Paroxetine protects against bleomycin-induced pulmonary fibrosis by blocking GRK2/Smad3 pathway

Drugs and reagents

Paroxetine (#HY-122272) and bleomycin (#HY-17565A) were obtained from MedChemExpress (MCE) Co. Ltd (Shanghai, China). Transforming growth factor-β1 (TGF-β1) (#SAB4502954) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies including Collagen I (#ab138492), α-SMA (#ab5831), GAPDH (#ab8245), GRK2 (#ab227825), P-Smad3 (#ab52903), Smad3 (#ab40854), P-Smad2 (#ab280888), and Smad2 (#ab40855) were provided by Abcam (Cambridge, United Kingdom). P-Smad3 (#10231-1-AP) was purchased from Proteintech Group, Inc (Rosemont, USA). The BCA assay kit was obtained from Pierce (Rockford, IL, USA).

Animals and treatment

C57/BL6 male mice (6-8 weeks) purchased from Experimental Animal Research Center, Hubei Center for Disease Control and Prevention were fed in a specific pathogen-free (SPF) room at the center. The laboratory was maintained under controlled temperatures (23° C) and a relative humidity of 46% with a 12 hour light/dark cycle.

Mice were randomly divided into 5 groups, the control group (saline), PF group (2 U/kg bleomycin), Paroxetine-Low (PRXT-L) group (2.5 mg·kg⁻¹·day⁻¹), PRTX-M (5 mg·kg⁻¹·day⁻¹), and PRTX-H (7.5 mg·kg⁻¹·day⁻¹). To mimic a murine pulmonary fibrosis model, mice were subjected to intratracheal administration of bleomycin (2 U/kg) after anesthetized with pentobarbital sodium (1%) on the basis of previous description [9]. The design scheme of the present experiment is displayed in Figure 1. From the beginning of bleomycin administration, different dosages of PRXT were administered intragastrically for 21 consecutive days. At the end of the study, pulmonary function and arterial blood gas were analyzed before lung tissues were harvested and weighed.

Figure 1. Flow chart presenting the experimental protocol of in vivo study.

The detection of pulmonary function and arterial blood gas

The pulmonary function of mice in the indicated groups was detected on the day 21^st using the FinePointe non-invasive testing system (FinePointe™ NAM, Data Sciences International, St. Paul, MN, USA). In detail, mice were fixed in a closed box equipped with an airflow monitoring apparatus on one end of the box. The non-invasive pulmonary function parameters including specific airway resistance (sRaw), specific airway conductance (sGaw), functional residual capacity (FRC), and minute volume (MV) were calculated from the airflow. Meanwhile, arterial blood was also analyzed via an automatic blood gas analyzer (ABL80, Radiometer, Copenhagen, Denmark) once it was collected from left ventricle puncture. The status of acid-base equilibrium of arterial blood gas was reflected by arterial oxygen partial pressure (PaO₂), arterial carbon dioxide partial pressure (PaCO₂), and sodium bicarbonate (HCO₃^-).

Cell culture and treatment

Primary murine lung fibroblasts from C57/BL6 mice (8 weeks) were isolated through collagenase-trypsin-DNase digestion according to previous description [10]. Lung fibroblasts seeded in 6-well cell culture dishes were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 μg/ml streptomycin, as well as 100 U/ml penicillin. Once the lung fibroblasts were passaged consecutively to the 3rd to 6th generations, they could be used for subsequent in vitro experiments. To induce the activation of lung fibroblasts, 10 ng/mL TGF-β1 was used to incubate for 48 hours. Meanwhile, different concentrations of PRXT (ranging from 0 to 50μM) were added to observe its effects on the activation of lung fibroblasts. Replication-defective adenoviral (Ad) vectors were employed to overexpress Smad3 in lung fibroblasts. In brief, lung fibroblasts were infected with Ad-Smad3 diluted in DMEM at 40 MOI. After infection for 24 hours, lung fibroblasts were starved with medium without FBS for 16 hours. After that, TGF-β1 and PRTX were added in medium.

