases and can cause dyspnea or even develop into respiratory failure. Currently, the average life expectancy of PF is 3 years after diagnosis.¹⁾ Its pathology is characterized primarily by tissue injury, which results in a highly conserved, tightly regulated inflammatory response and a dysregulated repair response.²⁾ However, this disease is heterogeneous, and failure to accurately distinguish between subtypes of PF can lead to ineffective treatment.³⁾ As a result, treatment regimens for PF are limited, and there is a significant unmet clinical need.⁴⁾ Therefore, developing novel medications has become urgent clinical issues for the treatment of PF.

Excessive epithelial–mesenchymal transition (EMT) has been demonstrated to be the key pathogenesis of PF. During EMT, excessive fibroblasts and myofibroblasts are produced throughout fibrosis,⁵⁾ and myofibroblasts can rapidly produce excess extracellular matrix (ECM) and exert traction on the ECM, resulting in lung tissue distortion.⁶⁾ Many natural products have been shown to alleviate PF through improving EMT: scutellarin inhibits bleomycin (BLM)-induced inflammation and EMT in PF through interacting with the nucleotide-binding domain of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)⁷⁾; tanshinone IIA improves silicon-induced PF by activating nuclear factor erythroid 2-related factor 2 (NRF2) and inhibiting EMT⁸⁾; and arctigenin can inhibit wingless-related integration site family member 3a/β catenin signaling pathway, thereby correcting overactivated EMT and paraquat-induced PF.⁹⁾ These studies suggest that improving EMT has great potential for treating PF.

In addition to EMT, autophagy plays a critical and complex regulatory role in the development of PF.¹⁰⁾ Autophagy, which is closely related to apoptosis, is a major cellular defense mechanism in eliminating the accumulation of damaged proteins or organelles.¹¹⁾ Autophagy is responsible for cellular homeostasis. Both impaired and excessive activation of autophagy could negatively affect cellular homeostasis, leading to the development of PF.^12,13) Analysis of lung tissue samples obtained from patients with idiopathic pulmonary fibrosis (IPF) showed sequestosome-1 (P62) accumulation, low microtubule-associated protein 1A/1B-light chain 3 (LC3)-II levels.¹⁴⁾ A downregulation of autophagy could also be found in transforming growth factor beta (TGF-β) 1-treated lung fibroblasts.¹⁴⁾ Pharmacological studies have found that metformin could inhibit silica-induced PF by activating autophagy in bronchial epithelial cells¹⁵⁾ and that dioscin could promote autophagy in macrophages in PF, thereby enhancing cell survival, alleviating oxidative stress, and reducing the release of proinflammatory factors.¹⁶⁾ However, study also reported that TGF-β1-induced autophagic degradation of lung epithelial cells triggers the development of EMT, ultimately leading to PF.¹⁷⁾ These results suggest that changes in autophagy levels in different cells during PF contribute differently to the disease and may even have opposite effects. Therefore, the effects of drugs on the altered levels of autophagy in specific cells during PF must be investigated.

Chlorogenic acid (CGA), derived from dicotyledons and ferns, has been demonstrated with anti-inflammatory, antibacterial, and free radical-scavenging effects.¹⁸⁾ Chen et al. found that CGA could enhance the anti-oxidative capacity of the body.¹⁹⁾ Wang et al. demonstrated that CGA binding to annexin A2 reduced the expression of anti-apoptotic genes downstream of NF-κB and inhibited A549 cell proliferation in vitro and in vivo.²⁰⁾ In addition, it has been found that CGA may exert therapeutic effects on PF by inhibiting endoplasmic reticulum stress.²¹⁾ However, the specific mechanism by which CGA treats PF still needs to be further investigated. In this study, we investigated whether the anti-PF effect of CGA is related to its autophagic function through in vivo and in vitro experiments. This has the potential to provide new ideas and potential drugs for delaying the development of PF.

