Arctigenin prevents monocrotaline-induced pulmonary arterial hypertension in rats

The hallmark features of the development of pulmonary arterial hypertension (PAH) include the proliferation of pulmonary vascular smooth muscle cells, oxidative stress, inflammation, and pulmonary artery remodeling. Arctigenin is a bioactive component of Arctium lappa that exerts anti-inflammatory and antiproliferative effects in several diseases; however, its effects on pulmonary arteries are still unclear. This study aimed to investigate the efficacy of arctigenin to prevent PAH. Rats injected with monocrotaline (MCT) progressively developed PAH. Arctigenin treatment (50 mg per kg per day, intra-peritoneally) ameliorated right ventricular systolic pressure and pulmonary arterial remodeling, decreased the expression of inflammatory cytokines, and limited the proliferation of pulmonary vascular smooth muscle cells in lungs. Mechanistically, arctigenin effectively inhibited the MCT-induced elevation of NLRP3, caspase-1, and interleukin 1-beta expression in the lungs. These results indicate that arctigenin ameliorates MCT-induced PAH, at least in part, through exerting its anti-inflammatory, antioxidant, and antiproliferative effects, which inhibit the NLRP3 inflammasome signal pathway in rats.


Introduction
Pulmonary arterial hypertension (PAH) is a progressive and fatal disease featuring structural changes in the pulmonary vasculature, which may induce a severe increase in pulmonary arterial pressure, right ventricular (RV) failure, and death. 1 Although the pathogenesis of PAH is still unclear, evidence suggests that PAH development is associated with inammatory activation, endothelial dysfunction, and vascular remodeling, including cellular proliferation in both the intima and media. 2 Current therapies for chronic PAH, such as endothelin receptor antagonists and phosphodiesterase inhibitors, are mainly designed to reduce pulmonary arterial resistance by inducing vasodilation. However, these approaches mainly induce symptomatic relief and do not have a satisfactory clinical outcome. 3 The proinammatory cytokine interleukin 1-beta (IL-1b) has a vital role in PAH. 4,5 The NLRP3 inammasome, consisting of the NLR protein NLRP3, an adapter ASC, and pro-caspase-1, has been implicated in the activation of IL-1b and PAH. 6 Arctigenin (ARC) is a bioactive component of Arctium lappa, which has various biological properties including anticancer activity, antioxidant activity, and anti-inammatory activity. 7 Our previous study reported that ARC may exert neuroprotective effects in experimental stroke through inhibiting neuroinammation. 8 Moreover, a recent study demonstrated that ARC attenuated lipopolysaccharide (LPS)-induced acute lung injury in rats by inhibiting oxidative stress and inammation. 9 Most recently, ARC was reported to attenuate stroke by inhibition of the NLRP3 inammasome. 10 Above all, pharmacokinetic study of arctigenin in rat has shown that optimal dose of ARC at 50 mg kg À1 may deliver delivers effective levels of the compound to the rats. 11 Given these ndings, the present study was designed to investigate whether ARC attenuates PAH in rats by inhibiting the NLRP3 inammasome. Rats develop severe PAH aer a single injection of monocrotaline (MCT), which mimics several key aspects of the primary and secondary forms of human pulmonary hypertension, including vascular remodeling, proliferation of pulmonary artery smooth muscle cells (PASMCs), oxidative stress, and upregulation of inammatory cytokines. We investigated whether ARC exerts benecial effects on PASMC proliferation and inammation.

Animal model
Animal use protocols were approved by the Nanjing University of Chinese Medicine Animal Care and Use Committee. Male Sprague-Dawley rats (200-250 g) were provided by the Nanjing University of Chinese Medicine Laboratory Animal Center (Nanjing, China). This research protocol was approved by the Institutional Animal Care Committee at Jiangyin Hospital of Traditional Chinese Medicine (201534).
Adult rats were randomly assigned to the following groups (n ¼ 6 animals in each group) to receive either vehicle or ARC: (1) control animals (Control); (2) MCT (60 mg kg À1 SC, once)treated animals (MCT); or (3) MCT-treated animals receiving ARC (50 mg per kg per day, ip) aer MCT treatment (ARC). For the MCT group, MCT was given once 24 h before ARC treatment and saline was administered instead of ARC in the same manner for 28 days. The dose of ARC was based on previous studies, in which 50 mg kg À1 of arctigenin pretreatment signicantly attenuated LPS-induced acute lung injury in mice. 9