H&E staining

Cleaned lung tissue specimens were fixed in 4% paraformaldehyde, followed by paraffin embedding and slicing into 4-μm-thick sections. Then the lung tissue sections were dewaxed and then rehydrated. The specimens were then stained with hematoxylin and eosin according to the reagent instructions. The staining of the sections was observed by light microscopy.

Masson staining

Lung tissue samples were first washed in PBS and then fixed in 4% paraformaldehyde. Paraffin-embedded specimens were sliced into 4-μm-thick section, which were then dewaxed and re-hydrated. Next, the lung tissue sections were stained with three stains according to reagent instructions, namely Hematoxylin, Ponceau S and Aniline Blue. Finally, images were captured through a light microscopy.

Reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA was first extracted from the tissue by the method trizol and tested for RNA purity and integrity. The extracted RNA was then reverse transcribed to produce cDNA according to the instructions of the corresponding kit. Using Light Cycler 480 SYBR Green Master Mix, the target fragment selected by the synthetic primers was amplified using the cDNA as a template, catalyzed by DNA polymerase, and normalized to Gapdh. PCR protocol comprises the following parts: an initial denaturation at 95° C for 5 minutes, followed by 30 cycles of 95° C for 20 seconds, 60° C for 25 seconds and 72° C for 45 seconds. Data were analyzed using the 2^-ΔΔcq method. Primer sets are provided in the Supplementary Table 1.

Western blot

Tissues and cells were harvested, lysed in RIPA buffer containing protease inhibitor cocktail and phosphatase inhibitor cocktail, sonicated and precipitated by centrifugation at 12,000 x g for 30 minutes at 4° C. The concentrations of protein were assessed using a bicinchoninic acid (BCA) protein assay kit. Protein samples (35 μg per lane) were separated on 10%-12% SDS-PAGE gels and transferred to PVDF membranes and blocked with 5% skimmed milk for 1 hour at room temperature. The membrane was then incubated with the appropriate primary and secondary antibodies at the appropriate level. The primary antibodies as well as their dilution ratio are shown as follows: Collagen I (1:1000), α-SMA (1:1000), GRK2 (1:500), GAPDH (1:1000), phosphorylated-Smad3 (p-Smad3) (1:500), Smad3 (1:500), p-Smad2 (1:500), Smad2 (1:1000), and Smad4 (1:500). The relative protein levels were normalized with the level of GAPDH. The membranes were scanned using an Odyssey infrared imaging system and the results were identified using Image J software.

Determination of oxidative stress

SOD activity, NADPH oxidase activity and MDA level in lung tissue samples and lung fibroblast were detected via commercial kits on the basis of the manufacturer's instructions.

Cell counting kit-8 (CCK-8)

A CCK-8 assay kit was employed to measure the cell viability of treated lung fibroblasts. A 100-ml cell suspension was isolated and added into a 96-well plate. Then the cell viability was detected using a commercial assay kit (#ab228554, Abcam) according to the instruction.

Data analysis

A log-rank test was used to draw survival curves. All data in our study are presented as mean ± standard deviation (SD) for the number (n) of experiments, which was analyzed using the software SPSS 23.3. Differences among three groups were analyzed by one way ANOVA with Tukey post hoc test while differences between two groups were analyzed by student’s unpaired t-test. Statistical difference was confirmed with P < 0.05.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

