Polydatin (purity >99%) and resveratrol (purity >99%) were purchased from Guangzhou Honsea Sunshine Biotech Co., Ltd. Bleomycin (BLM) hydrochloride was obtained from Zhejiang Hisun Pharmaceutical Co., Ltd. Pirfenidone was acquired from Dalian Meilun Biotechnology Co., Ltd. Recombinant human transforming growth factor-β1 was obtained from PeproTech Inc.; it was diluted and stored following the manufacturer's protocol. Sodium carboxymethylcellulose (CMC-Na) was supplied by Sigma-Aldrich (Merck KGaA). The assay kits for measuring hydroxyproline (HYP) and malondialdehyde (MDA) contents and total superoxide dismutase (T-SOD) and myeloperoxidase (MPO) activities were supplied by Nanjing Jiancheng Bioengineering Institute. Enzyme-linked immunosorbent assay (ELISA) kits for tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-13 were purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd. Reverse transcription primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), collagen type I α 1 (Col1α1), epithelial cell cadherin (E-cadherin) and α-smooth muscle actin (α-SMA) were provided by Sangon Biotech Co., Ltd. Antibodies against β-actin, transforming growth factor-β1 (TGF-β1), phosphorylated Drosophila mothers against decapentaplegic protein homolog 2/3 (p-Smad2/3), Smad2/3, phospho-extracellular-regulated protein kinases 1/2 (p-ERK1/2) and ERK1/2 were supplied by Affinity Biosciences. Horseradish peroxidase (HRP) and goat anti-rabbit immunoglobulin G (H+L) were purchased from EarthOx, LLC. All other chemicals and reagents were at least of the analytic grade.

Human type II alveolar epithelial cell line A549 (American Type Culture Collection) was purchased from iCell Bioscience Inc. It was cultured in Roswell Park Memorial Institute-1640 (RPMI-1640) medium (Gibco; Thermo Fisher Scientific, Inc.) containing 10% (v/v) fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 1% (v/v) penicillin/streptomycin solution (Gibco; Thermo Fisher Scientific, Inc.) at 37˚C, in a humidified atmosphere of 5% CO₂.

The A549 cells were seeded in a 96-well plate at a density of 4x10⁴ cells/ml at 100 µl per well. Then, they were treated with polydatin (0-120 µM) for 24, 48, 72 and 96 h. Next, 20 µl of thiazolyl blue tetrazolium bromide (MTT; 5 mg/ml) was added to each well. Following incubation for 4 h, MTT was removed and 150 µl of dimethyl sulfoxide (DMSO) was added to each well. The 96-well plate was shaken on a microplate reader (Thermo Fisher Scientific, Inc.) for 10 min to dissolve the crystals completely. The absorbance was recorded at 490 nm using a microplate reader. The experiment was repeated three times.

The A549 cells were seeded in a 6-well plate at a density of 4x10⁴ cells/ml at 2 ml per well. Then, they were treated with 0, 10, 30 and 90 µM polydatin combined with TGF-β1 (10 ng/ml) for 96 h. The cells were acquired after 96 h. TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was added to extract the total RNA according to the manufacturer's protocol.

A total of 42, six-week-old specific-pathogen-free male Sprague-Dawley rats (180-220 g) were provided by Guangdong Medical Laboratory Animal Center (certificate no. SYXK2018-0085). The rats were maintained in the animal experimental center of Guangzhou University of Chinese medicine with five animals housed per cage. They were placed at a constant temperature of 24˚C, relative humidity of 65±15% and a 12-h light/dark cycle. They were given standard food and free drinking water. Animal health and behavior were monitored every day. All experimental protocols were in accordance with the regulations of the Animal Protection and Use Committee of Guangzhou University of Chinese Medicine.

The rats were given adaptive feeding for 1 week before starting the experiment. They were randomly divided into seven groups: Sham control, model, pirfenidone, resveratrol and polydatin low-, medium- and high-dose groups, with six rats in each group. BLM-induced IPF was performed as described in a previous study (29). The rats were anesthetized with 50 mg/kg pentobarbital sodium by intraperitoneal injection. Following anesthesia, the anterior neck region of the rats was shaved and disinfected. Blunt dissection was made to expose the trachea. Then, 5 mg/kg BLM hydrochloride was intratracheally instilled into the rats in the six BLM-induced groups. The sham control group received the same treatment with sterile saline. The rats were shaken vertically for 3 min for a uniform distribution. The subcutaneous tissue and skin were carefully sutured.