Assessment of hemodynamics
Four weeks aer MCT injection, rats were anesthetized with pentobarbital (60 mg kg À1 , ip). Right ventricle systolic pressure (RVSP) was evaluated by right heart puncture with a microtip Millar pressure catheter. 12 Heart rate was measured by an echocardiography machine (VisualSonics, Canada). Aer hemodynamic analysis, pieces of the le lung and isolated segments of the small intrapulmonary arteries were removed for histological evaluation or biochemical measurements.

Measurement of RV hypertrophy
Aer dissection of the heart, the RV wall was weighed aer separation from the le ventricular (LV) wall and the ventricular septum. The ratio of the RV free wall weight divided by the length of the tibia was calculated as an index of RV hypertrophy (which is unaffected by changes in body weight or LV mass). Lung tissues were xed in 4% paraformaldehyde, embedded in paraffin, and sectioned. The sections were examined by light microscopy (Inverted Fluorescence Microscope, Olympus, Japan) aer hematoxylin and eosin (HE) staining. Pulmonary arteries with an external diameter of 25-100 mm were selected for morphometric analysis. 12 Medial wall thickness was calculated as follows: medial thickness (%) ¼ medial wall thickness/ external diameter Â 100. Twenty randomly selected vessels from each rat were evaluated, and the mean value was calculated.

Cell proliferation and apoptosis assays
Immunouorescent labeling for a-smooth muscle actin was performed to visualize PASMCs. Cell proliferation was also assessed in the walls of distal pulmonary vessels by use of a monoclonal antibody against proliferating cell nuclear antigen (PCNA). Distal pulmonary vessels were identied by morphology. The percentage of PCNA-positive cells was calculated in 10 randomly chosen elds. To assess the antiproliferative effects of ARC in vitro, primary human PASMCs were stimulated in vitro with platelet derived growth factor (PDGF; 10 ng mL À1 ) in the presence or absence of ARC (1 mg mL À1 ). The inhibitory effect of ARC on cell proliferation was tested with a CCK-8 kit. In brief, PASMCs were seeded on 96-well plates at 80% conuence and treated with PDGF with or without ARC. Viable cells were detected by CCK-8, where the absorbance was assessed at 450 nm with a microplate reader (TECAN, Salzburg, Austria). In addition, the effects of ARC on cell apoptosis of PASMCs were investigated by TUNEL and quanti-ed by ow cytometry.

Measurement of oxidative stress levels
Lung tissues were collected and homogenized in ice-cold RIPA lysis buffer with a Polytron homogenizer. The homogenates were centrifuged at 3000 rpm at 4 C for 15 min. The supernatant was collected for superoxide dismutase (SOD) and malondialdehyde (MDA) measurements. SOD activity was measured with SOD assay kits (Jiancheng Bio-Technology, Nanjing, China) and was expressed as U mg À1 protein. The lipid peroxide product MDA, a marker for lipid peroxidation and a stable indicator of oxidative stress, was examined by thiobarbituric acid (TBA) assay using an MDA kit (Jiancheng Bio-Technology, Nanjing, China) and expressed as nmol mg À1 protein.

Statistical analysis
Data were normalized to the respective control mean values and expressed as means AE SEM. Statistical analyses of data were performed by analysis of variance followed by the Scheffe's post hoc test for multiple comparisons. A value of P < 0.05 was considered statistically signicant. MCT-treated rats showed a reduced heart rate compared with Controls; however, no differences were found between untreated MCT and ARCtreated counterparts. Data are expressed as the mean AE SEM. *P < 0.05 vs. Control; #P < 0.05 vs. no ARC.