PRXT treatment preserved pulmonary function in bleomycin-induced mice

Mice injected with bleomycin (2 U/kg) presented with symptoms of pulmonary dysfunction evidenced by increased sRAW, decreased sGAW, reduced FRC, and increased MV in PF group compared with the Control group (Figure 2A–2D). Pulmonary function in PRXT-M group and PRXT-H group was significantly improved compared with that in the Control group, indicating that PRXT administration (5 mg·kg⁻¹·day⁻¹ and 7.5 mg·kg⁻¹·day⁻¹) attenuated bleomycin-induced pulmonary dysfunction. However, the lower PRXT dose (2.55 mg·kg⁻¹·day⁻¹) did not significantly prevent bleomycin-induced pulmonary dysfunction compared with the PF group (Figure 2A–2D). Arterial blood gas was further analyzed to examine pulmonary function and acid-base equilibrium. The results showed that PRXT administration (5 mg·kg⁻¹·day⁻¹ and 7.5 mg·kg⁻¹·day⁻¹) remarkedly restored PaO₂, PaCO₂, and HCO₃^- in bleomycin-induced mice while the lower PRXT dose (2.5 mg·kg⁻¹·day⁻¹) did not affect the status of arterial blood gas compared with the PF group (Figure 2E–2G). Taken together, these data demonstrated that PRTX administration ameliorated bleomycin-induced pulmonary dysfunction and preserved acid-base equilibrium of arterial blood.

Figure 2. PRXT treatment ameliorated lung pathological injury and improved survival rate in bleomycin-induced mice. (A–D) Analyses of pulmonary function parameters in the indicated groups including specific airway resistance (sRaw), specific airway conductance (sGaw), functional residual capacity (FRC), as well as minute volume (MV) (n=5). (E–G) Analyses of arterial blood gas including PaO₂, PaCO₂, and HCO₃^- in the indicated groups (n=5). *P < 0.05 vs. the Control group, ^#P < 0.05 vs. the PF group. PF, pulmonary fibrosis. PRTX-L, Paroxetine-Low dose (2.5 mg·kg⁻¹·day⁻¹). PRTX-M, Paroxetine-Medium dose (5 mg·kg⁻¹·day⁻¹). PRTX-H, Paroxetine-High dose (7.5 mg·kg⁻¹·day⁻¹).

PRXT treatment ameliorated lung pathological injury and improved survival rate in bleomycin-induced mice

H&E staining and Masson staining (Figure 3A) showed that bleomycin stimulation significantly promoted inflammatory response and collagen deposition in lung tissues, evidenced by higher inflammation score and fibrosis score compared with Control group (Figure 3B, 3C). PRXT administration (5 mg·kg⁻¹·day⁻¹ and 7.5 mg·kg⁻¹·day⁻¹) remarkedly inhibited inflammation score and fibrosis score in lung tissues from bleomycin-induced mice. In addition, we observed the effects of PRXT administration on 21-day survival rate in bleomycin-induced mice. The data showed that bleomycin stimulation remarkedly decreased survival rate in mice while PRXT administration (5 mg·kg⁻¹·day⁻¹ and 7.5 mg·kg⁻¹·day⁻¹) could restored 21-day survival rate in bleomycin-induced mice. Similarly, the lower PRXT dose (2.5 mg·kg⁻¹·day⁻¹) did not affect the survival rate in bleomycin-induced mice compared with the PF group (Figure 3D). These results indicated that PRXT treatment effectively reduced ameliorated lung pathological injury and improved survival rate in bleomycin-induced mice.

Figure 3. PRXT treatment ameliorated lung pathological injury and improved survival rate in bleomycin-induced mice. (A) Representative images of H&E staining and Masson staining in the indicated groups (n=5). (B, C) Inflammation score and fibrosis score based on H&E staining and Masson staining (n=5). (D) Survival rate of each group (n=10). *P < 0.05 vs. the Control group, ^#P < 0.05 vs. the PF group. PF, pulmonary fibrosis. PRTX-L, Paroxetine-Low dose (2.5 mg·kg⁻¹·day⁻¹). PRTX-M, Paroxetine-Medium dose (5 mg·kg⁻¹·day⁻¹). PRTX-H, Paroxetine-High dose (7.5 mg·kg⁻¹·day⁻¹).

