FIS (purity over 98%), Compound C (CC) and BLM were purchased from Selleck (Houston, United States). Hydroxyproline (HYP) Assay Kit was obtained from Nanjing Jiancheng Bio-Engineering Institute (Nanjing, China). Antibodies against GAPDH, AMPK, phospho-AMPK (p-AMPK), Smad3, phospho-Smad3 (p-Smad3), p65, and phospho-P65 were provided by Cell Signal Technology, Inc. (Massachusetts, United States). Antibodies against β-actin, TGF-β and alpha-smooth muscle actin (α-SMA) were purchased from Proteintech Group, Inc. (Wuhan, China). Antibodies against Collagen 1, p21 and p16 were gained from Abcam Technology Inc (Cambridge, United Kingdom).

Male C57BL/6J mice (25 ± 2 g), 8–10 weeks of age, were supplied by GemPharmatech Co. Ltd. (Jiangsu, China). All animals for this experiment were fed with standard diet and water at libitum and housed in a temperature-controlled room at 23 ± 2 °C and 50–60% humidity (12 h light/dark cycle) for 1 week before the experiments. All animals received care in accordance with the recommendations of the National Institutes of Health Guide for Care and Use of Laboratory Animals, and the experiment scheme was approved by the Committee on the Ethics of Animal Experiments of Sichuan University.

The animal model of BLM-induced pulmonary fibrosis was performed as described previously (Aoshiba et al., 2013). In brief, 70 mice were randomly divided into four groups. Control (CON) group: mice were given intra-tracheal administration of 50 μl of saline at day 0; FIS group: the saline-instilled mice were intragastrically administered with 100 mg/kg of FIS (suspended in 150 μl of 0.5% carboxy methyl cellulose sodium) starting at day 7 and treated once every other day for 3 weeks (Yousefzadeh et al., 2018); BLM group: mice were given intra-tracheal instillation of 50 μl of bleomycin (2.5 mg/kg); BLM + FIS group: On day 7 after BLM treatment, mice were orally administered with FIS once every other day for 3 weeks. On day 28, mice were euthanized by intraperitoneal injection of excessive pentobarbital sodium.

The BALF was harvested according to previously described (Wang et al., 2018). Briefly, the right lung was lavaged with 0.5 ml of iced phosphate-buffered saline (PBS) for three times, and fluid recovery is about 80% per time. The BALF were centrifuged at 1,000 g for 5 min, and then the supernatant was collected and stored at −80 °C.

The left lung tissues were fixed with 4% formalin, embedded in paraffin. Histological sections (6 μm) were stained with hematoxylin-eosin (HE) and Masson’s trichrome staining. According to previous literature, the dimensions of inflammation and pulmonary fibrosis were evaluated quantitatively (Szapiel et al., 1979). Immunohistochemistry was used to investigate the expression of p21. Paraffin-embedded lung sections were dewaxed with xylene, and endogenous peroxidase was removed with 3% H₂O₂. Antigen retrieval was performed by heating tissue sections, and non-specific binding sites were blocked with 5% BSA for 20 min. The primary anti-p21 antibody (1:200) was incubated at 4°C overnight, and the second antibody was incubated at room temperature for 1 h. Color was visualized via incubation with diaminobenzidine.

HYP content in lung tissues was determined by using a test kit according to the manufacturers’ instructions. About 30 mg of tissue samples were mixed with 1 ml of hydrolysase, placed in 60°C boiling water bath for about 20 min. After centrifugation of the supernatant at 3,500 rpm/min for 10 min, the absorbance at 550 nm was determined. HYP content was expressed as μg/mg.

A549 and human embryonic lung fibroblast (HELF) cells were purchased from American Type Culture Collection (Rockville, MD, United States). These cells were cultured in DMEM (Hyclone, United States) supplemented with 10% FBS (ZETA, United States) and 1× solution of penicillin-streptomycin (Hyclone) at 37°C in a humidified 5% CO₂ atmosphere.

To build the model of alveolar epithelial cell senescence and select the optimal concentration of BLM, A549 cells were stimulated with different doses of BLM for 3 days (Qiu et al., 2019). Next, we observed the roles of FIS and AMPK in alveolar epithelial cell senescence. Before BLM (5 μg/ml) was added to induce cell senescence, A549 cells in six-well plate were pretreated with or without 10 μM Compound C (CC, an inhibitor of AMPK) for 1 h (Liu et al., 2018). Subsequently, cells grew in the presence or absence of FIS (10 μM) for 3 days (Yousefzadeh et al., 2018).

In order to mimic SASP and observe its effects on epithelial-mesenchymal crosstalk, we used A549-derived conditioned mediums to further culture HELF cells. After treating with or without 5 μg/ml of BLM and 10 μM FIS for 3 days, the culture supernatants of A549 cells were replaced by fresh culture mediums without BLM, and A549 cells were cultured for another 2 days. Normal A549-derived conditioned mediums (N-CM), BLM-induced A549-derived conditioned mediums (B-CM) as well as BLM and FIS co-stimulated A549-derived conditioned mediums (BF-CM) were collected to analyze cytokines and further culture HELF cells with one half of concentration for 3 days. The protein expressions of collagen 1 and α-SMA in HELF cells were detected to reflect the fibrotic response.

