Stevioside attenuates bleomycin-induced pulmonary fibrosis by activating the Nrf2 pathway and inhibiting the NF-κB and TGF-β1/Smad2/3 pathways

Abstract

Objective: This study aimed to investigate the effects of stevioside (STE) on pulmonary fibrosis (PF) and the potential mechanisms. Methods: In this study, a mouse model of PF was established by a single intratracheal injection of bleomycin (BLM, 3 mg/kg). The experiment consisted of four groups: control group, BLM group, and STE treatment groups (STE 50 and 100 mg/kg). ELISA and biochemical tests were conducted to determine the levels of TNF-α, IL-1β, IL-6, NO, hydroxyproline (HYP), SOD, GSH, and MDA. Histopathological changes and collagen deposition in lung tissues were observed by HE and Masson staining. Immunohistochemistry was performed to determine the levels of collagen I-, collagen III-, TGF-β1- and p-Smad2/3-positive cells. Western blot analysis was used to measure the expression of epithelial-mesenchymal transition (EMT) markers, including α-SMA, vimentin, E-cadherin, and ZO-1, as well as proteins related to the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, nuclear transcription factor-κB (NF-κB) pathway, and TGF-β1/Smad2/3 pathway in lung tissues. Results: STE significantly alleviated BLM-induced body weight loss and lung injury in mice, decreased HYP levels, and reduced the levels of collagen I- and collagen III-positive cells, thereby decreasing extracellular matrix (ECM) deposition. Moreover, STE markedly improved oxidative stress (MDA levels were decreased, while SOD and GSH activity were enhanced), the inflammatory response (the levels of TNF-α, IL-1β, IL-6, and NO were reduced), and EMT (the expression of α-SMA and vimentin was downregulated, and the expression of E-cadherin and ZO-1 was upregulated). Further mechanistic analysis revealed that STE could activate the Nrf2 pathway and inhibit the NF-κB and TGF-β1/Smad2/3 pathways. Conclusion: STE may alleviate oxidative stress by activating the Nrf2 pathway, suppress the inflammatory response by downregulating the NF-κB pathway, and inhibit EMT progression by blocking the TGF-β1/Smad2/3 pathway, thereby improving BLM-induced PF.

Keywords:

• Stevioside
• pulmonary fibrosis
• oxidative stress
• inflammatory response
• epithelial-mesenchymal transition

1. Introduction

Pulmonary fibrosis (PF) is a chronic interstitial lung disease characterized by alveolar epithelial damage, alveolar inflammation and myofibroblast activation. The pathogenesis of PF is complex, and research has shown that oxidative stress, the inflammatory response, and epithelial-mesenchymal transition (EMT) are closely associated with the occurrence and progression of PF.^1–3 Oxidative stress can lead to PF by inducing epithelial cell damage and promoting apoptosis.⁴ Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key regulator of the antioxidant response in organisms and is a major transcriptional regulator that inhibits reactive oxygen species (ROS) production.⁵ Evidence suggests that reduced Nrf2 expression may be an important factor in the progression of PF.⁶ Heme oxygenase-1 (HO-1) is a cytoprotective enzyme that regulates oxidative damage by breaking down hemoglobin into bilirubin, Fe²⁺ and CO. HO-1 is a classical gene that is controlled by Nrf2.⁷ Studies have confirmed that Nrf2/HO-1 levels are strongly correlated with oxidative stress in BLM-induced PF.⁸ Chien et al. found that Sanghuangporus sanghuang could restore lung antioxidant defense and ameliorate BLM-induced lung fibrosis in mice by activating the Nrf2/HO-1 signaling pathway.⁹ Nuclear transcription factor-κB (NF-κB) is a multifunctional transcription factor that triggers inflammatory responses by activating the expression of inflammatory factors, chemokines, adhesion factors, and other factors.^10,11 The NF-κB signaling pathway is involved in the regulation of various fibrotic diseases.^12–14 Studies have reported significantly elevated oxidative stress parameters and inflammatory markers in PF patients compared to healthy individuals.¹⁵ Oxidative damage and inflammatory responses are also present in animal models of PF.^16,17 Transforming growth factor-β1 (TGF-β1) is a potent profibrotic factor that drives EMT by stimulating the phosphorylation of downstream Smad2/3, leading to the conversion of fibroblasts into myofibroblasts and increasing extracellular matrix (ECM) deposition, thereby accelerating PF progression.^18–20 Therefore, reducing oxidative stress, attenuating the inflammatory response, and inhibiting EMT may be effective strategies for treating PF.

