Luteolin attenuates PM2.5-induced inflammatory responses by augmenting HO-1 and JAK-STAT expression in murine alveolar macrophages

ABSTRACT

To explore the involved mechanisms and possible treatments of ambient PM2.5 exposure-induced lung inflammation, this work studied the activity of luteolin, a natural flavonoid which widely presents in many plant species, in murine alveolar macrophage MH S cells exposed to PM2.5. Results showed PM2.5 induced an inflammatory response, as evidenced by significantly increased TNF-α, IL-6, MCP-1 and Rantes levels. and induced iNOS, COX-2, and NF-κB protein expressions in MH-S cells. Moreover, luteolin pre-treatment reduced JAK2 and STAT1 but not STAT3 protein expressions in PM2.5-stimulated MH-S cells. Performing JAK2 inhibitor AG490 further showed reduced TNF-α and IL-6 productions as well as iNOS, COX-2, and NF-κB protein expressions. In addition, although PM2.5 exposure could elevate HO-1 expression basically, luteolin pre-treatment and AG490 administration further significantly enhanced HO-1 expression additionally. Collectively, these results revealed that luteolin inhibits inflammation through suppressing JAK2/STAT1/NF-κB pathway and enhancing HO-1 expression in PM2.5-challenged alveolar macrophage MH-S cells.

KEYWORDS:

• Ambient fine particulate matter (PM2.5)
• murine alveolar macrophages
• luteolin
• inflammatory response
• JAK/STAT/NF-κB
• HO-1

1. Introduction

Pollution by atmospheric particulate matter (PM) is a major public issue in urban areas, owing to its significant impact on human health (WHO, 2021). Particulate matter includes PM10, which can pass through the respiratory system, and PM2.5, with an aerodynamic diameter of < 2.5 μm, which can enter the gas-exchange region in the lungs. In particular, fine particles of PM2.5 can invade the bronchi and induce damages in the respiratory system, causing various health problems (Environmental Protection Agency, EPA). Epidemiological studies have revealed associations between PM exposure and pulmonary inflammation (Yang et al., 2020) or cardiovascular system disorders (Wang et al., 2020). In particular, prolonged exposure to PM2.5 increases the risk of cardiovascular (Hvidtfeldt et al., 2019) and respiratory death (Coleman et al., 2020). PM2.5 is classified as an important air pollutant owing to its bioaccumulation and its oxidative damage to humans (Ribeiro et al., 2016). Air pollution, including ambient PM2.5, has been shown to influence lung function development in vitro and in experimental studies, exacerbating asthma and causing other respiratory symptoms such as cough and bronchitis in children (Khalili et al., 2018) and adults (Lu et al., 2021). Many investigations have demonstrated that the induction of inflammatory responses by alveolar macrophages (AMs) upon exposure to PM2.5 is critical in the pathogenesis of inflammatory and allergic lung disease (Ma et al., 2017; Shoenfelt et al., 2009; Zhao et al., 2012). AMs play a critical role in inducing immune responses because they are phagocytes that absorb and degrade the inhaled small particles then introduce the foreign antigens to adaptive immune cells (Joshi et al., 2018).

Some studies have established lipopolysaccharides (LPS) that mimic bacterial infection (Geeraerts et al., 2017; Ostareck and Ostareck-Lederer, 2019), polluting PM2.5 in the air (Shoenfelt et al., 2009) and other innate immune response effectors, could activate macrophages by generating various cellular signals. Associated processes of infection generate many pro-inflammatory cytokines and inflammatory mediators, such as nitric oxide (NO) and prostaglandins (PG) which perform protective functions. Other studies have demonstrated that PM2.5 induces inflammatory responses (Jia et al., 2021) and oxidative stress in human bronchial epithelial 16HBE cells (Niu et al., 2020) and lung epithelial A549 cells in vitro (Huang et al., 2014; Zou et al., 2016). Increased levels of cyclooxygenase-2 (COX-2), a key enzyme that mediates PG synthesis, have also been detected in mice myocardial tissue upon co-exposure to SO₂, NO₂ and PM2.5 (Zhang et al., 2016).

