Paraquat (PQ, 1,1’-dimethyl-4,4’-bipyridylium), also known as methyl viologen, has been widely used as a broad-spectrum, highly effective and inexpensive herbicide in agriculture worldwide.^[1⇓-3] However, PQ is highly toxic to humans and animals owing to its high toxicity,^[4] which has aroused great concern from the public. PQ can be absorbed through a variety of ways, including oral, inhalational, and transdermal ways, leading to toxic effects on multiple organs.^[5] PQ primarily accumulates in the lungs (6-10 times higher than plasma concentrations)^[6] because it can be actively transported into lung alveolar epithelial cells and Clara cells by the polyamine uptake system.^[7] Without effective antidotes, PQ-induced acute lung injury (ALI) and subsequent progressive pulmonary fibrosis are the primary causes of death.^[8]

Although great attention has been given to PQ poisoning and some progress has also been made, the mechanisms of PQ toxicity remain to be further elucidated. Accumulated evidence has suggested that reactive oxygen species (ROS) play key roles in PQ poisoning. PQ, as an electron acceptor, can induce numerous ROS generated in cells,^[9] which further leads to direct oxidative stress.^[10] Additionally, ROS would also trigger cell apoptosis as signal transduction molecules.^[11] Thus, direct ROS inhibition by antioxidants might be a potential effective way to ameliorate PQ injury. Mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs) and p38 MAPKs, are a group of serine-threonine protein kinases. MAPKs are vital in signal transduction from the cell surface to nucleus in response to a diverse array of extracellular stimuli.^[12] In addition, MAPKs have been reported to be activated by ROS.^[13] Moreover, it has been proven that suppressing the ROS/p38 MAPK pathway could reduce PQ-induced lung injury.^[9,14]

Arctigenin, a natural lignan compound extracted from Arctium lappa L (A. Lappa), has attracted researchers’ attention for its promising therapeutic effects in the past several decades.^[15] Numerous studies have reported the beneficial biological activities of arctigenin, including anti-oxidase,^[16] anti-inflammation,^[17] and antitumor activities.^[17] More interestingly, our previous study demonstrated that arctigenin attenuated PQ-induced pulmonary fibrosis by suppressing epithelial-mesenchymal transition via the Wnt3a/β-catenin pathway.^[18] Additionally, arctigenin has been shown to protect against lipopolysaccharide-induced lung injury in rats^[19] and mice.^[20] However, whether arctigenin can attenuate PQ-induced ALI and whether the ROS/p38 MAPK pathway is involved are still unknown. Thus, our present study was designed to investigate the effects of arctigenin on PQ-induced ALI and the underlying mechanisms.

Human lung epithelial A549 cells were purchased from Fudan IBS Cell Center, Shanghai, China. Cells were cultured at 37 °C in 95% air and 5% CO₂ in Dulbecco’s modified Eagle’s medium (DMEM) (HyClone, South Logan, USA) with 10% (v/v) fetal bovine serum (Invitrogen, Germany) and 1% (v/v) penicillin/streptomycin (Invitrogen, Germany). Subculture was performed with 0.25% trypsin/ethylenediaminetetraacetic acid (EDTA) solution (Invitrogen, Germany) after washing the cell monolayers twice with phosphate-buffered saline (PBS). Trypsin was then inactivated, and the cells were seeded into fresh T25 flasks.^[21]

Cell viability was assayed using a cell counting kit-8 (CCK-8) kit (Beyotime, China) according to the manufacturer’s instructions.^[22] Briefly, A549 cells were cultured in a 96-well plate and treated with PQ (Sigma-Aldrich, USA) at the indicated concentrations for 24 h with or without arctigenin (MedChemExpress, USA), while PBS was used as a control. After treatment, 10 μL of CCK-8 solution per well was added. The cells were then incubated at 37 °C for another 30 min, and the amount of formazan dye generated by cellular dehydrogenase activity was measured by absorbance at 450 nm with a microplate reader.

TUNEL assays were performed with the one-step TUNEL apoptosis assay kit (Beyotime, China) following the manufacturer’s instructions. Briefly, A549 cells were fixed in 4% paraformaldehyde in PBS and then permeabilized with 0.1% Triton X-100, followed by incubation with TUNEL working solution. The 4’,6-diamidino-2-phenylindole (DAPI) was used to stain and visualize all nuclei. Cy3-labelled TUNEL-positive cells were detected by fluorescence microscopy.

Mitochondrial depolarization in A549 cells was measured using a 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethyl benzimidazolo-carbocyanine iodide (JC-1) probe (Beyotime, China) according to the information described previously.^[23] In brief, cells were incubated with JC-1 staining solution (5 μg/mL) at 37 °C for 20 min, followed by rinsing with JC-1 staining buffer twice. JC-1, a cationic carbocyanine dye, exhibits potential-dependent accumulation in healthy mitochondria, where it forms J aggregates (red), while it remains a monomer (green) upon mitochondrial depolarization. The fluorescence of red aggregates and green monomers was captured by fluorescence microscopy, while the fluorescence intensity was quantified using ImageJ software.

