Original Article
Ephedrine alleviates bleomycin-induced pulmonary fibrosis by inhibiting epithelial-mesenchymal transition and restraining NF-κB signaling
2023 Volume 48 Issue 10 Pages 547-556
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2023 Volume 48 Issue 10 Pages 547-556
Pulmonary fibrosis is a lethal and progressive pulmonary disorder in human beings. Ephedrine is a compound isolated from Ephedra and plays a regulatory role in inflammatory response. This study focused on the anti-pulmonary fibrosis effect of ephedrine and its potential molecular mechanism. After a mouse model of pulmonary fibrosis was established through bleomycin (BLM) induction, the survival percentage, body weight, and pulmonary index were measured. Hematoxylin-eosin staining and Masson’s trichrome staining for lung tissues were performed to observe the pathological alterations. The viability of lung epithelial BEAS-2B cells, intracellular production of reactive oxygen species, and the levels of pro-inflammatory cytokines were examined by cell counting kit-8 assays, 2ʹ,7ʹ-dichlorofluorescein diacetate (DCF-DA) staining, and enzyme-linked immunosorbent assay, respectively. Immunofluorescence staining was performed to determine E-cadherin and vimentin expression after BLM or ephedrine treatment. The mRNA and protein levels of cytokeratin-8, E-cadherin, α-SMA, and vimentin were subjected to quantitative polymerase chain reaction and immunoblotting. Experimental results revealed that ephedrine treatment rescued the repressive impact of BLM on BEAS-2B cell viability, and ephedrine inhibited BLM-induced overproduction of reactive oxygen species and inflammatory response in BEAS-2B cells. Additionally, ephedrine suppressed epithelial-mesenchymal transition (EMT) process stimulated by BLM treatment, as demonstrated by the reduced α-SMA and vimentin levels together with the increased cytokeratin-8 and E-cadherin levels in BLM + Ephedrine group. In addition, ephedrine inhibited NF-κB and activated Nrf-2 signaling in BLM-treated BEAS-2B cells. Moreover, ephedrine ameliorated pulmonary fibrosis in BLM-induced mice and improved the survival of model mice. In conclusion, ephedrine attenuates BLM-evoked pulmonary fibrosis by repressing EMT process via blocking NF-κB signaling and activating Nrf-2 signaling, suggesting that ephedrine might become a potential anti-pulmonary fibrosis agent in the future.
Pulmonary fibrosis is a serious and chronic lung disease, and patients with idiopathic pulmonary fibrosis have a median survival between 2 and 4 years (Shenderov et al., 2021; Ballester et al., 2019; Karampitsakos et al., 2017). Two drugs, pirfenidone and nintedanib, have been approved by the FDA for slowing down the deterioration of lung function (Sgalla et al., 2020). For now, lung transplantation is the only viable option to prolong the lives of patients with pulmonary fibrosis. Hence, to develop novel and effective agents for the treatment of pulmonary fibrosis is of utmost importance.
The features of pulmonary fibrosis include excessive extracellular matrix deposition, fibroblast foci accumulation, and chronic inflammation (Hewlett et al., 2018). Evidence suggests that epithelial-mesenchymal transition (EMT) process is a contributing factor to the development of pulmonary fibrosis (Yang et al., 2023; Junjie et al., 2023; Ding et al., 2023). EMT refers to a process that epithelial cells transit into cells of mesenchymal phenotype, such as fibroblasts (Kyung et al., 2018). It is estimated that a third of fibroblasts of pulmonary fibrosis are originally epithelial cells (Peng et al., 2020). During the process, E-cadherin in epithelial cells reduces while α-smooth muscle actin (α-SMA) and N-cadherin in mesenchymal cells increases (Kyung et al., 2018). Uncontrolled inflammatory injury and excessive oxidative stress, which are mutually connected, are key drivers of EMT and subsequent pulmonary fibrosis (Ma et al., 2020). Previous studies have shown that inhibiting inflammatory responses and excessive oxidative stress can directly prevent EMT and the development of pulmonary fibrosis (Ma et al., 2020).