MATERIALS AND METHODS

Materials

C57BL/6J mice (SCXK-2019-0010) were purchased from SPF Biotechnology (Beijing, China). A549 cell line was purchased from iCell Bioscience Inc. (Shanghai, China). CGA (B20782), BLM (B73357), dexamethasone (DXM, S17003) and 3-methyladenine (3-MA, B25357) were obtained from Yuan Ye Bio-Technology (Shanghai, China). Hydroxyproline (HYP) concentration (A030-2-1), and total protein concentration (A045-4-2) kits were obtained from Jian Cheng Institution (Nanjing, China). Enzyme-linked immunosorbent assay (ELISA) kits for interleukin (IL)-1β (EK201B), IL-6 (EK206), and tumor necrosis factor (TNF)-α (EK282) were purchased from Multi sciences (Hangzhou, China). Antibodies for alpha smooth muscle actin (α-SMA, 14395-1-AP), collagen-I (14695-1-AP), vimentin (10366-1-AP), epithelial-cadherin (E-cadherin, 20874-1-AP), LC3, 14600-1-AP), P62 (18420-1-AP), and beclin-1 (11306-1-AP) were purchased from Proteintech (Wuhan, China).

Animal Experiment

Seventy-five specific pathogen free (SPF)-grade C57BL/6J mice (22±2 g, 8 weeks old) were randomized into the control, BLM, DXM group, CGA low-dose (LD-CGA), and CGA high-dose (HD-CGA) groups, with 15 mice in each group. All mice were kept in SPF conditions with ad libitum access to water and food. This experiment was approved by the Ethics Committee of Sixth People’s Hospital of Nantong (20210007). Pain management protocols were used to reduce animal pain, and pain and distress relief were carefully monitored throughout the experiment.

PF was induced by a single intratracheal injection of 2.5 mg/kg of BLM. Mice in the control group received a single intratracheal injection of an equal volume of phosphate buffered saline (PBS). Starting on day 1 after intratracheal injection, the DXM group received 2 mg/kg of DXM via intragastric administration on a daily basis; the LD-CGA and HD-CGA groups received 60 and 120 mg/kg of CGA via intragastric administration on a daily basis, respectively; and the control and BLM groups received equal volumes of saline intragastrically as parallel controls. The entire drug administration process lasted for 21 d. On day 22 after intratracheal injection, all mice were sacrificed and samples were collected.

Bronchoalveolar Lavage Fluid (BALF) Collection and Examination

After the mice were sacrificed, the thorax was incised and the cervical trachea was fully exposed. After ligation of the left lung, the right lung was lavaged three times with PBS at 4 °C, 0.5 mL each time, and the recovered fluid was bronchoalveolar lavage fluid (BALF). BALF was then centrifuged at 150 × g for 15 min, and the total protein contents in the supernatant were analyzed. Cell pellets were counted using a hemocytometer plate.

Lung Tissue Histopathological Staining

After collecting BALF, the left lung was removed and fixed in 4% paraformaldehyde before being embedded in paraffin. Hematoxylin–eosin (H&E) and Masson’s trichrome staining were routinely performed. H&E staining for the evaluation of alveolitis was performed using the Szapiel scale.²²⁾ Masson staining to evaluate the degree of fibrosis was performed using a modified Ashcroft scale.²³⁾

Measurement of HYP in Lung Tissue

A sample of 30 mg of lung tissue was accurately weighed, and the level of HYP, the main component of collagen in the lung, was measured using a biochemical assay kit. The oxidation product produced by HYP in the presence of an oxidizing agent interacts with para-dimethylaminobenzaldehyde to produce a purplish-red color, which can be measured from its absorbance at 550 nm.

Immunohistochemical Staining of Lung Tissue

Paraffin sections were made of fixed lung tissues, which were cleaned and stained using PBS and clarified with distilled water. After antigen retrieval with sodium citrate and bovine serum albumin (BSA) blocking, immunohistochemical staining was performed. The sections were incubated overnight at 4 °C with rabbit anti-α-SMA (1 : 200) and rabbit anti-collagen-I (1 : 300). Thereafter, the sections were washed, incubated with secondary antibody (1 : 500), and washed again. The sections were then mounted after color development and hematoxylin counterstaining. Image Pro Plus 6.0 software was used to quantitatively analyze the expression in the positive regions.

Cell Culture

The A549 cells were cultured in complete medium contains 89% of dulbecco’s modified eagle medium (DMEM), 10% of fetal bovine serum, 1% of penicillin and treptomycin (100 U/mL) at a constant temperature and humidity (37 °C, 5% CO₂), and the cells were subcultured at a ratio of 1 : 3 when they reached 90% confluence.