To detect the level of superoxide anions, the dihydroethidium (DHE) (Beyotime, China) staining was performed following the manufacturer’s instructions. A549 cells were incubated with 5 μmol/L DHE at 37 °C for 30 min and then rinsed in PBS three times. Thereafter, the cells were analyzed by fluorescence microscopy, and the red fluorescence intensity was quantified using ImageJ software. Additionally, the 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) (Beyotime, China) assay was also carried out for the detection of oxidative species.

Immunoblotting studies were performed following procedures described in our previous reports with modifications.^[21,24] In brief, proteins were extracted from A549 cells using RIPA buffer (Beyotime, China) containing protease inhibitor cocktail (Roche, USA) and phosphatase inhibitor cocktail (PhosSTOP™, Sigma-Aldrich, USA). Protein samples (50 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then electrotransferred to nitrocellulose membranes. Then, the blocked membranes were incubated at 4 °C overnight with primary antibodies, including rabbit antibodies against p38 MAPK, phospho-p38 MAPK, ERK, phospho-ERK, JNK, and phospho-JNK (1:1000, Cell Signaling Technology, USA). Rabbit anti-GAPDH or anti-β-actin antibody (1:3000, Abways, China) was used as an internal control. The membranes were washed and then incubated with horseradish peroxidase (HRP)-labelled goat anti-rabbit or goat anti-mouse secondary antibody (1:10000, Jackson ImmunoResearch, USA) at room temperature for 1 h. The membranes were washed three times in tris-buffered saline with Tween-20 (TBST), and the bands were detected using enhanced chemiluminescence reagents (Millipore, USA) with a Tanon 5200 Chemiluminescent Imaging System (Tanon Science & Technology, China). The images were analyzed by ImageJ software to obtain integrated intensities.

Data were presented as the mean±standard deviation (SD). Statistical significance was determined by analysis of variance (ANOVA) followed by a post hoc Tukey’s test for multigroup comparison. Differences were considered statistically significant when a P-value <0.05.

To evaluate the protective effects of arctigenin in vitro, a PQ-induced A549 cell injury model was successfully established, and the results of the CCK-8 assay showed that A549 cell viability was inhibited by PQ in a dose-dependent manner (Figure 1A). Moreover, arctigenin showed no significant effect on the viability of A549 cells under control conditions (Figure 1B) but ameliorated PQ-induced inhibition of A549 cell viability in a dose-dependent manner (Figure 1C). Additionally, light microscopy indicated the protective effects of arctigenin on PQ-treated A549 cells, as reflected by cell number and morphology (Figure 1D). These data supported that arctigenin ameliorated PQ-induced A549 cell injury.

Cell death usually occurs upon toxic insults, and cell apoptosis plays an important role in PQ-induced lung injury.^[25] Thus, we further tested the effects of arctigenin on PQ-induced A549 cell apoptosis by TUNEL assay and JC-1 staining. The results of the TUNEL assay showed that arctigenin dramatically reduced the number of TUNEL-positive cells induced by PQ (Figure 2A). Consistently, the JC-1 staining results also revealed that PQ-induced mitochondrial membrane potential loss, a marker for early-stage apoptosis, was significantly ameliorated by arctigenin treatment (Figure 2B). The above data demonstrated that arctigenin alleviated PQ-induced cell apoptosis.

ROS are vital molecules in pathophysiological processes after PQ injury.^[26,27] On the one hand, ROS can directly cause oxidative stress damage,^[28] and on the other hand, ROS would serve as signaling molecules to trigger apoptosis.^[29] Therefore, we explored the role of arctigenin on ROS generation in PQ-treated A549 cells by DHE staining and DCFH-DA staining. The representative images and statistical results indicated that the ROS level was dramatically increased in A549 cells after incubation with PQ for 24 h, but co-incubation with arctigenin remarkably inhibited PQ-induced ROS generation (supplementary Figures 1 A and B). These results showed that arctigenin reduced PQ-induced ROS generation in A549 cells.

Generally, increased ROS production in cells would lead to the activation of MAPKs, a family of serine/threonine protein kinases that mediate fundamental biological processes and cellular responses to external stress signals.^[13,30] Moreover, the ROS/p38 MAPK pathway has been reported to participate in PQ-induced cell injury.^[9,14] Accordingly, we further tested whether arctigenin would affect the activation of MAPKs, and the immunoblotting results showed that arctigenin significantly suppressed PQ-induced p38 MAPK phosphorylation (supplemental Figure 2A). In addition, PQ also increased JNK but inhibited ERK phosphorylation, on which arctigenin had little effect (supplementary Figures 2B and C). These data suggested that the inhibition of p38 MAPK was mainly involved in the protective effects of arctigenin.

