Melatonin Alleviates Silica Nanoparticle-Induced Lung Inflammation via Thioredoxin-Interacting Protein Downregulation

Silica dioxide nanoparticles (SiONPs) have been increasingly used in various industries; however, this has raised concerns regarding their potential toxicity. SiONPs are also a major component in the Asian sand dust that causes pulmonary diseases among the general public. Melatonin exerts some inhibitory effects against lung inflammation. In this study, we explored the therapeutic properties of melatonin against lung inflammation using an SiONPs-induced lung inflammation murine model and SiONPs-stimulated H292 cells, human airway epithelial cell line, by focusing on the involvement of thioredoxin-interacting protein (TXNIP) in the modulation of the MAPKs/AP-1 axis. We induced an inflammatory response by exposing mouse lungs and the H292 cells to SiONPs and confirmed the anti-inflammatory effect of melatonin. Melatonin inhibited the expression of various inflammatory mediators, including TNF-α, IL-6, and IL-1β, in SiONPs-exposed mice and SiONPs-stimulated H292 cells; this inhibition contributed to a decline in inflammatory cell accumulation in the lung tissues. Furthermore, melatonin treatment decreased the expression of MAPKs and AP-1 by downregulating TXNIP, eventually decreasing the production of SiONPs-induced inflammatory mediators. Overall, these data suggest that melatonin reduces SiONPs-induced lung inflammation by downregulating the TXNIP/MAPKs/AP-1 signalling pathway, thereby supporting the use of melatonin as an effective approach to control SiONPs-induced lung inflammation.


Introduction
Silica dioxide nanoparticles (SiONPs) have been extensively used in biotechnology owing to the simple production process and ability to modify their shape and size [1]. The increasing use of SiONPs has raised concerns regarding their potential toxicity in humans. This is particularly true in an industrial setting where SiONPs can be inhaled and can be linked to serious health problems, including lung cancer, silicosis, emphysema, and chronic obstructive pulmonary disease (COPD) [2,3]. Inhaled SiONPs induce significant pulmonary inflammation, which can result in the activation of the MAPKs [4][5][6][7]. These responses further induce the activation of AP-1, resulting in accelerated inflammatory processes [8]. Furthermore, SiONPs are a major component in the Asian sand dust that affects northeast Asian countries, including China, Japan and Korea, especially in the spring, and are associated with the occurrence of various pulmonary diseases across Antioxidants 2021, 10, 1765 2 of 12 this region [9]. Thus, controlling the respiratory diseases caused by SiONPs is critical to maintaining human health.
Thioredoxin-interacting protein (TXNIP), an endogenous inhibitor of thioredoxin, is closely involved in the regulation of various inflammatory responses [10]. TXNIP contributes to the inflammation of the respiratory tract by interacting with the upregulated MAPKs pathway [11][12][13][14]. Recent studies have reported that exposure to SiONPs elevates the TXNIP expression, activating MAPKs and increasing pulmonary inflammation [15,16]. Therefore, inhibiting TXNIP/MAPKs signalling may be a potential strategy for treating SiONPs-induced pulmonary inflammation.
Melatonin is primarily released by the pineal gland and is known to exert a wide range of biological properties, such as anti-inflammatory and antioxidant properties [17][18][19]. Consequently, melatonin has been shown to exhibit some therapeutic effects on various respiratory diseases [20,21]. Exogenous melatonin alleviates inflammatory response by inhibiting MAPK signalling in the respiratory tract of asthmatic mice and attenuates smoke-induced pulmonary inflammation by inhibiting Erk-Sp1 expression. Furthermore, the anti-inflammatory effects of melatonin are associated with inhibition of TXNIP, as established in a cadmium-induced liver injury model [22]. However, the underlying mechanism of anti-inflammatory effects of melatonin against SiONPs-induced pulmonary inflammation via TXNIP regulation is not comprehensively understood.
The goals of this study were to explore the anti-inflammatory effect of melatonin on SiONPs-induced pulmonary inflammation and to elucidate the underlying mechanism of this hormone by focusing on its effects on TXNIP/MAPKs signalling.