Induction of EMT in A549 Cells Using TGF-β1

A549 cells were seeded at a density of 5 × 10⁵ cells/well in 6-well plates and incubated for 2 h in serum-free DMEM. Then, the cells were treated with TGF-β1 (5 ng/mL) for 24 h to induce EMT production.²⁴⁾

Cell Viability Assay

Cell viability assay is based on the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Mitochondrial dehydrogenase from viable cells reduces MTT to dark blue crystals. Briefly, A549 cells were seeded at a density of 5 × 10³ cells/well in 96-well plates and incubatefigd at 37 °C for 24 h. Cells were incubated with different concentrations of CGA (5, 10, 50, and 100 µM) for 24 h. At the end of the incubation, 10 µL of MTT solution was added to each well. After a further 4 h incubation, the medium was gently removed and 100 µL of dimethyl sulfoxide (DMSO) was added. The absorbance of the samples was then measured at 550 nm using a microplate reader.

Cell Experiment Grouping

In the study of the effect of CGA on EMT and autophagy in A549 cells, TGF-β1 was used to stimulate EMT in A549 cells and different concentrations of CGA were used for intervention. The experiment was divided into five groups: control, model, low-dose CGA, medium-dose CGA, and high-dose CGA. All cells underwent EMT induction except for cells in the control group. Meanwhile, the cells in low-dose CGA, medium-dose CGA, and high-dose CGA groups were treated with CGA at final concentrations of 5, 10, and 50 µM, respectively. As parallel controls, equal volumes of DMSO were added to the control group and the model group. Each group was cultured in three wells.

To determine whether CGA regulated the EMT of A549 via autophagy, CGA was combined with 3-MA, an autophagy inhibitor that could block the formation of autophagosomes and prevent the nucleation phase, thereby inhibiting autophagy.²⁵⁾ Rapamycin, an autophagy inducer, was selected as a positive control. The experiment was divided into five groups: control, model, CGA, rapamycin, and CGA + 3-MA. EMT induction was performed in all groups except for the control group, and the CGA group was given 50 µM of CGA, the rapamycin group was given 500 nM of rapamycin,²⁶⁾ the CGA + 3-MA group was given 50 µM of CGA and 5 mM of 3-MA intervention,²⁷⁾ and the control and model groups were given equal volumes of DMSO as parallel controls. Each group was cultured in three wells.

Quantitative Reverse Transcription PCR (RT-qPCR)

Total RNA was obtained from lung tissues and cells using the total RNA extraction kit (#DP419). The cDNA was obtained using a reverse transcription kit (#KR116-02). The mRNA expression of Vimentin (Vim) and cadherin 1 (Cdh1) was determined using RT-qPCR. Primer sequences were included in supplementary material. The relative expression of each target mRNA relative to β-actin was calculated based on the 2^−ΔΔCT method.

Western Blot

Lung tissues and cells were ultrasonically homogenized with radioimmunoprecipitation assay (RIPA) buffer to extract total protein. Bicinchoninic acid (BCA) protein assay kit was used to calculate the protein concentration in each sample. The extracted proteins were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene fluoride membranes. The membranes were blocked using a 5% skim milk powder solution for 2 h, followed by incubation with antibodies against vimentin (1 : 5000), E-cadherin (1 : 10000), beclin-1 (1 : 5000), LC3 (1 : 2000), and P62 (1 : 5000) overnight at 4 °C. The membrane was washed 3 times after incubation and then incubated with secondary antibodies for 2 h. After washing the membranes again and developing them with enhanced chemiluminescence, the bands were quantified and analyzed using Image J software. The intensity values are then used to calculate the relative expression of the protein of interest compared to β-actin. The formula for calculating the relative expression is: Relative expression = (intensity of protein of interest)/(intensity of β-actin). Additionally, the relative expression of LC3 was calculated as intensity of LC3-II divided by intensity of LC3-I.

Statistical Analysis

SPSS 22.0 software was used for statistical analysis. All data are expressed as mean±standard deviation (S.D.). Inter-group differences were analyzed by one-way ANOVA followed by Tukey’s honestly significant difference test for post hoc analyses. A difference of p < 0.05 was considered statistically significant.