Results
ARC prevents the MCT-induced increase in RVSP, RV hypertrophy, and pulmonary artery remodeling MCT-exposed rats developed signicant pulmonary hypertension 28 days aer MCT injection. ARC treatment signicantly prevented RVSP in MCT-injected rats over a 4 week period (Fig. 1A). In the MCT groups, signicant RV hypertrophy and increased medial thickness of pulmonary arteries developed as a consequence of PAH. These pathological changes were attenuated by ARC treatment (Fig. 1B-D). MCT-treated rats showed a reduced heart rate compared with Controls; however, no differences were found between untreated MCT and ARCtreated counterparts (Fig. 1E).

ARC inhibits the proliferation of PASMCs
In the MCT groups, a markedly increased smooth muscle cell mass was observed in the small pulmonary arteries at 28 days aer MCT injection. ARC treatment prevented the increase in PASMC mass in the vessels of MCT-treated rats ( Fig. 2A).
Medial hypertrophy of pulmonary resistance vessels occurred in parallel to an increased number of PCNA-positive proliferating vascular cells in MCT-induced pulmonary hypertension. However, in line with normalization of the PASMC mass, the number of PCNA-positive cells was signicantly reduced in ARC-treated animals ( Fig. 2B and C). In vitro experiments indicated that ARC prevented PDGF (10 ng mL À1 for 48 h) and stimulated the proliferation of cultured PASMCs (Fig. 3A). At the concentration range in which the antiproliferative effects were evident, ARC did not induce apoptosis in PASMCs (Fig. 3B).

ARC attenuates oxidative stress in the lungs
Ample evidence suggests that oxidative stress has a causal role in PAH. 13 The inhibition of oxidative stress by ARC (50 mg per kg per day) was associated with the preservation of SOD activity, as well as the downregulation of MDA. SOD activity was inhibited by MCT, and ARC treatment reversed this inhibition (Fig. 4A). Furthermore, as shown in Fig. 4B, MCT induced an elevation of lung MDA levels, whereas ARC inhibited this elevation.

ARC attenuates inammatory gene expression
The mRNA expressions of IL-1b, IL-6, MCP-1, and ICAM-1 in the lungs of MCT-treated rats were upregulated in MCT-induced PAH rats, whereas ARC treatment signicantly attenuated the expression of each inammatory marker (Fig. 5A-D).

ARC inhibits inammasome activation in MCT-induced PAH rats
It was reported that NLRP3 inammasome activation was tightly associated with PAH. As shown in Fig. 6, compared with the Control group, the MCT group had signicantly increased NLRP3, caspase-1 p20 (active subunit), and IL-1b protein levels, whereas ARC treatment reduced these protein levels in the lungs. These ndings indicated that ARC may reduce inammation in rats with MCT-induced PAH partly by inhibiting NLRP3 inammasome activation.