PRXT treatment suppressed collagen synthesis, inflammation and oxidative stress in bleomycin-induced mice

Western blot (Figure 4A–4C) showed that bleomycin stimulation significantly upregulated the protein levels of Collagen I and α-SMA in murine lung tissues. PRXT administration (7.5 mg·kg⁻¹·day⁻¹) could inhibit the protein expression of Collagen I and α-SMA compared with PF group. RT-PCR (Figure 4D–4F) also unveiled that the mRNA levels of Tgf-β, Collagen I, and α-SMA in lung tissues were upregulated by bleomycin stimulation, which could be restored by PRXT administration (7.5 mg·kg⁻¹·day⁻¹). Also, we measured oxidative stress levels in the indicated groups. Bleomycin stimulation could decrease SOD activity and enhance NADPH oxidase activity in lung tissues, apart from upregulating MDA levels (Figure 4G–4I). RT-PCR also showed that bleomycin stimulation could significantly downregulate the mRNA levels of Txn-1, Txnrd1, and Gpx4 in lung tissue (Figure 4J–4L). As expected, PRXT administration (7.5 mg·kg⁻¹·day⁻¹) could inhibit oxidative stress in lung tissues compared with PF group. Also, we accessed the effects of PRTX-H treatment on hepatic and renal function in mice. As shown in Supplementary Figure 1A, 1B, PRTX-H treatment for 21 days showed no effects on serum creatinine and serum ALT, suggesting the therapeutic dose of PRTX displayed no hepatotoxicity and renal toxicity. Taken together, these results suggested PRXT treatment suppressed collagen synthesis, inflammation and oxidative stress in mice with PF.

Figure 4. PRXT treatment suppressed collagen synthesis, inflammation and oxidative stress in bleomycin-induced mice. (A–C) Representative western blotting images of Collagen I and α-SMA and their quantification in murine lung tissues (n=5). (D–F) Relative mRNA expression levels of Tgf-β, Collagen I and α-SMA in murine lung tissues (n=5). (G) SOD activity (n=5). (H) NADPH oxidase activity (n=5). (I) MDA level (n=5). (J–L) Relative mRNA expression levels of Txn-1, Txnrd1, and Gpx4 in murine lung tissues (n=5). *P < 0.05 vs. the Control group, ^#P < 0.05 vs. the PF group. PF, pulmonary fibrosis. PRTX-H, Paroxetine-High dose (7.5 mg·kg⁻¹·day⁻¹).

PRXT promoted the expression of GRK2 and Smad3 in lung tissues from bleomycin-induced mice

One previous study has reported that GRK2 levels were increased in lungs and isolated fibroblast cells during pulmonary fibrosis and GRK2 inhibition could decrease extracellular matrix (ECM) accumulation by downregulating Smad3 expression [3]. Herein, we next detected the expression of GRK2 and TGF-β/Smads pathway. RT-PCR showed that bleomycin stimulation significantly upregulated the mRNA expression of Grk2. PRXT administration (7.5 mg·kg⁻¹·day⁻¹) could inhibit the mRNA level of Grk2 in lung tissues from bleomycin-induced mice (Figure 5A). Meanwhile, we detected the mRNA levels of Smad3, Smad2, and Smad4. The data showed that bleomycin stimulation showed no effects on the mRNA levels of Smad3, Smad2, and Smad4 in lung tissues from bleomycin-induced mice compared with Control group (Figure 5B–5D). PRXT administration (7.5 mg·kg⁻¹·day⁻¹) significantly inhibited the mRNA level of Smad3 but showed no effects on the mRNA levels of Smad2 and Smad4. Western blot further showed that bleomycin stimulation significantly upregulated the protein levels of GRK2, P-Smad3 and P-Smad2 but displayed no effects on the protein levels of Smad3, Smad2, and Smad4 (Figure 5E–5K). Intriguingly, PRXT administration (7.5 mg·kg⁻¹·day⁻¹) significantly downregulated the protein levels of GRK2, P-Smad3 and Smad3 in lung tissues from bleomycin-induced mice, suggesting that the inhibitory effects of PRXT on P-Smad3 may be implicated with the decrease of total Smad3 in bleomycin-induced lung tissues. Collectively, these data further unveiled that PRXT-mediated inhibition of GRK2 decreased the mRNA and protein expression of Smad3 during pulmonary fibrosis.