Stevioside (STE) is a diterpene glycoside extracted from Stevia rebaudiana, which is known for its high sweetness and low calorie content. Due to these advantages, STE has been used as a sweetener in the food industry in several countries. STE has been reported to possess anti-inflammatory, antioxidant and anticancer properties.^21–23 The literature has shown that STE can improve isoproterenol-induced myocardial fibrosis in mice and unilateral ureteral obstruction-induced renal fibrosis in mice, and the mechanism may be related to reducing oxidative stress and inhibiting the NF-κB/TGF-β1/Smad pathway.^24,25 Further­more, STE has inhibitory effects on thio­acetamide-induced liver fibrosis.²⁶ Based on these research findings, we presume that STE may have inhibitory effects on PF. Therefore, in this study, we used BLM to establish a mouse model of PF and investigated the protective effects of STE against PF and the potential mechanisms. This study aimed to provide a theoretical basis for the use of STE as a therapeutic agent for PF.

2. Materials and methods

2.1. Chemicals and experimental animals

Stevioside was purchased from Chengdu Lemeitian Pharmaceutical Technology Co., Ltd. (Chengdu, China, DSTDT001701). Bleomycin sulfate was purchased from Hefei Bomei Biotechnology Co., Ltd. (Hefei, China, LB171208). Sixty male C57BL/6 mice (weighing 20∼25 g) were obtained from Changsha Tianqin Biotechnology Co., Ltd. [Certificate number SCXK (XIANG) 2019-0014]. Each cage contained 5 mice, which were housed under specific aseptic conditions (23 ± 2 °C and 12 h light/12 h dark).

2.2. Establishment of the PF model and experimental groupings

The mice were randomly divided into four groups (n = 15): the control group, bleomycin group (BLM), and STE treatment groups (STE 50 and 100 mg/kg). Other than those in the control group, the mice received an intratracheal injection of BLM (3 mg/kg) to establish a PF model, and control mice were injected in the same way with saline.²⁷ The STE-treated groups were administered STE orally (50, 100 mg/kg/d) starting the day after the bleomycin injection, and the treatment was administered for 21 consecutive days. Mice in the control and BLM-treated groups were given an equal volume of distilled water by gavage. Hydroxyproline (HYP) levels in lung tissue were measured. Serum levels of superoxide dismutase (SOD), glutathione (GSH), malondialdehyde (MDA) and nitric oxide (NO) were determined. Bronchoalveolar lavage fluid (BALF) was collected to determine tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β) and IL-6 concentrations. Left lung tissue was used for pathological examinations, and the remaining lung tissue was stored at −80 °C for Western blot analysis.

2.3. HYP assay

A HYP test kit (Nanjing Jiancheng Bioengineering Institute, A030-2-1) was used to detect HYP levels in the lung tissue in each group. The OD values of each sample at 550 nm were recorded and calculated according to the formula in the instructions.

2.4. Determination of SOD, GSH, MDA and NO levels in serum

Serum levels of SOD (Nanjing Jiancheng Bioengineering Institute, A001-3-2), GSH (A006-2-1), MDA (A003-1-2), and NO (A012-1-2) were measured according to the kit instructions.

2.5. ELISA

BALF levels of TNF-α (Wuhan Elabscience Biotechnology Co., Ltd., E-EL-M0049c), IL-1β (E-EL-M0037c) and IL-6 (E-EL-M0044c) were determined using ELISA kits according to the manufacturer’s instructions. The OD values of each sample were measured at 550 nm, and the concentrations of TNF-α, IL-1β and IL-6 were calculated based on the absorbance versus the concentration calibration curve. The ELISA results are expressed as pg/mL.