Nuclear factor-κB (NF-κB) is known to be a transcription factor in the immune response process, which induces the expression of genes, including NOS and COX-2, to regulate immune system-associated cell proliferation (Lim et al., 2001). Shukla et al. showed that PM2.5 significantly increases the expression of inflammatory and cytokine genes by the NF-κB-dependent regulation (Shukla et al., 2000). Cytokines are activated by Janus kinase/signal transducer and transcriptional activator (JAK/STAT) transcription factors. Interleukin 6 (IL-6) mainly activates the JAK/STAT pathway (Lim et al., 2001; Okugawa et al., 2003) and has been proposed to contribute to the development of many inflammatory diseases, including anti-neuritis, rheumatoid arthritis (RA) (Liu et al., 2019) and Crohn's disease (Hammaker et al., 2019). Numerous studies have demonstrated that heme oxygenase-1 (HO-1) attenuates inflammation and modulates immune responses both in vitro and in vivo (Albarakati et al., 2020; Facchinetti, 2020; Li, Kakkar et al., 2018). Those studies focused on how luteolin inhibits LPS-induced inflammation by inducing HO-1 expression. Hence, the pharmacological induction of HO-1 expression has been proposed as a new potential strategy for managing various inflammatory diseases.

Luteolin (3’,4’,5,7-tetrahydroxyflavone) is a natural flavonoid that is found in many plants, such as chamomile, green peppers, celery and many medicinal herbs. Moreover, luteolin is one of the major flavonoids which distributes in the flower extract of Hieracium pannosum Boiss and the aerial part of Dracocephalum kotschyi (Gökbulut et al., 2017; Kamali et al., 2016). Luteolin has pharmacological effects and is currently known to have antioxidant, anti-inflammatory, antibacterial (Bustos et al., 2018) and anti-cancer properties (Imran et al., 2019 ). Studies have shown that luteolin exhibits anti-inflammatory activity in LPS treatment (Park et al., 2011; Wu et al., 2013 ) and virus infection (Liu et al., 2016) in vitro. However, the mechanisms of LPS or viral infection-caused inflammation may differ from that of ambient PM2.5 inhalation. In particular, whether luteolin reduces PM2.5-induced inflammation in alveolar macrophages has not been studied. In this work, a murine alveolar macrophage cell line, MH-S cells, was used to confirm whether PM2.5 can induce inflammatory responses and the effects of luteolin on the PM2.5-induced inflammation. This study characterised the anti-inflammatory effects of luteolin in alveolar macrophage MH-S cells exposed to PM2.5.

2. Materials and methods

2.1. Collection of PM2.5 and element analysis of PM2.5

A PM sample was collected between August 2017 and May 2018 in the South District of Taichung City by using a TISCH high-flow sampler (TE-6070) and a high-volume cascade impactor (TE-231, Tisch Environmental, Cleves, Ohio. USA). Air was sampled through the inlet of a sampler that was operated with a constant flow rate. Particulates with aerodynamic diameter < 10−2.5 μm (PM10−2.5) and < 2.5 μm (PM2.5) were collected using 5.63” × 5.38” and 8” × 10” quartz filters (Pall, USA) through an inertial particle separator, respectively. The filter paper was weighed using a high-precision micro analytical balance (Mettler Toledo XS105) before and after sampling at a specified temperature and humidity. A solution of the PM sample was prepared using the method of Zhang et al. (Zhang et al., 2017). The filter that contained on which was the PM sample was immersed in deionised water and sonicated for 30 min to extract the PM sample solution from the filter. The extracted PM sample was then stored at −4°C. A blank sample (unexposed filter) was prepared in the same manner as a control. After drying at 70°C, the PM2.5 was suspended in ultrapure water (d₂H₂O) at 5 mg/ml. To ensure the homogeneity of the solution and to minimise variance and error, every PM sample solution was sonicated for 3 min before analysis.