Ethics Statement
The Institutional Animal Care and Use Committee at the Chonnam National University approved the protocols used in this animal study (CNU IACUC-YB-2019-63).  AGT AGA CCT GCC CAG AC  GAC CTC TCT CTA ATC AGC CC  IL-6  ATG CAA TAA CCA CCC CTG AC  ATC TGA GGT GCC CAT GCT AC  IL-1β  AGC CAG GAC AGT CAG CTC TC  ACT TCT TGC CCC CTT TGA AT  GAPDH  CAA AAG GGT CAT CAT CTC TG  CCT GCT TCA CCA CCT TCT TG

Inflammatory Cell Count in Bronchoalveolar Lavage Fluid (BALF)
Mice were sacrificed 48 h post final SiONPs exposure and were subjected to tracheostomy. The BALF was obtained by infusing the lungs with cold PBS (0.7 mL) two times, which was subsequently collected using a tracheal cannula. Differential cell counts in the BALF were determined using a Diff-Quik ® reagent (IMEB Inc., San Marcos, CA, USA) as described by the manufacturer.

siRNA Transfection
Small interfering RNAs (siRNA) against the scrambled siRNA (4390843) control and TXNIP (4392420) were obtained from Ambion. Each siRNA (20 nM) was transfected into cells using Lipofectamine TM RNAiMAX (Invitrogen, Carlsbad, CA, USA) via the forward transfection method, as prescribed by the manufacturer. After suppression of endogenous TXNIP expression, cells were treated with 12.5 µg/mL SiONPs or PBS and harvested after 6 h. To investigate the protein expression, Western blot was performed as mentioned above.

Statistical Analysis
Data are presented as the means ± standard deviation. All statistical analyses were performed using GraphPad Prism 7 (San Diego, CA, USA). One-way analysis of variance (ANOVA) was performed, followed by the Bonferroni multiple comparison test. p < 0.05 was considered significant.

Melatoinin Reduces the Number of Inflammatory Cells of the BALF from SiONPs-Exposed Mice
Exposure to SiONPs showed markedly higher inflammatory cell counts of BALF when compared with those in the NC group ( Figure 1), with neutrophils and macrophages exhibiting significant elevation. DEX-treated animals exhibited lesser inflammatory cells than those in the SiONPs-exposed specimens. Moreover, there was a significant decrease in the number of inflammatory cells in the melatonin-treated animals compared with those in the SiONPs-exposed group, as observed in 40 mg/kg of melatonin ( Figure 1).

siRNA Transfection
Small interfering RNAs (siRNA) against the scrambled siRNA (4390843) control and TXNIP (4392420) were obtained from Ambion. Each siRNA (20 nM) was transfected into cells using Lipofectamine TM RNAiMAX (Invitrogen, Carlsbad, CA, USA) via the forward transfection method, as prescribed by the manufacturer. After suppression of endogenous TXNIP expression, cells were treated with 12.5 µ g/mL SiONPs or PBS and harvested after 6 h. To investigate the protein expression, Western blot was performed as mentioned above.

Statistical Analysis
Data are presented as the means ± standard deviation. All statistical analyses were performed using GraphPad Prism 7 (San Diego, CA, USA). One-way analysis of variance (ANOVA) was performed, followed by the Bonferroni multiple comparison test. p < 0.05 was considered significant.

Melatoinin Reduces the Number of Inflammatory Cells of the BALF from SiONPs-Exposed Mice
Exposure to SiONPs showed markedly higher inflammatory cell counts of BALF when compared with those in the NC group ( Figure 1), with neutrophils and macrophages exhibiting significant elevation. DEX-treated animals exhibited lesser inflammatory cells than those in the SiONPs-exposed specimens. Moreover, there was a significant decrease in the number of inflammatory cells in the melatonin-treated animals compared with those in the SiONPs-exposed group, as observed in 40 mg/kg of melatonin ( Figure  1).