Then, 3 days following surgery (to minimize suffering and distress of the rats), the sham control and model groups were given 0.5% CMC-Na orally, the pirfenidone group was given 50 mg/kg pirfenidone, the resveratrol group was given 40 mg/kg resveratrol and the polydatin low-, medium- and high-dose groups were given 10, 40 and 160 mg/kg polydatin, respectively, once a day for 28 days. Both polydatin and resveratrol were dissolved in 0.5% CMC-Na solution. As described in a previous study (30), on the 28th day following BLM administration, the degree of IPF in rats was obvious and the vital signs were affected. Therefore, this was chosen as the humane endpoint. Following the last administration, the animals were euthanized with 200 mg/kg pentobarbital sodium by intraperitoneal injection. When the rats were anesthetized, the blood of the rats was drained. After draining the blood and arresting the cardiac and respiratory functions, the lung tissues were rapidly stripped for further study.

The lung tissues were fixed with 4% paraformaldehyde for 24 h at room temperature. The lung tissues were dehydrated with an alcohol gradient (75% alcohol 4 h, 85% alcohol 2 h, 90% alcohol 2 h, 95% alcohol 1 h and anhydrous ethanol 1 h). Xylene is used to make the tissue transparent. They were then immersed in melted paraffin at 65˚C for 3 h, before embedding in a frame filled with molten paraffin and allowed to solidify at -20˚C. Paraffin-embedded tissue blocks were cut into 4-µm sections. These sections were stained with hematoxylin and eosin (H&E) reagent, hematoxylin (0.5%) for 5 min, followed by eosin (0.5%) for 5 min; or with Masson's trichrome reagent, sequentially with potassium dichromate (10%) overnight, iron-hematoxylin (1%) for 3 min, Ponceau acid (1%) for 10 min, phosphomolybdic acid (1%) stain for 3 min and aniline blue (1%) for 6 min; all at room temperature and observed under a light microscope. The lung injury scores were calculated according to the degrees of interstitial inflammation, inflammatory cell infiltration, congestion and edema. The scores on these indicators ranged 1-4. The final lung injury score was the sum of these scores (31). The lung fibrosis changes were evaluated according to the modified Ashcroft method (grades 0-8) (32). At scores 1-3, the alveoli were partly enlarged and rarefied. Fibrotic masses appeared from score 4. The lung structure was severely damaged with the confluence of single fibrotic masses at score 5. Most of the lung structure was not preserved at score 6. At score 7, the alveoli were partially covered by fibrotic masses and at score 8, complete occlusion occurred (33).

Total RNA of the rat lung tissues was extracted using TRIzol^® reagent. The total RNA of cells (5x10⁵) and lung tissues was reverse transcribed to cDNA (reaction volume, 20 µl) using the HiScript II Q RT SuperMix (Vazyme Biotech Co., Ltd.). An Ultra-trace UV-Visible spectrophotometer (Invitrogen; Thermo Fisher Scientific, Inc.) was used to detect the concentration and purity of RNA. The sample variation was amplified and quantified (reaction volume, 20 µl) using an SYBR quantitative polymerase chain reaction Master Mix kit (Vazyme Biotech Co., Ltd.). cDNA was subjected to a temperature of 95˚C for 30 sec; 40 cycles at 95˚C for 10 sec and 60˚C for 30 sec; and then 95˚C for 15 sec, 60˚C for 60 sec and 95˚C for 15 sec. The sequences of the primer are shown in Table I. GAPDH was used as an internal reference. RNA extraction, cDNA synthesis and qPCR were performed according to the manufacturer's protocols. The relative gene expression of Col I, E-cadherin and α-SMA was calculated using the following formulas: ΔΔCq=(Cq_sample-Cq_GAPDH)-(Cq_control-Cq_GAPDH); fold change=2^-ΔΔCq (34).

The content of HYP in lung tissues was measured according to the hydroxyproline assay kit protocol (35). The data are expressed as microgram of HYP per milligram wet lung weight (µg/mg tissue).