All PM2.5 samples were analyzed to determine the amounts of 22 metals (Ga, Ag, Cd, Sn, Ba, B, N, Ni, Co, Cr, As, Se, In, Hg, Pb, Cu, Al, Ca, Zn, Fe, Mg and Mn) and 17 PAHs (naphthalene (NAP), acenaphthylene (ACPY), acenaphthene (ACP), fluorene (FLU), phenanthrene (PHE), anthracene (ANTHR), fluoranthene (FLA), pyrene (PYR), benz(a)anthracene (BaA), chrysene (CHR), benzo(b)fluoranthene (BbF), benzo(k)fluoranthene (BkF), benzo(e)pyrene (BeP), benzo(a)pyrene (BaP), indeno (1,2,3-cd)pyrene (INP), dibenz(ah)anthracene (DBA) and benzo(ghi)perylene (BghiP)) that they contained. PAHs were quantified by gas chromatography with a flame ionisation detector (GC-FID) using a Perkins Elmer Auto-system Gas Chromatograph (Agilent 7890B GC/5977 MSD) with a capillary column (50 m × 0.32 mm × 0.17 μm, Hewlett Packard). Heavy metals were quantified using a flame atomic absorption spectrometer (PerkinElmer/NexION 300X, USA), as described in a previous study (Kuo et al., 2009).

2.2. Cell culture

MH-S cells (murine alveolar macrophage cell line) were maintained in RPMI medium which was supplemented with 2 mM glutamine, 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C under 5% CO₂.

2.3. Determination of cell viability and cytotoxicity

The MH-S cells were pre-treated with various concentrations of luteolin (80, 40, 20, 10 and 5 μM) (DMSO concentration in each well was 0.1%) for 1.5 h. Then, PM2.5 (25, 50 and 100 μg/ml) was added for 24 h. Cell viability was measured using a Cell Counting Kit-8 (CCK-8) (Dojindo molecular technologies, Inc.) accordingly (Liu et al., 2019). The absorbance (450 nm) of each well was measured using an ELISA plate reader (Multiskan Spectrum, Thermo Co., Vantaa, Finland). The lactate dehydrogenase (LDH) assay was performed using the LDH Cytotoxicity Assay Kit II (BioVision, Inc., Mountain View, CA) according to the manufacturer’s recommendation.

2.4. Determination of TNF-α, IL-6, MCP-1 and Rantes levels

The MH-S cells were pre-treated with various concentrations (20, 10 and 5 μM) of luteolin (Cat#L9283, Sigma-Aldrich, St. Louise, MO, US) or 15 μM of JAK 2 inhibitor AG490 (Cat#T3434, Sigma-Aldrich, St. Louise, MO, US) for 1.5 h following stimulated with PM2.5 (25, 50 and 100 μg/ml) at 37°C for 24 h. The amounts of TNF-α, IL-6, MCP-1 and Rantes in the supernatant were determined using ELISA kits (eBioscience, San Diego, CA).

2.5. Western blotting assay

The MH-S cell lysates were collected and analysed by western blotting with iNOS, COX-2, NFκB, pNFκB, JAK2, STAT1, STAT3, pJAK2, pSTAT1, pSTAT3, HO-1 and GAPDH antibodies (Santa Cruz Biotech) accordingly (Liu et al., 2019). Signals were visualised using an enhanced chemiluminescence system (ECL, Amersham Pharmacia Biotech).

2.6. Statistical analysis

All data are expressed as mean ± standard deviation (SD). For each pair of experiments, significant differences (set at P < .05) between the experimental group and the corresponding controls were evaluated by one-way analysis of variance (ANOVA).

3. Results

3.1. Analysis of components in PM2.5 samples

One mg sample of PM2.5 was analysed by Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) to measure the metal ion contents. Various toxic heavy metal ions, such as Zn, Fe, As, Cu, Mn, Pb, Ni and Ba, which may be severely toxic to mammalian cells and living organisms, were detected (Kuo et al., 2009; Zhang et al., 2017), as shown in Table 1. More than 10 μg/mg of Al, Ca, Zn, Fe and Mg were detected. More than 1 μg/mg of B, Pb, Cu, Mn, Cd, Ba, V, Ni, Cr, As and Se were also detected. Ag and Hg were not detected. No polycyclic aromatic hydrocarbons (PAHs) were detected at levels over the detectable limit in the PM samples, perhaps owing to the high temperature and dryness that prevailed during sample preparation.

Table 1. Concentrations of metal elements in PM2.5 were collected in the South District of Taichung City from August 2017 to May 2018. N.D., not detected.