2.6. HE and Masson staining

Mouse left lung tissues were fixed in 10% formaldehyde for 48 h, dehydrated with graded ethanol, embedded in paraffin and sectioned at a thickness of 4 μm. HE and Masson staining was performed according to the instructions of the HE staining kit (Fuzhou Phygene Scientific Co., Ltd., PH0516) and Masson staining kit (Hefei Bomei Biotechnology Co., Ltd., BMB1624). Lung tissue morphology and collagen deposition were observed by light microscopy.

2.7. Immunohistochemical staining

Immunohistochemical experiments were performed using a Rabbit IgG SABC Kit (Wuhan Boster Biological Technology Co., Ltd., SA2002) according to the method described by Wu et al.²⁸ In brief, antigen retrieval was performed with sodium citrate antigen repair buffer (covered and boiled for 4 min at 100 °C, low heat for 10 min). Endogenous peroxidase activity was eliminated by treatment with 3% H₂O₂ for 10 min at room temperature. The slides were incubated with 5% BSA for 1 h at 37 °C and then incubated with rabbit polyclonal primary antibodies against collagen I (Affinity, AF0134), collagen III (Affinity, AF5457), TGF-β1 (Abcam, ab155264) and p-Smad2/3 (Affinity, AF3367) overnight at 4 °C. The next day, the slides were incubated with goat anti-rabbit IgG for 1 h at 37 °C. Finally, an Enhanced HRP-DAB Chromogenic Kit (Beijing Tiangen Biochemical Technology Co., Ltd., PA110) was used for chromogenic analysis. The nuclei were stained with hematoxylin and then differentiated with 1% hydrochloric acid ethanol. The slides were mounted with resin. Positive cells were brown or brownish yellow and were observed by light microscopy.

2.8. Western blot analysis

Fifty milligrams of lung tissue was added to 625 µL (containing 1% protease inhibitor) of RIPA lysis buffer or cell nuclear and cytosolic protein extraction reagent (Shanghai Beyotime Biotechnology Co., Ltd., P0027) and lysed on ice for 10 min, and the protein concentration was determined using a BCA protein assay kit (Shanghai Beyotime Biotechnology Co., Ltd., P0010). Forty micrograms of protein per sample was separated by 10% SDS–PAGE gels (80 V constant pressure for 30 min, then adjusted to 120 V until the target proteins were separated) and transferred to PVDF membranes (Merck Millipore Ltd. Germany, 200 mA constant flow, 2 h). The blots were then blocked with 5% nonfat milk in TBST buffer for 1 h at room temperature and incubated with primary antibodies against alpha-smooth muscle actin (α-SMA, Beijing Bioworld Technology, BS70000), NF-kB p65 (Beijing Bioworld Technology, BS1257), vimentin (Santa Cruz, sc-6260), E-cadherin (Santa Cruz, sc-52328), zonula occludens-1 (ZO-1, Santa Cruz, sc-33725), Nrf2 (Affinity, AF0639), p-IkBa (Affinity, AF2002), Smad2/3 (Affinity, AF6367), p-Smad2/3 (Affinity, AF3367), PCNA (Affinity, AF0239), HO-1 (CST, #82206), TGF-β1 (Abcam, ab155264), and GAPDH (Shanghai Beyotime Biotechnology Co., Ltd., AF1186) at 4 °C overnight, followed by exposure to secondary antibodies for 2 h at room temperature. Finally, the proteins were visualized using an Immobilon Western Chemiluminescent HRP substrate kit (Sigma–Aldrich Trading Co., Ltd., WBKLS0100) and analyzed using ImageJ software.

2.9. Statistical analysis

The results are presented as the means ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism 8.0.1 software. One-way analysis of variance (ANOVA) was used for multiple comparisons between groups. Dunnett Tukey’s test was used for pairwise comparisons. p < 0.05 was considered statistically significant.