Figure 5. PRXT promoted the expression of GRK2 and Smad3 in lung tissues from bleomycin-induced mice. (A–D) Relative mRNA expression levels of Grk2, Smad3, Smad2, and Smad4 in murine lung tissues (n=5). (E–K) Representative western blotting images of GRK2, P-Smad3, Smad3, P-Smad2, Smad2, and Smad4 and their quantification in murine lung tissues (n=5). *P < 0.05 vs. the Control group, ^#P < 0.05 vs. the PF group. PF, pulmonary fibrosis. PRTX-H, Paroxetine-High dose (7.5 mg·kg⁻¹·day⁻¹).

PRXT inhibited TGF-β1-induced activation of lung fibroblasts in a concentration-dependent manner

Next, we observed the protective effects of PRXT on TGF-β1-induced lung fibroblasts. Figure 6A showed that PRXT (ranging from 0 to 50 μM) did not affect cell viability of primary lung fibroblasts. In TGF-β1 (10 ng/mL)-induced lung fibroblasts, 10, 20 and 50 μM of PRXT could significantly inhibit the cell viability and the mRNA level of α-SMA (Figure 6B, 6C). And 20 and 50 μM of PRXT also decreased the mRNA level of Collagen I in TGF-β1-induced lung fibroblasts (Figure 6D). Taken together, these data disclosed that PRXT inhibited TGF-β1-induced activation of lung fibroblast in a concentration-dependent manner.

Figure 6. PRXT inhibited TGF-β1-induced activation of lung fibroblasts in a concentration-dependent manner. (A) Cell viability in primary lung fibroblasts treated with different concentrations of PRTX (n=5). (B) Cell viability in TGF-β1-induced primary lung fibroblasts treated with different concentrations of PRTX (n=5). (C, D) Relative mRNA expression levels of α-SMA and Collagen I in primary lung fibroblasts (n=5). *P < 0.05 vs. the PBS group, ^#P < 0.05 vs. the TGF-β1 group. PRTX, Paroxetine.

Smad3 overexpression offset PRXT-elicited protection against TGF-β1-induced activation of lung fibroblasts

To further confirm Smad3 was involved in PRXT-elicited protection against TGF-β1-induced activation of lung fibroblast, we used adenovirus to overexpress the expression of Smad3 in lung fibroblasts (Figure 7A). In this part, we observed the role of PRXT (50 μM) on lung fibroblast stimulated with TGF-β1 for 49 hours. As expected, PRXT (50 μM) treatment significantly inhibited the activation of lung fibroblasts and the mRNA levels of Collagen I and α-SMA. By contrast, Smad overexpression could restore the cell viability and collagen synthesis in TGF-β1-induced lung fibroblasts. We also found that the cell viability and collagen synthesis in TGF-β1 group showed no difference compared with TGF-β1-PRXT-Ad-Smad3 group (Figure 7B–7D). Also, we detected the oxidative stress level in the indicated groups. Smad3 overexpression offset antioxidation of PRXT in TGF-β1-induced lung fibroblasts, evidenced by decreased SOD activity and increased NADPH oxidase activity (Figure 7E, 7F). Finally, we also found that Smad3 overexpression showed no effects on the protein level of GRK2 in TGF-β1-induced lung fibroblasts treated with PRXT, indicating that GRK2 served as the upstream protein of Smad3 (Figure 7G, 7H). Collectively, these data further demonstrated that Smad3 overexpression offset PRXT-elicited protection against TGF-β1-induced activation of lung fibroblasts.

Figure 7. Smad3 overexpression offset PRXT-elicited protection against TGF-β1-induced activation of lung fibroblasts. (A) Representative western blotting images of Smad3 in primary lung fibroblasts transfected with Ad-Smad3. (B) Cell viability in the indicated groups (n=5). (C, D) Relative mRNA expression levels of Collagen I and α-SMA in the indicated groups (n=5). (E, F) SOD activity and NADPH oxidase activity (n=5). (G, H) Representative western blotting images of GRK2 and its quantification in the indicated groups (n=5). (I) Chemical structure of PRXT and the possible mechanisms of the protective effects of PRXT in pulmonary fibrosis. Arrowheads denote the upregulate while the “T” ending lines represent downregulate.