SA-β-Gal staining was performed by using the senescence β-Galactosidase staining kit (Beyotime, Shanghai, China) as previously described (Tian et al., 2019). Frozen lung tissue sections and cell samples were fixed with 4% formaldehyde for 10–15 min at room temperature, washed three times with PBS for 5 min each time, and then placed in freshly prepared staining solution overnight at 37°C. To better show lung structure, the tissue sections were further stained with eosin. SA-β-Gal-positive cells (bluish green color) were counted in three random microscopic fields and expressed as % of total cells.

Lung tissues or cells were collected and lysed in ice-cold RIPA lysis buffer containing fresh protease and phosphatase inhibitor cocktails (MedChemExpress, United States). Protein samples were separated on 10% or 12.5% SDS-PAGE gels, and then transferred to methanol-activated PVDF membranes (Merck Millipore, Germany). After 1 h blocking with 5% nonfat milk, these membranes were incubated with primary antibodies overnight at 4°C. After membranes were washed three times, the corresponding secondary antibody was added for incubation at room temperature for 1–2 h. Protein expression was visualized using ECL (GE Healthcare, United Kingdom), and band intensities were measured using ImageJ software 6.0.

Total RNA was extracted from lung tissues using TRIzol reagent (Invitrogen Life Technologies, United States), reverse-transcribed to complementary DNA (cDNA) using ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo, Japan). Relative gene expression was quantified using iTaq Universal SYBR Green Supermix (Bio-Rad, United States). Table 1 shows the primer sequences, relative gene expression levels were normalized to β-actin and calculated using the 2^−ΔΔCt method.

The levels of IL-1β, IL-6, TGF-β, MMP-9, and TNF-α in BALF and culture mediums were detected by ELISA assay following the manufacturer’s instructions (NeoBioscience, Shenzhen, China).

Statistical analyses were performed using GraphPad Prism version 6.0 (GraphPad software, United States). All the original data were showed as mean ± SD. One-way analysis of variance and repeated-measures analysis of variance followed by the Tukey’s multiple comparison test were used for comparisons of multiple groups. The survival curve of each group was drawn using the Kaplan-Meier method. Values of p < 0.05 were considered statistically significant.

After intra-tracheal instillation of BLM, significant weight loss, survival rate reduction and increased wet/dry weight ratio of lung were noticed (Figures 1A–C). The seventh day after BLM administration is considered to be the beginning of the fibrotic phase (Li et al., 2019), FIS was intragastrically administered at day 7 for 21 days. BLM-induced weight loss and the increase in the wet/dry weight ratio were attenuated by FIS administration. There was no significant difference in the survival rate between the BLM group and BLM + FIS group, but a trend was revealed that FIS treatment would improve the survival rate of mice (Figure 1B).

In terms of histopathology, no significant pathological alteration in lung architecture was observed in the CON and FIS group. However, HE staining showed obvious morphologic changes of lung in the BLM group, including thickening of alveolar septum, alveolar structure destruction, and heavy inflammatory cell infiltration in alveolar space and pulmonary interstitium (Figure 1D). In addition, the lung tissues of BLM treated mice also displayed an increase in collagen deposition, which was evident from the increase of blue fiber bundles in Masson-stained lung sections (Figure 1D). As expected, treatment with FIS markedly reduced inflammatory cell infiltration, interstitial thickness and collagen deposition. In accordance with these observations, there was a statistically significant increase in the inflammation and fibrosis scores in the BLM group, while protection of FIS on lung microstructure was clearly reflected in the lower scores in the BLM + FIS group (Figures 1E,F).

Massive collagen secreted by myofibroblasts is the major source of ECM, while α-SMA is a biomarker protein of myofibroblasts (Li et al., 2019). HYP is one of the main components of collagen. As shown in Figures 2B–D, FIS administration obviously reduced the expressions of collagen 1 and α-SMA both at mRNA and protein levels as well as HYP content (Figure 1G) in lung tissues, suggesting that FIS suppressed transdifferentiation of fibroblasts to myofibroblasts and therefore decreased accumulation of collagen. Taken together, these data indicate that FIS can effectively alleviate the development of BLM-induced PF.