Metal	PM2.5 (μg/mg)
Ga	0.067
Ag	N.D.
Cd	0.217
Sn	0.066
Ba	0.755
B	1.31
V	0.213
Ni	0.508
Co	0.032
Cr	0.439
As	0.284
Se	0.158
In	0.003
Hg	N.D.
Pb	2.08
Cu	3.90
Al	10.6
Ca	27.8
Zn	22.9
Fe	30.2
Mg	13.5
Mn	3.46

3.2. PM2.5 exposure induces pro-inflammation mediators in MH-S cells

Upon viral infection, pro-inflammatory cytokines such as tumour necrosis factor (TNF)-α, interleukin (IL)-6 and monocyte chemoattractant protein (MCP)-1 were produced in mouse immune RAW264.7 cells (Liu et al., 2016). Chemokine regulated upon activation, normal T cell expressed and presumably secreted (Rantes) is reportedly significantly generated by PM10 but not by PM2.5 stimulation in mouse peritoneal macrophages (Shoenfelt et al., 2009). In this study, the murine alveolar macrophage MH-S cells were stimulated with various concentrations of PM2.5 (25, 50 and 100 μg/ml) for 24 h to study the issue. Compared to the mock group, PM2.5 induced significant levels of TNF-α and IL-6 in the culture medium (Figure 1). The productions of MCP-1 and Rantes were also elevated by PM2.5 stimulation, although their endogenous levels were high. These data strongly support the claim that after 24 h of exposure, PM2.5 (> 25 μg/ml) induces the inflammation responses of MH-S cells.

Figure 1. PM2.5 induces the production of pro-inflammation mediators in MH-S cells. Cells were untreated (mock) or treated with various concentrations of PM2.5 (25, 50 and 100 μg/ml) for 24 h. Then, the cultured media were collected to determine the levels of TNF-α, IL-6, MCP-1 and Rantes using enzyme-linked immunosorbent assay (ELISA). Data are representative of at least three independent experiments and values are expressed in mean ± SD (n ≥ 3). *P < .05 (significant difference compared with mock cells).

3.3. PM2.5 exposure induces iNOS, COX-2 and NFκB expressions in MH-S cells

Genes of NF-κB, iNOS and COX-2 are well known to be induced during the inflammatory reaction. Studies have revealed that iNOS and COX-2 have a clear role in mediating inflammatory responses in many diseases that are caused by bacterial or viral infection (Lim et al., 2001; Liu et al., 2016). A previous study also shown that there was cumulative p-IKBα in PM-treated bone marrow-derived macrophages, indicating that PM could activate NF-κB signalling (Li, Wu et al., 2018). Therefore, the effects of PM2.5 on the expressions of iNOS, COX-2 and NFκB proteins in murine alveolar macrophage MH-S cells were studied. In this experiment, MH-S cells were treated with PM2.5 at doses of 25, 50 or 100 μg/ml for 24 h, and PM2.5 was found to enhance the expressions of iNOS, COX-2 and NFκB proteins in a dose-dependent manner (Figure 2). Notably, the 100 μg/ml concentration of PM2.5 caused robustly induction of iNOS, COX-2 and NFκB proteins, we therefore, applied 100 μg/ml of PM2.5 in the following experiments. Thus, PM2.5 stimulation increases iNOS, COX-2 and NFκB expressions in MH-S cells.

Figure 2. Exposure to PM2.5 induces expressions of iNOS, COX-2 and NFκB in MH-S cells. Cells were untreated (mock) or treated with various concentrations of PM2.5 (12.5, 25, 50 and 100 μg/ml) for 24 h. Then, harvested cell lysates underwent western blotting to detect iNOS, COX-2, NFκB and GAPDH proteins. Data are representative of at least three independent experiments and values are expressed in mean ± SD (n ≥ 3). *P < .05 (significant difference compared with mock cells).

3.4. Cell viabilities and cytotoxicity of luteolin to MH-S cells

Studies have shown that luteolin can inhibit the inflammatory cytokines inductions by LPS (Albarakati et al., 2020; Boengler et al., 2008) or a virus (Liu et al., 2016) in mouse macrophage RAW264.7 cells. Luteolin has not been tested to determine whether it has anti-inflammatory effects in PM2.5-induced MH-S cells. The cytotoxicity of various luteolin concentrations in MH-S cells and the corresponding cell viabilities were analyzed using CCK-8 (Figure 3(A)) and LDH (Figure 3(B)) assay. The results represented that less than 20 μM of luteolin had no detectable toxic effect on MH-S cells; therefore, 20 μM of luteolin was used in the following works.