According to recent studies, the compounds extracted from Chinese herbal medicines have the potential to alleviate tissue damage and pulmonary fibrosis induced by abnormal inflammatory responses and EMT (Jia et al., 2019; Wang et al., 2021). Ephedrine is a compound found in plants of the genus Ephedra, which is known for its sympathomimetic property. Ephedrine is commonly used as an adrenergic agent (Magkos and Kavouras, 2004) and has been shown to regulate the inflammatory response in various diseases such as acute liver failure, allergic asthma, and cerebral ischemic stroke (Shi et al., 2021; Wu et al., 2014; Laccourreye et al., 2015). For example, ephedrine hydrochloride exerts protective effects on mice undergoing lipopolysaccharide challenge by promoting the secretion of anti-inflammatory factors such as interleukin (IL)-10 and reducing pro-inflammatory cytokines, such as tumor necrotic factor (TNF)-α (Zheng et al., 2012). Additionally, ephedrine hydrochloride represses peptidoglycan-triggered inflammatory injury by enhancing IL-10 secretion and reducing the production of pro-inflammatory cytokines in an experimental mouse model of peritonitis via regulating the PI3K/Akt/GSK3β pathway (Zheng et al., 2013). Moreover, ephedrine has been demonstrated to reduce nephrotoxicity and hepatotoxicity by inhibiting oxidative damage and genotoxicity in cisplatin-treated mice (Sioud et al., 2020). Furthermore, administration of ephedrine significantly alleviates histological impairments and pulmonary indexes in mice with chronic obstructive pulmonary disease by suppressing oxidative injury, inflammation, and apoptosis via blockage of endoplasmic reticulum stress (Wang et al., 2022). However, the role and mechanism of ephedrine in pulmonary fibrosis have not been clarified yet.
Consequently, the study was designed to investigate the functions of ephedrine in pulmonary fibrosis by establishing a mouse model of pulmonary fibrosis through intratracheal instillation of bleomycin (BLM). Additionally, an in vitro cell model of pulmonary fibrosis was established by treating human lung epithelial BEAS-2B cells with BLM to explore the molecular mechanism of ephedrine for mitigating pulmonary fibrosis. The study may extend our understanding of ephedrine in the alleviation of pulmonary fibrosis.
Human lung epithelial cells BEAS-2B were procured from the ATCC (Manassas, VA, USA). BEAS-2B cells were grown in Dulbecco's modified Eagle’s medium/nutrient mixture F-12 (icell-0005, iCell, Shanghai, China) supplemented with 10% fetal bovine serum (76294-180, AVANTOR, Australia) at a temperature of 37°C with 5% CO2. For cell treatment, BEAS-2B cells were pre-treated with 10 μg/mL ephedrine or dimethyl sulfoxide (DMSO) for 2 hr followed by 50 μM BLM treatment for 24 hr.
Cell counting Kit-8 assayBEAS-2B cells with ephedrine or DMSO treatment were plated into a culture plate (96-well, 1 × 104/well) for cell culture. At indicated timepoints (0, 3, 6, 12, 24, 48, and 72 hr), the diluted cell counting kit-8 (Dojindo, Japan) was added to the culture plate for another 2 hr cell incubation. The absorbance at 450 nm was measured using a spectrophotometer to evaluate cell viability.
Cellular reactive oxygen species (ROS) detectionThe oxidative-sensitive fluorescent probe 2ʹ,7ʹ-dichlorofluorescein diacetate (DCF-DA; Sigma-Aldrich, MO, USA) was adopted to detect intracellular ROS production. Cells with designated treatments were washed twice with phosphate buffered saline and were exposed to 5 µM DCF-DA for 0.5 hr at 37°C in the absence of light. A microplate reader was used to evaluate superoxide generation. The degree of green fluorescence observed reflects ROS level within the cells.
Biochemical measurementTo determine the concentrations of IL-8, IL-6, and TNF-α in the supernatant of BEAS-2B cells, human IL-8 ELISA kit (EK-H10356, EK-Bioscience, enzyme research, Shanghai, China), human IL-6 ELISA kit (EK-H10353, EK-Bioscience), and human TNF-α ELISA Kit (ab181421, Abcam, Cambridge, UK) were used following the protocols provided by the manufacturer.