RESULTS

CGA Improves BLM-Induced PF in Mice

After 21 d of intratracheal BLM injection, CGA treatment improves the survival rate of BLM-induced PF mice. The survival rates were higher in DXM and CGA-treated groups compared with the BLM group. The survival rate of Control, BLM, DXM, LD-CGA, HD-CGA groups were 100, 53.33, 66.67, 66.67, 73.33% (Fig. 1a). The mice showed significant fibrotic pathology in lung. H&E staining results revealed that the lungs of mice in the BLM group showed severe distortion with increased alveolar lumen and significantly increased interstitial lung cells, inflammatory cell infiltration, and fibrous tissue hyperplasia (Figs. 1b, d). Masson staining showed significant collagen deposition in the lung interstitium (Figs. 1c, e). The level of HYP was significantly elevated in the lung tissues of the mice in BLM group, suggesting the presence of a large amount of collagen in the lung tissues (Fig. 1f). Immunohistochemical results showed that in lung tissues from control mice, α-SMA was mainly expressed in the pulmonary vascular wall, while collagen-I was barely detectable. However, in the BLM group, substantial α-SMA expression was observed in the lung parenchyma and significant collagen-I expression was observed in the area of thickened interalveolar septa (Figs. 1g, h)

Fig. 1. CGA Reduces BLM-Induced Inflammatory Response and Fibrosis in Mouse Lung Tissue

Mice were first administrated with intratracheal injection of BLM (2.5 mg/kg) once to induce PF and were then treated with different doses of CGA. (a) CGA treatment improves the survival rate of PF mice (n = 15 per group). (b) H&E staining showed that CGA improved pathological changes in the lung tissue of PF mice, with blue arrows indicating inflammatory cell infiltration and red arrows indicating collagen. (c) Masson trichrome staining of lung tissue, with arrows indicating collagen. (d–f) Szapiel score (d), modified Ashcroft score (e), and hydroxyproline concentration assay (f) showed significant therapeutic effects of CGA on inflammation and collagen deposition. (g, h) Immunohistochemical staining and quantification showed that CGA treatment significantly reduced increased α-SMA and collagen-I in PF mice. (i, j) CGA treatment also reduced the number of cells (i) and the amount of total protein (j) in BALF. n = 15, 8, 10, 10, and 11 for the five groups respectively. ^##: p < 0.01, compared to the control group; *: p < 0.05, compared to the BLM group; **: p < 0.01 compared to the BLM group.

In addition, the BALF of mice in the BLM group was significantly different from that of the control group. The total cell count in the BLM group was significantly increased than that in the control group (Fig. 1i). Total protein quantification showed that the total protein concentration of BALF in BLM group was elevated compared with the control group (Fig. 1j). These results suggest significant cell and protein exudation into the alveolar space.

In contrast, the above pathological changes were significantly improved after CGA or DXM treatments, and no significant differences were found between the HD-CGA and DXM groups.

CGA Inhibits EMT and Promotes Autophagy in BLM-Induced PF in Mice

The mesenchymal cell indicator vimentin and the epithelial cell indicator E-cadherin were both widely-used indicator proteins of EMT. RT-qPCR results showed that the mRNA levels of Vim in lung tissues were also up-regulated, while the mRNA expressions of Cdh1 were significantly decreased. Both CGA and DXM had corrective effects on these changes (Fig. 2a). These results suggest that CGA could inhibit EMT. Western blot results also showed the same trends. The expression of vimentin in lung tissues was higher and the expression of E-cadherin was lower in the BLM group than those in the control group. CGA and DXM treatment could decrease the vimentin expression and increase the E-cadherin expression in PF mice. (Figs. 2b, c). In addition, a series of autophagy-associated proteins in mouse lung tissues were measured to find the changes in autophagy during PF. The protein levels of both LC3 and beclin-1 were decreased, while P62 level was significantly elevated in the lung tissue of BLM mice compared with the control group, suggesting disrupted autophagic flux. In contrast, both LC3 and beclin-1 were elevated after treatment with CGA and DXM, whereas P62 was significantly reduced, suggesting improved autophagic flux (Figs. 2d, e).

Fig. 2. CGA Inhibits EMT and Promotes Autophagy in BLM-Induced PF in Mice

(a) RT-qPCR results showed that the mRNA levels of Vim and Cdh1, which were altered in the PF model, were improved after CGA treatment. (b, c) Western blot analysis showed that CGA treatment decreased vimentin and increased E-cadherin expression in the lung tissue of PF mice. (d, e) The levels of autophagy-related proteins (beclin-1, LC3-II, and P62) in lung were tested by Western blot. CGA treatment increased beclin-1 and LC3 and decreased P62 expression in the lung tissue of PF mice. Vim: vimentin; Cdh1: cadherin 1. (n = 3 per group).