Figure 3. Cell viabilities of MH-S cells treated with various concentrations of luteolin. MH-S cells were treated with various concentrations of luteolin (Lut; 80, 40, 20, 10 and 5 μM) for 24 h. (A) The cell viabilities were measured using a CCK-8 assay and (B) the supernatants of cultures were collected and subjected to LDH assay. Data are representative of at least three independent experiments and values are expressed in mean ± SD (n ≥ 3). *P < .05 (significant difference compared with mock cells).

3.5. Effects of luteolin on TNF-α, IL-6, MCP-1 and Rantes production in PM2.5-stimulated MH-S cells

As shown in Figure 2, PM-2.5 induced the dose-dependent upregulation of inflammatory factors in cells that had been treated only with PM2.5. The effects of luteolin on TNF-α, IL-6, MCP-1 and Rantes productions in the PM2.5-induced pro-inflammatory responses of murine alveolar macrophages were subsequently studied. The results revealed that the levels of TNF-α (Figure 4(A)) and IL-6 (Figure 4(B)) were significantly induced by PM2.5 (100 μg/ml) while markedly reduced in the cells pre-treated with increased concentrations of luteolin. Compared to a mock-treated group, the PM2.5 (100 μg/ml)-upregulated MCP-1 (Figure 4(C), Mock) and Rantes (Figure 4(D), Mock) were suppressed in the luteolin-treated group (20 μM). These results suggest that luteolin inhibits the productions of pro-inflammatory factors TNF-α, IL-6, MCP-1 and Rantes in PM2.5-treated MH-S cells. Thus, luteolin contributes to the blockage of PM2.5-induced inflammation.

Figure 4. Inhibitory effects of luteolin on the production of inflammatory mediators in PM2.5-exposed MH-S cells. MH-S cells were untreated (mock) or pre-treated with various concentrations of luteolin (5, 10 and 20 μM) for 1.5 h followed by stimulated without (Mock) or with PM2.5 (100 μg/ml). After 24 h, cultured media were collected and analysed using ELISA to determine the levels of (A) TNF-α, (B) IL-6, (C) MCP-1 and (D) Rantes. Data are representative of at least three independent experiments and values are expressed in mean ± SD (n ≥ 3). *P < .05 (significant difference compared with mock cells). N.D., not detected.

3.6. Luteolin inhibits the expressions of iNOS, COX-2 and NF-κB in MH-S cells stimulated with PM2.5

The regulating effects of luteolin on the expressions of iNOS, COX-2 and NFκB in MH-S cells that are challenged by PM2.5 were studied. By comparison with the mock group, there were robust expressions of iNOS, COX-2 and NFκB proteins in PM2.5-stimulated (100 μg/ml) MH-S cells (Figure 5, line 2), as was shown in Figure 2. However, pre-treatment with luteolin significantly reduced iNOS, COX-2, NFκB and pNFκB protein expressions in MH-S cells in a luteolin dose-dependent pattern (5, 10 and 20 μM) under PM2.5 stimulation (Figure 5). Thus, luteolin suppresses PM2.5-induced pro-inflammatory iNOS, COX-2 and NFκB in MH-S cells.

Figure 5. Luteolin inhibits expressions of iNOS, COX-2 and NF-κB in PM2.5-stimulated MH-S cells. MH-S cells were untreated (mock) or pre-treated with various concentrations of luteolin (5, 10 and 20 μM) for 1.5 h, and then untreated (mock) or treated with PM2.5 (100 μg/ml). After 24 h, harvested cell lysates were blotted to detect iNOS, COX-2, NFκB, pNFκB and GAPDH proteins. Data are representative of at least three independent experiments and values are expressed in mean ± SD (n ≥ 3). *P < .05 (significant difference compared with mock cells).