The activities of T-SOD and MPO and the contents of MDA in lung tissues were determined by the hydroxylamine, hydrogen peroxide and thiobarbituric acid methods. The levels were determined by the colorimetric method following the manufacturer's protocols (Nanjing Jiancheng Bioengineering Institute).

Saline was added to the lung tissues (100 mg) at a ratio of 1:9. The tissues were then homogenized and centrifuged to remove the supernatant. TNF-α (cat. no. ml002859), IL-6 (cat. no. ml102828) and IL-13 (cat. no. ml003012) ELISA kits (Shanghai Enzyme-linked Biotechnology Co., Ltd.) were used to detect the expression of these proteins following the manufacturer's protocols.

To collect the total proteins, the lung tissues were homogenized using preconfigured radioimmunoprecipitation assay (RIPA) cracking liquid [containing RIPA lysis buffer, phenylmethanesulfonyl fluoride (PMSF), cocktail and phosphatase inhibitors A and B at ratios of 100:1:2:1:1]. The supernatant was collected following centrifugation at 14,000 x g for 10 min at 4˚C. The concentrations of proteins were measured using a bicinchoninic acid assay kit. Proteins were loaded at 50 µg per lane. The proteins were dispersed using 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes. The membranes were blocked for 1 h at room temperature in Tris buffer solution-0.1% Tween 20 (TBST) containing 5% skimmed milk powder. Subsequently, they were incubated with specific primary antibodies at 1:1,000 dilution [p-ERK1/2 (cat. no. AF1015), ERK1/2 (cat. no. AF0155) and β-actin (cat. no. AF7018)] and 1:2,000 dilution [p-Smad2/3 (cat. no. AF3367), Smad2/3 (cat. no. AF6367) and TGF-β1 (cat. no. AF1027)] overnight at 4˚C. The membranes were incubated for 1 h at room temperature in TBST containing HRP and goat anti-rabbit immunoglobulin G secondary antibody (cat. no. E03012001; EarthOx Life Sciences). The protein bands were detected using an enhanced chemiluminescence advanced kit (GE Healthcare). Quantity One (v4.6.2; Bio-Rad Laboratories, Inc.) was used for densitometry.

SPSS software (version 23.0, IBM Corp.) was used for data analysis. One-way analysis of variance and Fisher's least significant difference test were used to analyze the significance of different groups. Values represented as mean ± standard deviation P<0.05 was considered to indicate a statistically significant difference. ChemDraw Professional v16.0 was used to draw chemical structures. GraphPad Prism software (v7; GraphPad Software, Inc.) was used to draw the graphs.

The results demonstrated that the viability of A549 cells after administering polydatin (10-120 µM) for 24, 48, 72 and 96 h was more than 90%. This suggested that polydatin had low toxicity to A549 cells at 10-120 µM (Fig. 2).

Based on the results of the cell toxicity experiment, different concentrations (0, 10, 30 and 90 µM) of polydatin combined with TGF-β1 (10 ng/ml) were subsequently selected to investigate the effect of their independent or combined use on the phenotypic transformation of A549 cells for 96 h. The results showed no significant difference in cell morphology between the control and polydatin-administered groups. The epithelial cell phenotype in these two groups was anserine nephrite type. After 96 h, significant morphological differences were observed between the TGF-β1-treated and control groups; a number of the cells became spindle shaped in the TGF-β1 intervention group. Little difference in cell morphology was found between the polydatin (10 µM) combined with TGF-β1 group and the TGF-β1 intervention group during the experiment. However, the polydatin (30 and 90 µM) combined with TGF-β1 group showed significant morphological differences 96 h following the intervention; only a few cells were spindle shaped (Fig. 3).

The results of phenotypic observation showed that the morphological differences among the groups were most obvious at 96 h following administration. Therefore, at 96 h following culture, total RNA was extracted from the cells in the polydatin and TGF-β1 alone or combined administration groups. The epithelial or mesenchymal marker gene was detected to reflect the EMT of the cells. The RT-qPCR results demonstrated no significant difference in the changes in the expression levels of collagen I (Col I), E-cadherin and α-SMA between the control and polydatin (30 µM) treatment groups. However, the expression of E-cadherin was significantly downregulated in the TGF-β1 (10 ng/ml) intervention group (P<0.01) and the expression of α-SMA and Col I was significantly increased (P<0.01), showing statistically significant differences compared with the control group. TGF-β1 (10 ng/ml) intervention combined with polydatin (30 and 90 µM) administration demonstrated significant differences in the expression of Col I, E-cadherin and α-SMA compared with TGF-β1 (10 ng/ml) intervention alone (P<0.01). The administration of polydatin inhibited the reduction in E-cadherin and the increase in Col 1 and α-SMA induced by TGF-β1. This inhibition displayed a dose-dependent trend (Fig. 4).