CGA Inhibits EMT and Promotes Autophagy in the Fibrosis of A549 Cells

In order to deeply investigate the changes in EMT and autophagy during PF, we selected a lung epithelial cell line A549 as the subject. MTT assay results showed that the cell viability of A549 cells did not significantly change at concentrations of 5, 10, and 50 µM CGA (Fig. 3a). In addition, 24 h of 5 ng/mL TGF-β1 treatment was used to induce EMT in A549, while 5, 10, and 50 µM CGA were added at the same time. A TGF-β1-only intervention group and a no TGF-β1 intervention group were also established. After treated with TGF-β1, cells exhibited mesenchymal-like cell morphology, such as fusiform shape and loose intercellular junctions, while 10 and 50 µM of CGA treatment could reduce the TGF-β1-induced mesenchymal-like changes (Fig. 3b). RT-qPCR analysis indicated that TGF-β1 induction increased the mRNA level of Vim and decreased the mRNA level of Cdh1 in comparison to the control group (Fig. 3c). Likewise, Western blot results demonstrated that TGF-β1 induction significantly increased vimentin level and decreased E-cadherin level in A549 cells (Figs. 3d, e). Furthermore, detection of autophagy-related proteins revealed that TGF-β1 induction decreased beclin-1 and LC3 levels and increased P62 level in A549 cells (Figs. 3f, g). The treatments of 10 or 50 µM CGA corrected these changes to varying degrees, with 50 µM CGA being the most effective.

Fig. 3. CGA Inhibits TGF-β1-Induced EMT in A549 Cells and Promotes Cellular Autophagy

A549 cells were treated with 5 ng/mL TGF-β1 to induce EMT. Different concentrations of CGA were also added to study the effects on EMT and autophagy. (a) The effects of CGA on cell viability in different concentrations. (b) CGA treatment could reduce the TGF-β1-induced mesenchymal-like changes in A549 cell morphology to varying degrees. (c) RT-qPCR results showed that mRNA levels of Vim and Cdh1 altered by TGF-β1 induction were improved after CGA treatment, with downregulated Vim expression and upregulated Cdh1 expression in cells. (d, e) Western blot analysis indicated that CGA decreased the level of vimentin and increased the level of E-cadherin in A549 cells. (f, g) Western blot analysis revealed that CGA treatment increased beclin-1 and LC3-II, and decreased P62 expression in cells. Vim: vimentin; Cdh1: cadherin 1. n = 3 per group. ^##: p < 0.01, compared to the DMSO group; *: p < 0.05, compared to the TGF-β1 group; **: p < 0.01 compared to the TGF-β1 group.

Inhibition of Autophagy Eliminates the Inhibitory Effect of CGA on EMT in A549 Cells

To verify whether CGA inhibits EMT in lung epithelial cells by promoting autophagy, autophagy inhibitor 3-MA was used then to study the effect of CGA on EMT and autophagy in TGF-β1-induced A549 cells. In addition, the autophagy inducer rapamycin was selected as a positive control drug to treat TGF-β1-induced A549 cells to compare the effect of CGA with rapamycin. Both CGA and rapamycin were able to reduce TGF-β1-induced cell morphological changes, while 3-MA treatment abolished the effects of CGA on cell morphology (Fig. 4a). RT-qPCR results indicated that both CGA and rapamycin treatment improved the changed mRNA levels of Vim and Cdh1 caused by TGF-β1 induction, while the effects of CGA could be abolished by 3-MA (Fig. 4b). Likewise, Western blot results demonstrated that both CGA and rapamycin were able to down-regulate the vimentin, and up-regulate the E-cadherin. The effects of CGA could also be abolished by 3-MA (Figs. 4c, d). Furthermore, the changed levels of autophagy-related proteins could be improved by CGA or rapamycin. The effects of CGA were abolished by 3-MA as expected (Figs. 4e, f). These results demonstrated that both CGA and rapamycin were able to improve EMT and disturbed autophagic flux in vitro. When 3-MA was added, CGA lost its corrective effect on autophagy defects and failed to reduce EMT, suggesting that the inhibitory effect of CGA on EMT was exerted primarily through the regulation of autophagy.