3. Results

3.1. STE alleviated weight loss and lung injury in mice with BLM-induced PF

Within one week of BLM injection, body weight in the BLM group was significantly lower than that in the control group (p < 0.01) (Figure 1a). Although the body weights in the STE-treated groups were reduced, the degree of the reduction was less than that in the BLM-treated group. However, from Day 8 to Day 21, the body weights of mice with BLM-induced PF gradually stabilized, and STE treatment increased body weights to varying degrees (p < 0.05 or p < 0.01), which were still lower than those in the control group.

Figure 1. Effect of STE on bleomycin-induced pathological changes. (a) Effect of STE on body weight. (b) HE staining of the lung (200×). n = 6 for each group. *p < 0.01 vs. control; ^#p < 0.05 and ^##p < 0.01 vs. BLM.

HE staining showed that the alveolar structure was relatively normal, with thin intervals and no obvious inflammatory responses in the control group. After BLM stimulation, the tissue structure was disrupted, the septa became thickened, and many inflammatory cells accumulated in the alveolar cavity (Figure 1b). However, treatment with STE partially mitigated BLM-induced pathological injury and the inflammatory response in lung tissue. These results suggest that STE can alleviate weight loss and lung injury in mice with BLM-induced PF.

3.2. STE mitigated BLM-induced PF

Masson staining showed that there was little collagen in the pulmonary interstitium in control group mice, whereas a large area of blue staining was present in the pulmonary interstitium of BLM-treated mice, indicating high collagen production (Figure 2a). STE decreased BLM-induced collagen deposition in the pulmonary interstitium. We also determined HYP levels in lung tissue. As shown in Figure 2b, the BLM-treated group showed a significant increase in HYP levels compared to the control group (p < 0.01), while STE decreased HYP levels in a dose-dependent manner (p < 0.05 or p < 0.01). To investigate the effect of STE on ECM deposition in BLM-induced PF, we focused on collagen I and collagen III, which are primary markers of the ECM. Immunohistochemical staining revealed that the levels of type I and type III collagen-positive cells were increased in the BLM group compared with the control group (Figure 2c). However, STE treatment inhibited the increase in type I and type III collagen expression induced by BLM. Therefore, STE may reduce HYP levels, decrease ECM deposition, and thereby improve BLM-induced PF.

Figure 2. Effect of STE on pulmonary fibrosis. (a) Masson staining of the lung (200×). (b) The levels of HYP in the lung. (c) Immunohistochemical staining of collagen I and collagen III (400×). n = 6 for each group. *p < 0.01 vs. control; ^#p < 0.05 and ^##p < 0.01 vs. BLM.

3.3. STE inhibited BLM-induced oxidative stress

To investigate whether the antifibrotic effect of STE is related to its antioxidant activity, we measured the levels of SOD, GSH and MDA in mouse serum. As shown in Figure 3, BLM stimulation markedly reduced SOD (Figure 3a) and GSH (Figure 3b) activities and enhanced MDA (Figure 3c) activity compared to those in the control group (p < 0.01). STE treatment differentially increased SOD and GSH levels and decreased MDA overproduction induced by BLM (p < 0.01 or p < 0.05). These results demonstrate that STE can suppress BLM-induced oxidative stress in PF.

Figure 3. Effect of STE on oxidative stress. (a-c) Serum levels of SOD, GSH and MDA. n = 6 for each group. *p < 0.01 vs. control; ^#p < 0.05 and ^##p < 0.01 vs. BLM.

3.4. STE relieved the BLM-induced inflammatory response

To evaluate the effect of STE on BLM-induced inflammation, we measured the levels of TNF-α, IL-1β and IL-6 in BALF and NO in serum. As shown in Figure 4, the levels of TNF-α (Figure 4a), IL-1β (Figure 4b), IL-6 (Figure 4c) and NO (Figure 4d) were markedly elevated in the BLM group compared to the control group (p < 0.01). However, treatment with STE decreased the release of inflammatory cytokines and mediators induced by BLM to varying degrees (p < 0.05 or p < 0.01). These data indicate that STE protects against BLM-induced inflammation in PF.