ImmunoblottingFor protein extraction, BEAS-2B cells were treated with RIPA buffer containing 1% protease inhibitor and 1% phosphatase inhibitor at 4°C for 0.5 hr, followed by centrifugation at 12,000 × g for 15 min at 4°C to obtain the supernatant. The concentration of protein was determined using a BCA Protein Assay Kit (PA002-01A, Novoprotein, Suzhou, China) as per the manufacturer's protocol. For immunoblotting, the proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). The membranes were then blocked with non-fat milk powder for 10-15 min at room temperature, followed by incubation with primary antibodies against NF-κB (ab283688, 1:1,000), Nrf-2 (ab137550), Keap-1 (ab119403), p-NF-κB (ab194927), Lamin B (ab229025), and GAPDH (ab181602) at 4°C overnight. Next, the membranes were further incubated with HRP Anti-Rabbit IgG antibody (1:2,000). Immunoreactive blots were visualized using enhanced chemiluminescence (Millipore, USA). The dilution of GAPDH and secondary antibodies was 1:10,000, while the remaining antibodies were diluted to 1:1,000. All antibodies were purchased from Abcam (Cambridge, UK). The signal intensity of the target bands was analyzed using ImageJ software (NIH, USA), with GAPDH as a loading control.
Immunofluorescence stainingImmunofluorescence staining was conducted in accordance with a previous study (Min et al., 2022). The treated cells were fixed with 4% paraformaldehyde and blocked with 3% bovine albumin for 15 min. Then, the cells were incubated with primary antibodies targeting cytokeratin-8 (ab154301, 1:500, Abcam), E-cadherin (ab287970, 1:250, Abcam), α-SMA (ab202509, 1:200, Abcam), and vimentin (ab137321, 1:200, Abcam) overnight. Then, the cells were incubated with goat anti-rabbit secondary antibodies (ab150077, 1:500, Abcam) in the dark for 60 min. After washing, cell nuclei were stained with DAPI for 10 min. A fluorescence microscope (Nikon, Tokyo, Japan) was used to observe these cells.
Reverse transcription quantitative polymerase chain reaction (qPCR)The total RNAs in BEAS-2B cells were extracted using the RNAex Pro Reagent (Accurate Biology, Hunan, China) and then reverse transcribed into complementary DNAs using the Evo M-MLV RT Kit (Accurate Biology) following the product manuals. These complementary DNAs served as templates for qPCR, and qPCR was performed using the SYBR Premix Ex Taq Kit on the StepOne PCR system (Applied biosystems, Thermo Fisher Scientific, USA). The expression of cytokeratin-8, E-cadherin, α-SMA, and vimentin were normalized to the expression of GAPDH. The primer sequences used for qPCR are listed below:
cytokeratin-8 (forward: 5′-ACAAGGTAGAGCTGGAGTCTCG-3′, reverse: 5′-AGCACCACAGATGTGTCCGAGA-3′);
α-SMA (forward: 5′-ACTGAGCGTGGCTATTCCTCCGTT-3′, reverse: 5′-GCAGTGGCCATCTCATTTTCA-3′);
vimentin (forward: 5′-AGGCAAAGCAGGAGTCCACTGA-3′, reverse: 5′-ATCTGGCGTTCCAGGGACTCAT-3′);
E-cadherin (forward: 5′-GCCTCCTGAAAAGAGAGTGGAAG-3′, reverse: 5′-TGGCAGTGTCTCTCCAAATCCG-3′);
GAPDH (forward: 5′-TTGGTATCGTGGAAGGACTCA-3′, reverse: 5′-TGTCATCATATTTGGCAGGTT-3′).
Establishment of mouse model with pulmonary fibrosisMale C57BL/6J mice (8-week-old, 23-30 g, Vital River Laboratory Animal Technology, Beijing, China) were housed in a controlled environment (40-65% humidity, 25 ± 1°C, 12 hr/12 hr light/dark cycle) with free access to food and water. All experimental procedures and animal care were conducted following the guidance of the Ethics Committee of Wuhan Hospital of Traditional Chinese Medicine for minimizing the suffering of animals. The mice were stabilized for 3 days and then were divided into four groups (n = 12/group) at random: Con + DMSO, Con + Ephedrine, BLM + DMSO, and BLM + Ephedrine.