Prior studies have shown that BLM treatment can effectively induce cellular senescence in alveolar epithelial cells (Aoshiba et al., 2003). Cyclin-dependent kinase inhibitors p16 and p21, as well as SA-β-Gal activity are often used to confirm the appearance of senescence (Aoshiba et al., 2013; Qiu et al., 2019). As shown in Figure 2, both p16 and p21 gene expressions were apparently up-regulated in BLM-induced fibrotic mouse lungs, p21 protein was also significantly increased compared with the control group. Unfortunately, due to lacking of an ideal p16 antibody for mice (Demaria et al., 2014; Lehmann et al., 2017), we could not analyze p16 on protein level. In addition, using immunohistochemical staining, we found that the positive staining of p21 was mainly located in lung structural cells that morphologically resembled alveolar epithelial cells in BLM-induced mice (Figure 2A). Subsequently, we evaluated SA‐β‐Gal activity, a widely used indicator for detecting senescent cells, which will be dyed bluish green color. As previous findings demonstrated, an increase in the number of blue-green stained senescent cells was observed in fibrotic lung tissues, whereas that was barely seen in normal lungs (Figure 2A). However, both the strong SA-β-gal positive staining and the elevated expression of p21 and p16 induced by BLM were significantly alleviated by treatment with FIS compared to the BLM group.

To further explore the effects of FIS on inflammatory reaction, several common cytokines were analyzed by ELISA, which have also been suggested to be part of the SASP (Aoshiba et al., 2013; Qiu et al., 2019). In Figure 3, the levels of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α), profibrogenic cytokine (TGF-β) and metalloproteinase-9 (MMP-9) in the BALF of BLM-induced mice were significantly higher than those in healthy mice. In contrast, intervention with FIS dramatically decreased the release of these cytokines, except for MMP-9. Together, these results indicate that FIS can attenuate BLM-induced alveolar epithelial cells senescence and inflammatory responses in vivo.

TGF-β/Smad3 signaling plays a critical role in the development of pulmonary fibrosis. The phosphorylation of Smad3 was used to reflect Smad3 activity. As shown in Figures 3A,B, compared with the CON and FIS group, the protein levels of TGF-β and phospho-Smad3 (p-Smad3) were significantly increased in the BLM group. However, the increase was effectively reduced in the BLM + FIS group.

The activity of AMPK were evaluated by measuring the phosphorylation of Thr172 of AMPK. As illustrated in Figure 3, the levels of phosphorylated AMPK (p-AMPK) were obviously lower in the BLM group as compared to the CON and FIS group. However, FIS evidently increased the expression of p-AMPK thus to enhance AMPK activity in lungs compared with the BLM group.

Similarly, we detected the phosphorylation levels of p65 component of NF-κB to reflect NF-κB activity. Elevated levels of phospho-p65 (p-p65) were detected in the BLM group as compared to the CON and FIS group, while p-p65 protein levels in the BLM + FIS group were obviously lower than that in the BLM group (Figures 3A,B). These results suggest that FIS can suppress TGF-β/Smad3 signaling and regulate the activity of AMPK and NF-κB.

Refer to previous literatures (Aoshiba et al., 2003; Qiu et al., 2019; Tian et al., 2019), we used BLM to build a cellular senescence model. Different concentrations of BLM were used to stimulate A549 cells for 3 days. Treatment of A549 cells with BLM caused a dose-dependent increase in protein expression of p21 (Figure 4C), and this increase paralleled with a distinct, flat, and enlarged cell morphology (Figure 4A). Meanwhile, with the increase of BLM concentration, the SA‐β‐Gal activity was also up-regulated. However, there was no significant change between 5 and 10 μg/ml of BLM (Figure 4B). Therefore, the following experiments were continued with 5 μg/ml of BLM.

Further study found that the activity of SA‐β‐Gal in BLM-induced senescent A549 cells was remarkably increased as compared to the CON and FIS group, while FIS prevented this change (Figures 5A,B). Consistent with the results of in vivo experiments, the decreased protein expressions of p-AMPK and increased protein levels of p-p65 were detected in BLM-induced senescent A549 cells (Figure 5D), which were obviously improved by FIS, whereas such changes were reversed by AMPK inhibitor Compound C (CC). Similarly, FIS notably inhibited p21 and p16 protein expressions, which were partially blocked by CC (Figure 5C). These findings confirme that FIS can effectively relieve alveolar epithelial cell senescence partly through activating AMPK and inhibiting NF-κB activity.

Several widely-used cytokines, IL-1β, TNF-α, IL-6 and TGF-β were detected to reflect SASP from A549 cells in this study (Aoshiba et al., 2013; Tian et al., 2019). In Figure 6A, the levels of IL-1β, TNF-α, TGF-β, and IL-6 in the BLM-induced A549-derived conditioned mediums (B-CM) were higher than those in the normal A549-derived conditioned mediums (N-CM). All the above inflammatory cytokines, except for TNF-α, were significantly ameliorated in the BLM and FIS co-stimulated A549-derived conditioned mediums (BF-CM). As shown in Figures 6B–D, exposure of HELF cells to conditioned mediums for 3 days, increased protein expressions of collagen 1 and α-SMA were detected in B-CM stimulated HELF cells as compared to N-CM cultured HELF cells, whereas BF-CM obviously decreased the protein expressions of collagen 1 and α-SMA in HELF cells as compared to B-CM. These results suggest that FIS can ameliorate SASP from senescent alveolar epithelial cells, and therefore reduce transdifferentiation of fibroblasts to myofibroblasts as well as collagen deposition in fibroblasts.