Figure 4. Effect of STE on inflammatory mediators in BALF and serum. (a-c) The levels of TNF-α, IL-1β and IL-6 in BALF. (d) The levels of NO in serum. n = 6 for each group. *p < 0.01 vs. control; ^#p < 0.05 and ^##p < 0.01 vs. BLM.

3.5. STE blocked BLM-induced EMT

To validate the role of EMT in BLM-induced PF, we performed Western blot analysis to determine the protein expression of EMT markers. As shown in Figure 5, BLM upregulated the expression of the mesenchymal markers α-SMA and vimentin and downregulated the epithelial markers E-cadherin and ZO-1 compared to those in the control group (p < 0.01). In contrast, STE treatment ameliorated the BLM-induced changes in the expression of EMT markers (p < 0.05 or p < 0.01). These findings show that STE can inhibit EMT in BLM-induced PF.

Figure 5. Effect of STE on epithelial-mesenchymal transdifferentiation markers. (a) The expression of α-SMA, vimentin, E-cadherin and ZO-1 was measured by Western blotting. (b) Relative density values showing the expression of α-SMA, vimentin, E-cadherin and ZO-1. n = 3 for each group. *p < 0.01 vs. control; ^#p < 0.05 and ^##p < 0.01 vs. BLM.

3.6. STE activated the Nrf2 pathway in the lung tissue of mice with BLM-induced PF

To further elucidate the molecular mechanism by which STE alleviates BLM-induced oxidative stress, we examined the protein expression of Nrf2 and HO-1 in mouse lung tissues. The Western blot results showed that BLM significantly decreased the expression levels of Nrf2 and HO-1 compared to those in the control group (p < 0.01) (Figure 6). In contrast, STE had the opposite effect (p < 0.05 or p < 0.01). These data indicate that STE can suppress BLM-induced oxidative stress by activating the Nrf2 pathway.

Figure 6. Effect of STE on the Nrf2 pathway. (a) The protein levels of Nrf2 and HO-1 were measured by Western blotting. (b) Relative density values showing the expression of Nrf2 and HO-1. n = 3 for each group. *p < 0.01 vs. control; ^#p < 0.05 and ^##p < 0.01 vs. BLM.

3.7. STE suppressed the NF-κB pathway in the lung tissue of mice with BLM-induced PF

To investigate whether the NF-κB pathway was involved in the anti-inflammatory effect of STE, the protein expression levels of NF-κB p65 in the nucleus and p-IκBα in the cytoplasm were determined. As shown in Figure 7, nuclear translocation of NF-κB p65 and cytoplasmic expression of p-IκBα were notably increased in the BLM-treated group compared with the control group (p < 0.01), and treatment with STE attenuated this change to different extents (p < 0.05 or p < 0.01). These results suggest that STE can mitigate the BLM-induced inflammatory response by inhibiting activation of the NF-κB pathway.

Figure 7. Effect of STE on the NF-κB pathway. (a) The nuclear translocation of NF-κB p65 and cytoplasmic expression of p-IκBɑ were measured by Western blotting. (b) Relative density values showing the expression of NF-κB p65 and p-IκBɑ. n = 3 for each group. *p < 0.01 vs. control; ^#p < 0.05 and ^##p < 0.01 vs. BLM.

3.8. STE inhibited the TGF-β1/Smad2/3 pathway in the lung tissue of mice with BLM-induced PF

To further reveal the mechanisms by which STE protects against EMT in BLM-induced PF, we examined the expression of TGF-β1 and the phosphorylation level of Smad2/3 by immunohistochemistry and Western blotting. Immunohistochemical staining showed that the levels of TGF-β1- and p-Smad2/3-positive cells were increased in BLM-treated lungs compared with those in the control group, and these levels were decreased by STE treatment (Figure 8a). Similarly, Western blot analysis showed that BLM markedly increased the protein expression of TGF-β1 and Smad2/3 phosphorylation (p < 0.01), whereas treatment with STE attenuated these effects (p < 0.05 or p < 0.01) (Figure 8b and c). These results demonstrate that STE can inhibit BLM-induced EMT by blocking the TGF-β1/Smad2/3 pathway.