Pulmonary fibrosis was induced in mice by intratracheal instillation of BLM as described previously (Peng et al., 2020). The mice in the BLM + Ephedrine and BLM + DMSO groups were given ephedrine (10 mg/kg) or the same amount of DMSO once daily for 7 days via intragastric administration, followed by intratracheal administration of 50 μL BLM (3.0 mg/kg) for 14 days. The mice in Con + DMSO and Con + Ephedrine groups intragastrically received either 10 mg/kg ephedrine or the same amount of DMSO. All mice in each group were sacrificed on the 16th day after BLM treatment.
Pulmonary index determinationAfter euthanasia of the mice, the harvested pulmonary tissues were rinsed in cold isotonic saline and then immediately weighed. Pulmonary index was calculated according to the following formula: Pulmonary index = pulmonary wet weight/body weight × 100%.
Histopathological analysisThe lung tissues were treated with 4% polymethylaldehyde and examined for histopathology. Subsequently, the tissues were embedded in optimal cutting temperature compound and cut into 3-μm-thick sections using a freezing microtome. These sections were stained using haematoxylin and eosin (H&E) and Masson's trichrome staining. Three arbitrary areas were chosen in one section, and a total of three sections were selected randomly from each mouse for histopathological analysis.
Statistical analysisSPSS 19.0 software (SPSS Inc., Chicago, IL, USA) was used for statistical analyses. Data are presented as the mean ± standard errors of the mean (SEM), with p-value less than 0.05 deemed as statistical significance. Differences between two groups were compared using student’s t-test. One-way analysis of variance followed by a post hoc Tukey’s test was used for evaluation of differences among multiple groups. All experiments were biologically repeated three times.
To probe into the role of ephedrine (Fig. 1A) in pulmonary fibrosis, an in vitro cell model of pulmonary fibrosis was constructed by stimulating BEAS-2B cells with BLM. First, the impacts of ephedrine on the viability of BLM-induced BEAS-2B cells at different timepoints were detected. The results of cell counting kit-8 assays delineated that 50 μM BLM time-dependently inhibited BEAS-2B cell viability and significant reduction of cell viability was happened at 48 hr and 72 hr. Additionally, 10 μg/mL ephedrine notably reversed the repressive impact of BLM on cell viability at 48 hr and 72 hr (Fig. 1B). Substantial evidence has demonstrated that oxidative stress and inflammation are closely associated with the progression of pulmonary fibrosis. Hence, the production of intracellular ROS was examined using DCF-DA staining, and the results illustrated that BLM induced a high ROS level in BEAS-2B cells and the increase in ROS production was significantly eliminated by ephedrine and BLM co-treatment (Fig. 1C-1D). Additionally, the augmented concentration of IL-8, IL-6 and TNF-α in the supernatants of BLM-stimulated BEAS-2B cells was markedly suppressed upon co-treatment of ephedrine and BLM (Fig. 1E-1G). All these findings indicated that ephedrine attenuates oxidative injury and inflammation in BLM-stimulated BEAS-2B cells.
Ephedrine inhibits BLM-induced ROS overproduction and inflammatory response in BEAS-2B cells. (A) Ephedrine chemical structure is provided. (B) BEAS-2B cells co-treated with 50 μM BLM and 10 μg/mL ephedrine were cultured for 0, 3, 6, 12, 24, 48, and 72 hr, followed by examination of cell viability using the cell counting kit-8 kit. (C-D) BEAS-2B cells were pre-treated with 10 μg/mL ephedrine for 2 hr and then treated with 50 μM BLM for 24 hr. ROS production was evaluated by DCF-DA staining. (E-G) The levels of IL-8, IL-6, and TNF-α in cells treated with ephedrine and/or BLM were measured by corresponding ELISA kits. *p < 0.05, **p < 0.01 vs Con + DMSO group; +p < 0.05, ++p < 0.01 vs BLM + DMSO group.