In vitro experiments confirmed that polydatin significantly inhibited the TGF-β1-induced EMT of alveolar epithelial cells, which plays a crucial role in the pathogenesis of fibrosis. Therefore, a model of bleomycin-induced pulmonary fibrosis in vivo was established to verify whether polydatin had a protective effect on bleomycin-induced pulmonary fibrosis in rats. H&E staining revealed the most intuitive response to histopathological changes. The fibers in the tissue were stained blue by Masson trichome stain, directly reflecting the degree of organ fibrosis.

H&E staining showed that the model group had severe pulmonary structure collapse, alveolar structure consolidation, marked edema and congestion and inflammatory cell infiltration compared with the sham control group. Fortunately, pirfenidone (PFD), resveratrol (Res) and polydatin interventions reduced the pathological injury of lung tissue. They suppressed the alveolar wall damage and reduced inflammatory cell infiltration, congestion and edema (P<0.01). The effect of polydatin was dose-dependent. At the same dose, polydatin and resveratrol produced similar effects. However, higher doses of polydatin provided a stronger protective effect. All of these results are shown in Fig. 5A and B. Masson trichrome staining results showed that the lungs of rats treated with BLM had alveolar septal thickening and collapse, massive collagen deposition and fibrous hyperplasia compared with the sham control group (P<0.05). However, collagen deposition in the lungs was significantly reduced by pirfenidone (PFD), resveratrol (Res) and polydatin (P<0.01). Polydatin exerted the effect in a dose-dependent manner (P<0.05). The results are displayed in Fig. 5C and D.

The results demonstrated that the content of HYP, a collagen deposition marker, significantly increased in the model group compared with the sham control group (P<0.01). However, pirfenidone (PFD) and resveratrol (Res) downregulated the expression of HYP (P<0.01). Polydatin also significantly blocked this induction in a dose-dependent manner (P<0.01; Fig. 6).

Fig. 7 shows that BLM significantly stimulated the gene expression of interstitial markers α-SMA and Col I and decreased the gene expression level of epithelial marker E-cadherin compared with rats having no BLM administration (P<0.01). However, pirfenidone (PFD), high- and medium-dose polydatin and resveratrol (Res) treatment significantly ameliorated the magnitude of all these changes at the mRNA level (P<0.01).

BLM administration significantly elevated the levels of pro-inflammatory factors TNF-α, IL-6 and IL-13 in the lung homogenate compared with the sham control group. However, pirfenidone (PFD), resveratrol (Res) and polydatin (40 and 160 mg/kg) significantly decreased the elevated TNF-α, IL-6 and IL-13 levels (P<0.01) compared with the model group. Polydatin (10 mg/kg) also mitigated the lesions, but its effect was less compared with the other two doses of polydatin (P<0.01; Fig. 8).

Fig. 9 shows that BLM administration in rats caused increased oxidative stress as manifested by a significant decrease in T-SOD, and a significant increase in MDA levels and MPO activities in the lung tissues compared with the sham control group (P<0.01). Nevertheless, pirfenidone (PFD), resveratrol (Res) and polydatin (40 and 160 mg/kg) significantly overcame the BLM-mediated decrease in T-SOD and inhibited the BLM-mediated increase in MDA levels and MPO activities in the lung tissue (P<0.01) compared with the model group. Polydatin (10 mg/kg) also exerted the effect to a certain extent (P<0.05).

Fig. 10 shows that following BLM injection, the expression level of TGF-β1 and the phosphorylation of its downstream signals Smad2/3 and ERK1/2 were significantly increased in rats. Promisingly, polydatin was able to inhibit the elevation of the expression level of TGF-β1 and the phosphorylation of Smad2/3 and ERK1/2 in a dose-dependent manner (P<0.05).