Figure 8. Effect of STE on the TGF-β1 and p-Smad2/3 pathways. (a) Immunohistochemical staining of TGF-β1 and p-Smad2/3 (400×). (b) The protein levels of TGF-β1, Smad2/3 and p-Smad2/3 were measured by Western blotting. (c) Relative density values showing TGF-β1 expression and phosphorylation of Smad2/3. n = 3 for each group. *p < 0.01 vs. control; ^#p < 0.05 and ^##p < 0.01 vs. BLM.

4. Discussion

PF is an incurable chronic inflammatory disease of the lung that is characterized by excessive matrix formation, structural remodeling, loss of lung function and ultimately death.²⁹ The population that is susceptible to PF includes elderly males who smoke and have hypertension, diabetes, or other complications.^30,31 The incidence, mortality, and prevalence of PF have been increasing annually, posing significant challenges to the global public health system.^32,33 Currently, the pathogenesis of PF remains unclear, and effective treatment options for PF are limited. In recent years, numerous studies have suggested that herbs can have antifibrotic properties.³⁴ STE is one of the main components of Stevia rebaudiana. Several studies have shown that STE is safe, has no side effects, and can be broken down by the intestinal flora into steviol, which can be excreted from the body.^35–37 Stevioside inhibits lipopolysaccharide (LPS)-induced oxidative stress and inflammation in acute lung injury mice and liver injury rats.^38,39 Additionally, STE can inhibit LPS-induced EMT in NRK-52E cells by downregulating the expression of proteins in the NF-κB/TGF-β1/Smad signaling pathway.⁴⁰ Therefore, this study used oxidative stress, the inflammatory response and EMT as starting points to explore the therapeutic role of STE in PF and elucidate its mechanisms.

The BLM-induced PF animal model is characterized by robust features and good reproducibility, making it widely used in pulmonary fibrosis-related research.⁴¹ In this study, we established the PF mouse model by a single intratracheal injection of bleomycin. The results showed that mice in the BLM group exhibited significant weight loss, lung tissue damage, increased interstitial thickness, and the accumulation of inflammatory cells in the alveolar cavity. However, STE alleviated weight loss and lung injury induced by BLM. Further experiments revealed that HYP levels in lung tissues, as well as the levels of type I collagen and type III collagen-positive cells, were increased in the BLM-treated group. These changes were improved by STE treatment, indicating that STE can reduce HYP levels, decrease ECM deposition and thereby ameliorate BLM-induced PF.

Studies have indicated that ROS generated by oxidative stress are involved in the pathogenesis of BLM-induced PF.^42,43 Bleomycin leads to excessive ROS production, resulting in an imbalance between oxidation and antioxidant defense. However, ROS can accelerate the progression of PF by reducing the activity of antioxidant enzymes, promoting the activation of growth factors, and influencing the expression of matrix metalloproteinases (MMPs) and protease inhibitors.⁴⁴ SOD and GSH are important members of the antioxidant defense system and are responsible for decomposing superoxide radicals into hydrogen peroxide and clearing them from the body. MDA, which is a marker of lipid peroxidation, leads to irreversible tissue damage. Nrf2 is a central regulator of the oxidative/antioxidative balance and maintains cellular redox homeostasis by stimulating the expression of downstream antioxidant substances such as HO-1 and glutathione peroxidase-1 (Gpx1).^45,46 Swamy et al. demonstrated that bleomycin could exacerbate oxidative stress in PF by inhibiting activation of the Nrf2/HO-1 signaling pathway.⁸ In the current study, we discovered that the protein expression of Nrf2 and HO-1 was downregulated in mouse lung tissues in the BLM group. Additionally, the activities of SOD and GSH were decreased, while the generation of MDA was increased in mouse serum after exposure to BLM, which was consistent with the findings reported by Liu et al.⁴⁷ However, STE treatment activated the Nrf2 pathway and alleviated oxidative stress. Jiang et al. also found that STE could ameliorate LPS-induced intestinal mucosal damage in chickens by inhibiting oxidative stress by activating the Nrf2/HO-1 pathway.⁴⁸ Based on these results, we presume that STE can regulate the oxidative/antioxidative balance by increasing Nrf2 pathway activity, thereby alleviating BLM-induced PF.