To further investigate the role of ephedrine in pulmonary fibrosis, the effect of ephedrine on BEAS-2B cell morphology was observed. It was clearly observed in Fig. 2A that after BLM challenge, BEAS-2B cell morphology was changed from oval or round shape into long spindle shape with the enlarged intracellular space, and the change mediated by BLM was reversed by ephedrine treatment. Then, the influence of ephedrine on EMT process in BEAS-2B cells post BLM treatment was investigated. It is well-known that cytokeratin-8 and E-cadherin are biomarkers of epithelial cells, while α-SMA and vimentin are biomarkers of mesenchymal cells (Zhang et al., 2015). Immunofluorescence staining showed that there were enhanced fluorescence of vimentin and the weakened fluorescence of E-cadherin in BLM-treated cells, and the changes in vimentin and E-cadherin fluorescence intensity were reversed by ephedrine supplement (Fig. 2B). Consistently, BEAS-2B cells with BLM treatment showed low protein and mRNA expression of cytokeratin-8 and E-cadherin and high protein and mRNA expression of α-SMA and vimentin (Fig. 2C-2G). As expected, the levels of these EMT-related markers altered by BLM stimulation were rescued by ephedrine treatment (Fig. 2C-2G). Taken together, ephedrine restrains BLM-evoked fibrosis and EMT process of pulmonary epithelial cells.
Ephedrine restrains fibrosis and EMT in BLM-induced BEAS-2B cells. Cells were divided into four groups: Con + DMSO, Con + Ephedrine, BLM + DMSO, and BLM + Ephedrine. (A) The morphology of BEAS-2B cells was observed. (B) The expression of E-cadherin and vimentin in BEAS-2B cells was detected by immunofluorescence staining. (C-G) Immunoblotting and PCR analysis were performed for measuring protein and mRNA expression of cytokeratin-8, E-cadherin, α-SMA, and vimentin in BEAS-2B cells in four indicated groups. **p < 0.01 vs Con + DMSO group; +p < 0.05 vs BLM + DMSO group.
It has been reported that pulmonary fibrosis can be ameliorated by inactivating NF-κB (Peng et al., 2020). Nrf-2 activation contributes the transcription of antioxidants and thereby inhibits ROS production as well as consequent oxidative injury to pulmonary tissues (Harvey et al., 2011). Ephedrine has been demonstrated to have anti-inflammatory property by inhibiting NF-κB and activating Nrf-2 in rat models of cerebral ischemia injury (Shi et al., 2021; Li et al., 2021). Therefore, we wondered whether ephedrine can control NF-κB and Nrf-2 pathways in BLM-treated pulmonary epithelial cells. As presented in Figure 3A, ephedrine evidently decreased the phosphorylated level of NF-κB and reduced Keap-1 protein level while notably elevating Nrf-2 protein expression in BLM-exposed BEAS-2B cells. Consistently, nuclear translocation of NF-κB was obstructed by ephedrine while that of Nrf-2 was promoted by ephedrine in BEAS-2B cells in exposure to BLM (Fig. 3B). These data suggested that ephedrine can inhibit the NF-κB signaling and activate the Nrf-2 pathway in BLM-exposed BEAS-2B cells.
Ephedrine blocks NF-κB and activates Nrf-2 signaling in BLM-stimulated BEAS-2B cells. Mice in Con and BLM groups were treated with 10 μg/mL ephedrine or the same volume of DMSO for 24 hr. (A) The protein expression of p-NF-κB, NF-κB, Nrf-2, and Keap-1 in BEAS-2B cells in four experimental groups was examined by immunoblotting. (B) The protein expression of NF-κB and Nrf-2 in the nucleus of BEAS-2B cells in four groups was quantified using immunoblotting.
Afterwards, the impact of ephedrine on BLM-induced pulmonary fibrosis was explored in vivo. As Fig. 4A shows, the average survival rate of mice in BLM + DMSO group was reduced when compared to that in Con + DMSO or Con + Ephedrine group, and ephedrine treatment improved the survival of pulmonary fibrosis model mice. Additionally, the body weight of BLM-treated mice was significantly reduced in comparison to that in control groups (Fig. 4B). However, the loss of body weight caused by BLM administration was notably recovered by ephedrine (Fig. 4B). Moreover, there was a significant increase in pulmonary index in BLM-treated mice due to the reduction in body weight and increase in wet lung weight (Fig. 4C). However, the increase in pulmonary index was markedly reduced in model mice received ephedrine administration due to the elevated body weight and reduced wet lung weight (Fig. 4C). Overall, ephedrine alleviates BLM-triggered lung injury in vivo and ephedrine with safe dosage exerts damage to mice.