3.7. Luteolin suppresses PM2.5-induced activation of JAK/STAT pathway in MH-S cells

A review by Boengler et al. reveals that the JAK/STAT signalling pathway has an important role in inflammation and is activated by its upstream inflammatory factors stimulation such as IL-6 and NF-κB (Boengler et al., 2008). The activation of STAT1 and STAT3 has been reported to stimulate IL-6 expression (Cheon et al., 2011; Xu et al., 2020). A recent study has established that PM2.5 significantly increases IL-6 and IL-8 expression and triggers inflammatory responses (Wang et al., 2021). As shown in Figure 1, PM2.5 enhanced the IL-6 secretion of MH-S cells. Therefore, the issue of whether PM2.5 induces inflammatory responses in MH-S cells via the JAK/STAT pathway was studied. The expressions of phosphorylated JAK2 (pJAK2) and pSTAT1 were markedly increased in PM2.5-stimulated MH-S cells. Pre-treatment with luteolin significantly suppressed pJAK2, JAK2, pSTAT1 and STAT1 but not STAT3 and pSTAT3 expressions in PM2.5-challenged cells (Figure 6). Thus, luteolin inhibits JAK2/STAT1 signalling in MH-S cells.

Figure 6. Luteolin inhibits JAK2 and STAT1 but not STAT3 expressions in PM2.5-stimulated MH-S cells. MH-S cells were untreated (mock) or pre-treated with various concentrations of luteolin (5, 10 and 20 μM) for 1.5 h and then untreated (mock) or treated with PM2.5 (100 μg/ml). After 24 h, cell lysates were blotted to detect JAK2, STAT1, STAT3, pJAK2, pSTAT1 and pSTAT3 proteins. Data are representative of at least three independent experiments and values are expressed in mean ± SD (n ≥ 3). *P < .05 (significant difference compared with mock cells).

We next applied JAK2 inhibitor AG490 and found that AG490 significantly reduced both PM2.5-mediated TNF-α and IL-6 production (Figure 7(A)) in MH-S cells. Adding AG490 also inhibited iNOS, COX-2 and NF-κB protein expressions in MH-S cells under PM2.5 exposure (Figure 7(B)). These findings revealed that JAK/STAT signalling is required for induce TNF-α and IL-6 secretion in PM2.5-exposed MH-S cells. Accordingly, the expressions of iNOS, COX-2 and NF-κB are activated in part by JAK2.

Figure 7. JAK2 inhibitor AG490 reduces the levels of inflammatory mediators in PM2.5-stimulated MH-S cells. MH-S cells were untreated (mock) or pre-treated with JAK inhibitor AG490 (15 μM) for 1.5 h and then untreated (mock) or treated with PM2.5 (100 μg/ml). After 24 h, cultured media were assayed using ELISA to determine levels of (A) TNF-α and IL-6, and (B) cell lysates were blotted to detect iNOS, COX-2, NFκB, pNFκB and GAPDH proteins. Data are representative of at least three independent experiments and values are expressed in mean ± SD (n ≥ 3). *, P < .05 (significant difference compared with untreated cells).

3.8. HO-1 expression is induced by PM2.5 and further augmented by luteolin through the JAK/STAT signalling pathway in MH-S cells

Several studies have reported that flavonoids, an agent exhibits antioxidant, anti-inflammatory and anti-apoptotic activities, induces cellular HO-1 expression (Liu et al., 2016; Song and Park, 2014; Sung and Lee, 2015). Under various concentrations of PM2.5 (0, 25, 50 and 100 μg/ml) stimulation, the same results in this study as a previous study using human proximal tubule epithelial HK-2 cells (Huang et al., 2020) that HO-1 protein level was increased in MH-S cells (Figure 8(A)). Next, the MH-S cells were pre-treated with various concentrations of luteolin following exposed to PM2.5 (100 μg/ml) to investigate the expression of HO-1. The HO-1 protein level was significantly increased in the PM2.5-treated cells to the extent that depended on the luteolin dose (5, 10 and 20 μM) (Figure 8(B)). Additionally, applying the JAK inhibitor (AG490) further increased HO-1 expression in cells treated with PM2.5 (Figure 8(C)). Collectively, the data revealed that HO-1 upregulation is mediated by the inhibition of the JAK/STAT signalling pathway.