In addition to oxidative stress, the inflammatory response is a critical step in the development of PF.⁴⁹ When the body experiences inflammation, a series of proinflammatory factors, such as IL-6, IL-1β, and TNF-α, are released, and the levels of inflammatory mediators, such as iNOS/NO, are elevated. These inflammatory factors and mediators directly or indirectly participate in tissue fibrosis.^50,51 NF-κB is a key factor that regulates proinflammatory cytokines and inflammatory mediators. Under normal physiological conditions, NF-κB forms an inactive complex with IκB-α as a dimer and is present in the cytoplasm. When cells are stimulated, IκB kinases are activated, promoting the phosphorylation and ubiquitination of IκB, accelerating the degradation of IκB-α, and releasing the NF-κB dimer from the inactive complex, which allows it to translocate to the nucleus and trigger the transcription of target genes such as TNF-α, IL-1β, and IL-6.^52,53 In the present study, we found that the nuclear translocation of NF-κB p65 and the cytoplasmic expression of p-IκBα were significantly increased in the lung tissue of mice in the BLM group, and the levels of TNF-α, IL-1β and IL-6 in BALF and NO in serum were also markedly increased. STE treatment inhibited activation of the NF-κB pathway and reduced the release of inflammatory factors and mediators. The results demonstrate that STE can protect against BLM-induced PF by attenuating the inflammatory response by downregulating the activity of the NF-κB pathway.

EMT is accompanied by an increase in the expression of the mesenchymal markers α-SMA and vimentin and a decrease in the expression of the epithelial markers E-cadherin and ZO-1.⁵⁴ TGF-β1 is an EMT inducer that is involved in fibrosis in the lung, heart and kidney by promoting downstream Smad2/3 phosphorylation.^55,56 In this study, we used two different approaches to determine the levels of TGF-β1 expression and Smad2/3 phosphorylation, and the results showed that the expression of TGF-β1 and Smad2/3 phosphorylation were notably elevated in the BLM-treated group. We also observed that BLM upregulated α-SMA and vimentin expression and downregulated E-cadherin and ZO-1 expression, thereby inducing EMT. Conversely, STE treatment markedly decreased TGF-β1 expression and Smad2/3 phosphorylation and interfered with the EMT process. Thus, STE can improve PF by suppressing BLM-induced EMT by blocking the TGF-β1/Smad2/3 pathway.

5. Conclusion

Taken together, these results suggest that STE can have multiple protective effects in the treatment of BLM-induced pulmonary fibrosis. STE attenuates oxidative stress by activating the Nrf2 pathway and inhibits the inflammatory response by downregulating the activity of the NF-κB pathway. In addition, STE interferes with EMT by blocking the TGF-β1/Smad2/3 pathway. These pathways work synergistically to mitigate lung injury and decrease HYP levels and ECM deposition in the lung interstitium, which improves BLM-induced PF. The exact mechanism needs to be further investigated.

Ethics approval and consent to participate

The authors declare that the data supporting the findings of this study are available within the article. All procedures were performed in accordance with the protocol outlined in the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996) and were approved by the Committee on the Ethics of Animal Experiments of Wannan Medical College.

Declaration of interest

No potential conflict of interest was reported by the authors.

Data availability statement

The data set used and analyzed in this study can be obtained from the corresponding author upon reasonable request.

Additional information

Funding

This work was supported by the Innovation Training Program for College Students in Anhui Province in 2021 (No. S202110368079).