Ephedrine mitigates lung injury in BLM-induced mice. The mice were given ephedrine (10 mg/kg) or the same amount of DMSO once daily for 7 days via intragastric administration, followed by being intratracheally administrated with 50 μL BLM (3.0 mg/kg) for 14 days. (A) Survival rate of mice in each group was recorded at day 5, 10, and 15. (B) Body weight of mice in each group was measured every 2 days within 2 weeks. (C) The pulmonary index (pulmonary wet weight/body weight) in the indicated experimental groups was measured. N = 12/group. *p < 0.05, **p < 0.01 vs Con + DMSO group; +p < 0.05 vs BLM + DMSO group.
Histopathological assessment of pulmonary sections was performed to observe the effect of ephedrine on lung fibrosis in vivo. As shown in Fig. 5, representative images of H&E staining revealed that the pulmonary sections of control mice displayed normal alveolar spaces and alveolar septum thickening with normal reticular distribution around the alveoli. However, after BLM treatment, septum thickening was increased, alveolar collapse happened, and there were interstitial infiltration by fibroblasts and inflammatory cells in pulmonary tissues (Fig. 5). According to Masson’s trichrome staining, collagen deposition as well as perialveolar and interstitial fibrosis were existed in pulmonary tissue sections of BLM-stimulated mice, which is different from pulmonary tissues of the control group where reticulin were naturally distributed around the bronchi and alveoli (Fig. 5). Fortunately, these lesions in pulmonary sections were alleviated by daily administration of ephedrine to BLM-treated mice (Fig. 5).
Ephedrine ameliorates BLM-induced pulmonary fibrosis. The mice were randomly divided into four groups: Con + DMSO, Con + Ephedrine, BLM + DMSO, and BLM + Ephedrine. H&E and Masson’s trichrome staining for observing the pathological changes of lung tissues. N = 12/group.
Pulmonary fibrosis is a fatal lung disease with variable etiologies and limited treatment options (Parimon et al., 2021). Recently, more attention has been paid to the therapeutic effects of Chinese herbal medicines and their derived agents on pulmonary fibrosis (Jia et al., 2019). Ephedrine is the primary active constituent in ephedra and has been shown to exert an ameliorating effect on chronic obstructive pulmonary disease (Wang et al., 2022). In this study, we explored the anti-pulmonary fibrosis effect of ephedrine on BEAS-2B cells and C57BL/6J mice treated with BLM. Our results first manifested that ephedrine mitigated BLM-induced pulmonary fibrosis as evidenced by the inhibition of oxidative stress, inflammatory response, and EMT process in BLM-stimulated BEAS-2B cells via restraining NF-κB signaling and activating Nrf-2 signaling in vitro and the mitigation of lung injury in vivo. The findings suggest that ephedrine might become a potential agent for anti-pulmonary fibrosis treatment.
Oxidative stress, definitively engaged in pulmonary fibrosis pathogenesis (Ornatowski et al., 2020), is an unbalanced process predominantly caused by oxidant overproduction and the inhibited antioxidant ability of cells, leading to the generation of ROS, including peroxynitrite, hypochlorous acid, hydroxyl radical, superoxide radical, and hydrogen peroxide (Phan et al., 2021). It has been reported by previous studies that ROS can induce inflammatory response and elicit a crucial role in EMT and pulmonary fibrosis (Abais et al., 2015). Pulmonary fibrosis develops gradually through repeated cycles of damage and repair induced by inflammation and oxidative stress. Hence, the most effective way to combat pulmonary fibrosis is to reduce inflammation and oxidative injury (Ma et al., 2020). As an agent with anti-inflammatory and anti-oxidative stress potentials, ephedrine has been demonstrated to alleviate the hyperresponsiveness of lung tissue caused by reductions in airway remodeling and inflammatory response in asthmatic rats (Zhai et al., 2021). Ephedrine prevents mice against smoking-induced chronic obstructive pulmonary disease by suppressing ROS generation and inflammation (Wang et al., 2022). Our study illuminated that ephedrine evidently ameliorated BLM-induced pulmonary injury and improved the survival of BLM-treated mice in vivo. Additionally, ephedrine significantly reduced intracellular ROS production and the levels of IL-8, IL-6, and TNF-α in BLM-stimulated BEAS-2B cells. Collagen deposition is one of the characteristics of epithelial repair and is the root of fibrosis process after oxidative stress and inflammation (Hytti et al., 2015; Bitterman, 1992). In this study, through histopathological analysis, we found the notably attenuated deposition of fibrillar collagen in BLM-treated mice receiving ephedrine treatment. These findings indicated that ephedrine mitigates lung fibrosis by inhibiting oxidative injury and inflammatory response.