4. Discussion

Frequent exposure to higher levels of PM2.5 is associated with a greater risk of cardiovascular and respiratory death. Many investigations have studied the pathological effects and the corresponding mechanisms of environmental air pollution on the airway epithelial cells (Chowdhury et al., 2018; Honda et al., 2021). In addition, it is also reported that alveolar macrophages induce pro-inflammatory mediators when cells are exposed to ambient PM, which is a major factor in inducing lung inflammation (Shoenfelt et al., 2009). However, the role of PM in human sickness remains largely unclear. This work provides evidence that PM2.5 induced inflammatory responses in lung alveolar MH-S macrophages by activating iNOS, COX-2 and NFκB expressions. Applying luteolin, a phytochemical agent which have antioxidant and anti-inflammatory properties, inhibited the pro-inflammatory cytokine production through regulating HO-1 expression. These results enhance the potential use of luteolin as a clinical application in patients with lung inflammation.

LPS-inducible genes are reportedly differentially regulated in macrophages that are derived from different tissues, such as microglia cells in nervous tissue, alveolar macrophages in the lungs, Langerhans cells in the skin and Kupffer cells in the liver (Drummond and Lionakis, 2019). However, alveolar macrophages have a key role in inflammatory and immune responses in the lung. One study has claimed that type II alveolar cells may be sensitive to PM2.5-induced oxidative stress and cause inflammatory responses in adult mice (Zhang et al., 2016). In this study, PM2.5 induced the expression of several inflammatory factors (IL-6, TNF-α, MCP-1 and Rantes) in murine alveolar macrophages MH-S cells (Figure 1). Bletilla striata ethanol-extract is reported to significantly reduce the PM2.5-induced inflammatory cytokines (TNF-α, IL-6 and MCP-1) productions in RAW264.7 cells by down-regulating NF-κB activation and MAPK signalling pathways (Li et al., 2019). To reduce the inflammation, we applied luteolin, an important plant-derived flavonoid polyphenolic compound which is widely exist in many herbs and vegetables (Gökbulut et al., 2017; Kamali et al., 2016), to PM2.5-exposed MH-S cells. Luteolin has been shown to be able to inhibit LPS-induced inflammation responses in murine macrophages RAW264.7 cells (Zhang et al., 2018) and brain epithelial SH-SY5Y cells (Zhu et al., 2014). We found that luteolin pre-treatment reduced the levels of TNF-α, IL-6, MCP-1 and Rantes (Figure 4) in PM2.5-simulated MH-S cells. Our previous finding also revealed that luteolin significantly reduces the IL-6 and MCP-1 levels in virus-infected RAW264.7 cells (Liu et al., 2016). In addition, luteolin could decrease the protein expressions of iNOS, COX-2 and NF-κB in MH-S cells under PM2.5 stimulation (Figure 5), suggesting that luteolin represents strong anti-inflammatory activity in PM2.5-exposed MH-S cells.

Recent studies have found that LPS activates STAT1 and STAT3 signalling pathways and that phosphorylated STAT dimers are the major transcription factors that promote inflammatory responses (Choi et al., 2014 ; Kim et al., 2017). A study of neonatal rat cardiomyocytes suggests that cooking oil fumes-derived PM2.5 stimulation induces significant inflammation by activating the JAK/STAT3 and NF-κB signalling pathways (Kim et al., 2017). Furthermore, JAK inhibitor AG490 and STAT3 inhibitor Stattic were used in this study to verify the role of the JAK/STAT pathway in PM2.5-induced inflammatory response. Results revealed that AG490 and STAT3 inhibitors reduce the levels of the iNOS, COX-2 and NF-κB proteins (Figure 7(B)), as well as the secreted TNF-α and IL-6 (Figure 7(A)). These results indicated that the JAK/STAT pathway was involved in the PM2.5-induced inflammatory response in MH-S cells. In addition, luteolin is reported to suppress JAK2, STAT1 and STAT3 phosphorylation in RAW264.7 cells that were infected by viruses (Liu et al., 2019). To determine how luteolin attenuates the PM2.5-induced inflammatory responses in MH-S cells, the levels of the phosphorylated forms of JAK2, STAT1 and STAT3 were measured (Figure 6). Interestingly, the results showed that luteolin inhibits the phosphorylation of JAK2 and STAT1 relative to the untreated group under PM2.5 exposure, but it does not significantly affect the phosphorylation of STAT3. These data suggest that luteolin inhibits PM2.5-induced inflammation through JAK2 and STAT1 but not STAT3 activation in MH-S cells. Further investigation is required for investigating the critical role of STAT3 on luteolin treatment in MH-S cells under PM2.5 stimulation.