Discussion
ARC, an extract from Arctium lappa L., has a variety of pharmacological activities in the systemic circulation, such as cytoprotective, antioxidative, anti-inammatory, and vasoprotective properties. 14 Herein, we demonstrate that ARC treatment signicantly prevented pulmonary artery remodeling, the reversed mean pressure of pulmonary artery elevation, and RV hypertrophy in MCT-treated rats. Moreover, our results suggest that ARC may attenuate the progression of MCT-induced PAH by reducing the expression of inammatory cytokines, inhibiting the proliferation of pulmonary arterial smooth muscle cells, decreasing oxidative stress, and downregulating the NLRP3 signal pathway in lungs.
In agreement with human pulmonary hypertension, marked medial wall thickening was observed in MCT-induced pulmonary hypertension rats. We found a marked reduction in the progression of pulmonary hypertension and medial wall thickening in response to ARC treatment ( Fig. 1A-C). Accordingly, adaptive hypertrophy in the right ventricle of MCT-treated rats was also prevented by ARC (Fig. 1D). Therefore, the present results indicate that ARC treatment prevents pulmonary arterial remodeling and exerts a cardiopulmonary protective effect, at least in the MCT-induced PAH rat model.
The aberrant proliferation of PASMCs is a pathological hallmark of PAH. 15 The present study shows that smooth muscle cell proliferation was markedly increased in pulmonary resistance vessels of MCT-treated animals. ARC treatment induced near normal vessel morphology and inhibited pulmonary arterial smooth muscle proliferation ( Fig. 2A-C). Our in vitro study demonstrated that ARC prevented PDGF-induced proliferation in cultured PASMCs without increasing apoptotic cell death in PASMCs (Fig. 3). Therefore, it is likely that the inhibition of pulmonary arterial smooth muscle proliferation and vascular remodeling is predominantly attributed to the direct effect of ARC.
Increasing evidence supports an important role for inammation in the development and progression of human This journal is © The Royal Society of Chemistry 2019 pulmonary hypertension and experimental animal models. 16 For example, in various forms of clinical pulmonary hypertension, there is increasing recognition of inammation on the basis of evidence including increased plasma levels of inammatory cytokines and the pulmonary inltration of inammatory cells. 17 Similarly, in animal models of pulmonary hypertension, increased numbers of macrophages have been described in mouse and rat lungs. 17 These processes might contribute to vascular remodeling, collagen deposition, and PASMC proliferation and migration in PAH, and eventually lead to pulmonary resistance and subsequent right heart failure. IL-1b is a prototypic multifunctional cytokine that has a vital role in the pathogenesis of PAH. 18,19 In agreement with previous ndings, IL-1b was increased in MCT-treated rats, and other proinammatory cytokines were also upregulated (Fig. 4). In addition, ARC, a natural anti-inammatory agent, has shown apparent protective effects in LPS-induced acute lung injury mouse model at a dose of 50 mg per kg per day. 9 Here, we found that ARC treatment in MCT-treated rats normalized the expression of inammatory cytokines (Fig. 4).
In addition to inammatory reactions, incremental evidence suggests that oxidative stress may impair the pulmonary vascular endothelium and induce the proliferation of vascular muscle cells in rat models of PAH. 3 Moreover, increased oxidative stress was observed in PAH patients, while the reduction of oxidative stress was associated with an attenuation of the clinical symptoms. 20 It was previously reported that ARC reduced oxidative stress in many diseases, in line with our nding that treatment with ARC signicantly downregulated MDA levels and increased SOD activity (Fig. 5A and B). Therefore, the antioxidative effects of ARC may also contribute to blocking the progression of MCT-induced PAH in rats.
The mechanisms by which the antioxidative effects of ARC interfere with inammatory processes in the lung are not well understood. Recently, several studies have highlighted the role of NLRP3 inammasome in several types of lung injury. NLRP3-decient mice showed a suppressed inammatory response and blunted lung epithelial cell apoptosis in hyperoxia-induced acute lung injury. 21 In addition, ROS is central to NLRP3 activation, which is critical for the release of caspase-1 and IL-1b. 6,22 Of note, the roles of inammation, caspase-1, and IL-1b in the pathogenesis of experimental PAH have been reported by many studies. 23 Recently it was shown that ARC effectively blocked NRLP3 activation in neurons, thus protecting oxygen glucose deprived neurons. 10 In accordance with previous studies, our present study implies that ARC inhibits the NLPR3 inammasome in MCT-treated rats. On the basis of the respective roles of oxidative stress, inammation, and IL-b in the pathogenesis of PAH, as well as the interactions between reactive oxidative species, the inammasome, and IL-1b, we propose that the antioxidative effect of ARC on PAH may contribute to the inhibition of the NLRP3 inammasome via oxidative stress inhibition.
In conclusion, we demonstrated that ARC treatment prevented the development of MCT-induced PAH by signicantly attenuating pulmonary vascular remodeling, increasing RVSP, RV hypertrophy, and cardiomyocyte enlargement. These bene-cial effects of ARC might be attributed to antiproliferative, anti-inammatory, and antioxidative actions, which are mediated, at least in part, via inhibition of the NLRP3 inammasome signaling pathway. Future studies are needed to conrm these specic mechanisms and to determine whether ARC treatment can reverse or delay established PAH.

Conflicts of interest
None.