Our study provides evidence that arctigenin protects against PQ-induced lung epithelial injury in vitro. Arctigenin ameliorated cell viability inhibition and cell apoptosis induced by PQ. Mechanistically, arctigenin may exert its protective effects by suppressing PQ-induced activation of ROS/p38 MAPK signaling (supplementary Figure 3).

Arctigenin is the main active ingredient from A. Lappa, a herbal medicine widely used in China. In recent years, arctigenin has been extensively studied for its pharmacological properties, and numerous studies have reported the beneficial effects of arctigenin on anti-inflammation,^[31] antioxidative stress,^[16,17] antitumor,^[32] and antineuronal degeneration.^[33] PQ poisoning has been frequently reported in recent years. As a highly toxic herbicide, PQ causes toxicity to multiple organs, including the lung, kidney, liver, and brain, among which the lung is the most targeted.^[34] However, there is still little effective treatment for PQ-induced lung injury and subsequent fibrosis, which leads to high morbidity and frequently to death. A previous experiment has proven that arctigenin can ameliorate PQ-induced pulmonary fibrosis via suppressing the epithelial-mesenchymal transition.^[18] Additionally, arctigenin has also been demonstrated to alleviate the lipopolysaccharide-induced lung injury in rats^[19] and mice.^[20] In line with these previous studies, our present work also supports the benefits of arctigenin against PQ-induced lung epithelial injury, as reflected by the CCK-8 cell viability assay. This finding implies that arctigenin also protects against PQ-induced ALI and the early stage of PQ-induced lung toxicity. Thus, arctigenin might be considered a potential candidate drug for the treatment of PQ-induced lung poisoning.

We next investigated the potential mechanisms by which arctigenin exerted its protective effect against PQ-induced lung epithelial injury. It is generally considered that cell death would occur upon toxic insults, and apoptosis is one of the classical ways by which PQ causes lung epithelial cell death.^[24] Although arctigenin has been shown to induce apoptosis in several types of tumors and therefore has potential antitumor activity,^[35] the effect of arctigenin on PQ-induced lung epithelial cell apoptosis remains unknown. Thus, we tested the role of arctigenin in PQ-induced A549 cell apoptosis via a TUNEL assay and mitochondrial membrane potential assay, and the results showed that arctigenin dramatically reduced PQ-induced A549 cell apoptosis, which is not consistent with what has been found previously in tumors. This may result from the fact that these are two categories of diseases, the tumors and the organ injuries, because other studies in myocardial ischemia-reperfusion injury^[36] and brain injury^[37] have also demonstrated the anti-apoptosis properties of arctigenin. However, the underlying mechanisms of these differences remain to be further explored.

ROS, including the superoxide anion (O₂^-), hydrogen peroxide (H₂O₂) and the hydroxyl radical (HO•), are a group of chemical species that are formed upon incomplete reduction of oxygen.^[38] While PQ exerts its toxicity mainly through a process of redox cycling with superoxide anion production,^[39] ROS generation and oxidative stress have a central role in PQ poisoning. More importantly, excessive ROS would trigger the apoptotic signaling pathway.^[11,29] MAPKs, a group of serine-threonine protein kinases, play important roles in signal transduction in response to diverse extracellular stimuli.^[12] MAPKs have also been reported to be activated by ROS.^[13] In addition, it has been proven that PQ-induced activation of p38 MAPK plays a key role in the apoptotic pathway of A549 cells and that inhibition of ROS/p38 signaling could ameliorate PQ-induced lung injury.^[9,14] Thus, in our present study, we further checked whether ROS/p38 MAPK signaling was involved in the protective effect of arctigenin against PQ-induced lung epithelial injury. We found that arctigenin remarkably reduced PQ-induced ROS generation and p38 MAPK phosphorylation. This finding supports the previous concept that ROS/p38 signaling is vital in PQ toxicity.

However, we also realized that one of the limitations of our present work was the lack of in vivo experiments since only human lung epithelial A549 cells were used. It should be noted that what has been found in these cell experiments might not be equally applicable in animals or even in the clinic; thus, it would be more stringent to use in vivo studies to verify the protective effects of arctigenin.

Our present study shows that arctigenin attenuates PQ-induced lung epithelial A549 cell injury in vitro by suppressing PQ-activated ROS/p38 MAPK-mediated cell apoptosis, and arctigenin might be considered a potential candidate drug for PQ-induced ALI.

Funding: This work was supported by the National Natural Science Foundation of China (82172182 and 82102311); Social Development Projects of Jiangsu Province (BE2017720); Natural Science Foundation of Jiangsu Province (BK20190247); Science Foundation of Jiangsu Health Commission (H2018039); and Jiangsu Postdoctoral Research Foundation (2018K048A and 2020Z193).

Ethical approval: No animal or human subjects were involved.

Conflicts of interest: None.

Contributors: CL, ZRS, and MMW performed the experiments; ZZY, WZ, and YR provided help during the experiments; XQH, RL, and QL contributed to the manuscript revision and data curation; SNN supervised all experiments.

All the supplementary files in this paper are available at http://wjem.com.cn.