Fig. 4. Inhibition of Autophagy Eliminated the Inhibitory Effect of CGA on EMT in A549 Cells

A549 cells were treated with 5 ng/mL TGF-β1 to induce EMT. Fifty micromolar CGA with absence or presence of autophagy inhibitor 3-MA (5 mM) were added to verify the effect of CGA on EMT and autophagy. In addition, 500 nM rapamycin (an autophagy inducer) was used as a positive control. (a) Light microscopy analysis showed that both CGA and rapamycin were able to reduce the TGF-β1-induced cell morphological changes. 3-MA could abolish the effect of CGA. (b) RT-qPCR results showed that CGA treatment failed to improve Vim and Cdh1 mRNA levels when 3-MA was present. (c, d) Western blot assays revealed that the EMT-related proteins vimentin and E-cadherin, which could have been improved by CGA treatment, were eliminated by the autophagy inhibitor 3-MA. (e, f) Western blot assays revealed that the autophagy-related proteins beclin-1, LC3, and P62, which could have been improved by CGA treatment, were eliminated by the autophagy inhibitor 3-MA. Vim: vimentin; Cdh1: cadherin 1. All data are presented as mean±S.D. (n = 3 per group). ^##: p < 0.01, compared to the DMSO group; *: p < 0.05, compared to the TGF-β1 or TGF-β1+CGA group; **: p < 0.01 compared to the TGF-β1 or TGF-β1+CGA group.

DISCUSSION

In the current study, our results found that CGA alleviated lung structural damage and fiber deposition in BLM-induced PF in mice. ECM provides the necessary structural framework for the cells that determines the tissue structure of the lung, and also provides mechanical support for lung function.^28,29) The lung ECM is used to synthesis major structures such as the basement membrane of epithelial cells, the basement membrane of endothelial cells and the interstitial matrix.³⁰⁾ The main components of ECM include collagen, elastin, glycoproteins and proteoglycans. Therefore, the balancing the synthesis and degradation of ECM is essential for maintaining the normal morphology of lung tissue.³¹⁾ In addition, ECM is a key regulator of fibrosis, in which fibroblasts and myofibroblasts play important roles.^13,32) Myofibroblast can rapidly synthesize and accumulate excessive ECM.³³⁾ When hexokinase is activated in the cytosol, it produces a large amount of pyruvate which is then metabolized to lactate and then released extracellularly to lower pH and thus activate potential TGF-β. When pyruvate can be metabolized via the tricarboxylic acid cycle (TCA cycle), it causes a buildup of succinate, which increases α-SMA and collagen expression and promotes cell proliferation, resulting in fibrosis. Therefore, the increase of matrix hardness in PF can be achieved by forcing the activation of TGF-β. On the contrary, increased glutaminase activity leads to a massive breakdown of glutamine to glutamate, which is then metabolized to α-ketoglutarate via the TCA cycle, thereby enhancing collagen stability and inhibiting apoptosis.³⁴⁾ DXM is widely used in anti-fibrotic therapy as it protects the lungs from fibrosis by inhibiting the production of inflammatory mediators.^35,36) DXM treatment was found to reduce TGF-β1 levels and decrease TGF-β1-induced Smad2 phosphorylation and abnormal activation of the JAK/signal transducer and activator of transcription (STAT) pathway.³⁷⁾ Early treatment of PF with DXM can substantially reduce lung cell proliferation and collagen deposition and help to inhibit lung fibrosis.³⁸⁾ In the present study, DXM was found to inhibit EMT development and promote autophagy in BLM-induced PF mice. This may be other possible mechanisms for DXM treatment of PF.

Besides, our results revealed that CGA could improve EMT through reducing the level of the mesenchymal indicator vimentin and increasing the level of the epithelial indicator E-cadherin in vivo and in vitro. ECM acts not only with lung fibroblasts but also with alveolar epithelial cells. EMT can be induced in alveolar epithelial cells when the stiffness of the matrix is increased. ECM is deposited in response to abnormal pathway activation due to epithelial damage, the most important of which is EMT.³⁹⁾ EMT is a key driver in the normal repair of damaged lung epithelial cells. During this process, epithelial cells lose some of their features and indicators, and gain indicators of mesenchymal cells.⁴⁰⁾ Vimentin is a key component of cytoskeleton of mesenchymal cells and the major mesenchymal marker. Vimentin expression is low in normal epithelial cells. When EMT occurs, epithelial cytokeratin is transformed into a vimentin-based cytoskeleton with mesenchymal cell morphology and achieves wandering ability.⁴¹⁾ E-cadherin is a key component of adhesion junctions and is essential in maintaining cell adhesion and epithelial phenotype. The downregulation of E-cadherin leads to the reduction of intercellular adhesion and the loss of cell polarity. Therefore, E-cadherin is the major epithelial marker.⁴²⁾ However, repeated injury combines with persistent inflammation and hypoxia along these highly specific repair pathways, disrupting the normal repair process and resulting in persistent fibrosis.⁴³⁾