Uncontrolled inflammation and excessive oxidative stress are mutually connected and become key drivers of EMT and subsequent pulmonary fibrosis (Ma et al., 2020). EMT is a dynamic process in which epithelial cells transdifferentiate from epithelial phenotype to mesenchymal phenotype. While EMT process is fundamental in body development, the process in a disordered state may contribute to diseases such as organ fibrosis and cancer (Akrida et al., 2022; Marconi et al., 2021). The process of EMT is characterized by the significant alterations in cell morphology, increases in mesenchymal markers (e.g., vimentin and α-SMA) and decreases in epithelial markers (e.g., cytokeratin-8 and E-cadherin) (Zhang et al., 2015). Accumulating evidence highlights the important role of EMT in the pathogenesis of pulmonary fibrosis (Liu et al., 2022). In the present study, long spindle shape of BEAS-2B cells with large intracellular space was observed after BLM induction. Importantly, BEAS-2B cell shape in the context of BLM and ephedrine treatment was recovered to be oval or round. Additionally, the increases in vimentin and α-SMA as well as the decreases in cytokeratin-8 and E-cadherin caused by BLM exposure were reversed upon the treatment with ephedrine. These results suggested that ephedrine mitigates BLM-induced pulmonary fibrosis in vitro via inhibition of EMT.
As an extensively studied nuclear transcription factor, NF-κB is activated by either endogenous or exogenous stimuli. The activated NF-κB translocates from the cytoplasm to the nucleus and contributes to the transcription of factors implicated with inflammatory response, such as IL-6 and TNF-α (Liu et al., 2017). NF-κB activation is also closely associated with the pathogenesis of multiple pulmonary diseases, such as occupational lung disease and acute lung injury (Wang et al., 2021). The gradually increased expression of NF-κB in parallel with inflammation and pulmonary fibrosis development indicates its implication in the pathogenesis of lung fibrosis. In the present study, ephedrine treatment reduced NF-κB protein expression in the nucleus of BEAS-2B cells exposed to BLM induction, implying that ephedrine inhibits nucleus translocation of NF-κB and thus attenuates inflammatory response in BLM-induced BEAS-2B cells. Our finding is in line with that of a previous study revealing the mitigative impact of ephedrine on inflammatory responses and NF-κB inactivation in a rat model of focal cerebral ischemia (Shi et al., 2021).
Oxidative stress acts as a vital inducer of pulmonary fibrosis. Nrf-2 is a critical transcription factor for modulating oxidative stress via activation of downstream antioxidant proteins. It has been reported that Nrf-2 hinders EMT process in pulmonary fibrosis by inhibiting Snail expression and Numb expression (Zhou et al., 2016; Zhang et al., 2018). Additionally, ephedrine treatment upregulates nuclear Nrf-2 levels in lipopolysaccharide-induced BV2 microglial cells (Li et al., 2021). The present work revealed the reduced protein expression of Nrf-2 in the nucleus of BLM-stimulated BEAS-2B cells, and nuclear Nrf-2 level was rescued by ephedrine treatment. These results illuminated that ephedrine has the potential to restrain oxidative stress in BLM-treated BEAS-2B cells by promoting nuclear Nrf-2 translocation.
However, some limitations in this study are also worth mentioning. First, since the molecular mechanisms are complicated, the upstream molecules or other related signaling pathways associated with the ephedrine/NF-κB axis still need further exploration. Second, sample size would be expanded to enhance persuasion of our findings in subsequent experiments.
Conclusively, our study reveals that ephedrine suppresses BLM-induced pulmonary fibrosis by inhibiting EMT, oxidative stress and inflammatory response. Moreover, ephedrine exerts its protective role by activating the Nrf-2 signaling and blocking the NF-κB signaling. These data revealed that ephedrine has the potential to be a protective candidate for the treatment of pulmonary fibrosis.
We appreciate for the support provided by the Wuhan Municipal Health Commission (No.WZ22C58) for the study.
Conflict of interestThe authors declare that there is no conflict of interest.