Heme oxygenase-1 (HO-1) is an inducible gene whose expression is known to protect cells from death. HO-1 is commonly regarded as an anti-inflammatory and immune-suppressive enzyme in many types of cells (Naito et al., 2014). Recently, luteolin has been reported to be an effective inducer of HO-1, which has an anti-inflammatory effect by LPS-induced RAW164.7 macrophages to the extent that depends on the dose, resulting in iNOS inhibition (Song and Park, 2014). Ge et al. have shown that exposure to PM2.5 causes the phosphorylation of JNK, triggering an inflammatory response and oxidative stress by reducing the level of HO-1/Nrf-2 in RAW264.7 cells (Chenxu et al., 2018 ). The cellular results herein revealed that exposure to PM2.5 greatly upregulated HO-1 and luteolin significantly augmented HO-1 expression in PM2.5-stimulated MH-S cells (Figure 8). Notably, the finding is novel and warrants further investigation that the inhibition of the JAK/STAT signalling pathway upregulated the HO-1 level.

Figure 8. HO-1 expression is enhanced by luteolin and JAK inhibitor AG490 treatments. (A) Induction of HO-1 expression by PM2.5 (25, 50 and 100 μg/ml) for 24 h. (B) MH-S cells were untreated (mock) or pre-treated with various concentrations of luteolin (5, 10 and 20 μM) or (C) JAK inhibitor AG490 (15 μM) for 1.5 h following stimulated with PM2.5 (100 μg/ml). After 24 h, cell lysates were blotted to detect HO-1 and GAPDH protein expressions. Data are representative of at least three independent experiments and values are expressed in mean ± SD (n ≥ 3). *, P < .05 (significant difference compared with mock cells).

5. Conclusions

In summary, the effects of luteolin on the PM2.5-induced inflammatory responses in MH-S cells were studied. When PM2.5 infiltrates the alveoli, the resulting stress induces HO-1 to protect the lung from harmful free radicals whose production is induced by heavy metals. Meanwhile, an immune response is initiated by JAK and NFκB activation. Phosphorylated NFκB can be detected and translocated to nuclei to promote iNOS and COX-2 expressions. Phosphorylated STAT1/3 also induces the productions of TNF-α, IL-6, MCP-1 and Rantes. Newly synthesised cytokines are secreted, strengthening the inflammatory response. As we have demonstrated before, the active JAK/STAT signalling downregulated HO-1 expression and weakened the anti-oxidative protection. In this study, we also found that the HO-1 expression was upregulated by luteolin treatment and further increased by JAK/STAT signalling inhibitor in cells stimulated with PM2.5. In addition, luteolin treatment reduced the expressions of iNOS, COX-2, TNF-α, IL-6, MCP-1 and Rantes, controlling the inflammatory responses. This work provided clear evidence and the molecular basis for the anti-inflammatory mechanism of luteolin in PM2.5-exposed murine alveolar macrophage MH-S cells (Figure 9). It thus supports the potential of luteolin as a phytochemical agent in the management of inflammatory responses that are triggered by exposure to PM2.5.

Figure 9. Proposed role of luteolin in regulating inflammatory responses in PM2.5-stimulated alveolar macrophage MH-S cells. Proposed role of luteolin in regulating inflammatory responses in PM2.5-stimulated alveolar macrophage MH-S cells.

Acknowledgments

The authors thank the Instrument Resource centre of Chung Shan Medical University for providing the chemiluminescence/fluorescence imaging analyser.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This study was supported by Chung Shan Medical University (NCHU-CSMU-10613 and NCHU-CSMU-10708) and the Ministry of Science and Technology, Taiwan (MOST106-2320-B-040-023-MY3 and MOST109-2320-B-040-019-MY3).