Activation of autophagy has been shown to improve BLM-induced PF by inhibiting EMT in lung epithelial cells. Epithelial cells can induce EMT production in the presence of blocked autophagic flux and impaired autophagic function.⁴⁴⁾ In addition, epithelial cells with impaired autophagic flux cause chronic inflammation due to the inability to clear large amounts of misfolded proteins, which contributes to EMT in epithelial cells.⁴⁵⁾ Therefore, normal autophagic flow activity prevented endothelial-to-mesenchymal transition and the development of PF. Annexin A2 (ANXA2) is a specific bleomycin target to induce pulmonary fibrosis by impeding autophagic flux.⁴⁶⁾ BLM binding to ANXA2 not only prevents transcription factor EB from initiating autophagic flow, leading to defective autophagy, but also mediates the inflammatory response.⁴⁷⁾ Defective autophagy causes α-SMA overexpression and increases ECM deposition.⁴⁸⁾ These results suggest that physiological autophagy of lung epithelial cells is beneficial in inhibiting the progression of PF. Studies have shown that IL-37 can exert antifibrotic effects by enhancing autophagy, inhibiting TGF-β1-induced inflammation, and reducing the proliferation of lung fibroblasts in PF.⁴⁹⁾ This demonstrates the feasibility of intervening in the PF process through autophagy regulation. Both in vitro and in vivo experiments showed that CGA treatment could increase LC3-II and beclin-1 levels and decrease P62 level in PF mice lung tissues and TGF-β1-induced A549 cells. Beclin-1 is an important autophagy initiator protein that promotes the extension of autophagic membranes, which in turn converts LC3-I to LC3-II by binding to phosphatidylethanolamine, causing autophagosomes to expand and close.^50–52) Both beclin-1 and LC3-II were decreased in PF.⁵³⁾ Thereafter, LC3-II binds to P62 in autophagic vesicles, resulting in autophagic vesicles that fuse with lysosomes.^54,55) Increased level of P62 suggests a block in autophagic flux in PF.⁵⁶⁾ Increased beclin-1, LC3-II, and decreased P62 could indicate an increase in autophagy. The results of present study suggest that CGA can improve autophagy blockade both.

To further validate the role of CGA in the treatment of PF by activating autophagy to inhibit EMT, we selected rapamycin as a positive control and added the autophagy inhibitor 3-MA along with the CGA treatment. Rapamycin is a widely used activator of autophagy, and 500 nM rapamycin could induce autophagy in A549 cells.⁹⁾ 3-MA is a commonly used inhibitor of autophagy, and studies have shown that 5 mM 3-MA could significantly inhibit autophagy in A549 cells.⁵⁷⁾ The results showed that both CGA and rapamycin promoted autophagy and inhibited the TGF-β1-induced EMT process. In addition, the effects of CGA on TGF-β1-induced EMT and autophagy were abolished after 3-MA intervention. The promotion of autophagy was demonstrated to be the main pathway through which CGA exerts its effect of reducing EMT in epithelial cells.

In summary, CGA has significant therapeutic effects on PF, which are primarily attributed to the promotion of epithelial cell autophagy and reduction of EMT. This study demonstrates that modulating autophagy may be a novel approach to PF treatment and identifies CGA as a potential medication for the treatment of PF.

Acknowledgments

This work was supported by Nantong Health Care Commission Research Project (MA2020020).

Author Contributions

XJM carried out the experiments and XMX performed manuscript writing. JM and YLW provided experimental help. ZYH and TTW performed data analysis. JQZ and YW performed visualization. HZ provided ideas and technical guidance for the whole work and reviewed and edited the manuscript, JJH supervised the experiments. All authors contributed to the article and approved the submitted version.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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