Melatonin Ameliorates Pathological Changes in the Respiratory Tract of SiONPs-Exposed Mice
Exposure to SiONPs noticeably increased inflammatory cell accumulation (34.70 ± 6.03%, p < 0.01) in the respiratory tract as compared with that in the NC group (3.37 ± 1.22%) ( Figure 3). However, the accumulation in these tissues markedly declined in the DEX-treated mice (15.02 ± 1.04%, p < 0.01) in comparison with that in the SiONPs group. Melatonin-treated animals also exhibited a notable decrease in inflammatory cell accumulation (Mel 20: 26.73 ± 2.75%, p < 0.05) in comparison with that in the SiONPs group, which was also evident in the 40 mg/kg melatonin specimens (Mel 40: 21.80 ± 2.59%, p < 0.01).

Melatonin Ameliorates Pathological Changes in the Respiratory Tract of SiONPs-Exposed Mice
Exposure to SiONPs noticeably increased inflammatory cell accumulation (34.70 ± 6.03%, p < 0.01) in the respiratory tract as compared with that in the NC group (3.37 ± 1.22%) ( Figure 3). However, the accumulation in these tissues markedly declined in the DEXtreated mice (15.02 ± 1.04%, p < 0.01) in comparison with that in the SiONPs group. Melatonin-treated animals also exhibited a notable decrease in inflammatory cell accumulation (Mel 20: 26.73 ± 2.75%, p < 0.05) in comparison with that in the SiONPs group, which was also evident in the 40 mg/kg melatonin specimens (Mel 40: 21.80 ± 2.59%, p < 0.01).

Melatonin Ameliorates Pathological Changes in the Respiratory Tract of SiONPs-Exposed Mice
Exposure to SiONPs noticeably increased inflammatory cell accumulation (34.70 ± 6.03%, p < 0.01) in the respiratory tract as compared with that in the NC group (3.37 ± 1.22%) ( Figure 3). However, the accumulation in these tissues markedly declined in the DEX-treated mice (15.02 ± 1.04%, p < 0.01) in comparison with that in the SiONPs group. Melatonin-treated animals also exhibited a notable decrease in inflammatory cell accumulation (Mel 20: 26.73 ± 2.75%, p < 0.05) in comparison with that in the SiONPs group, which was also evident in the 40 mg/kg melatonin specimens (Mel 40: 21.80 ± 2.59%, p < 0.01). Melatonin ameliorated inflammatory responses in the respiratory tract of SiONPs-exposed mice. Lung tissue was stained with H&E stain to assess airway inflammation (magnification: x200). Degree of inflammation was quantified using image analyzer (IMT i-Solution). Scale bars = 50 µm. ## p < 0.01, vs. the NC; * p < 0.05, ** p < 0.01, vs. the SiONPs.

Melatonin Inhibits TXNIP/MAPK/AP-1 Pathway in the Lungs of SiONPs-Exposed Mice
Exposure to SiONPs resulted in a clear increase in TXNIP expression (Figure 4a,b). However, DEX-and melatonin-treated mice exhibited a notable decline in TXNIP expression compared with that on SiONPs exposure. Additionally, SiONPs exposure markedly elevated phosphorylation of ERK, JNK, and p38, which was accompanied with an increase in the AP-1 expression. However, these significantly decreased following melatonin treatment. These observations were also consistent with those of the TXNIP expression assays.
Exposure to SiONPs resulted in a clear increase in TXNIP expression (Figure 4a,b). However, DEX-and melatonin-treated mice exhibited a notable decline in TXNIP expression compared with that on SiONPs exposure. Additionally, SiONPs exposure markedly elevated phosphorylation of ERK, JNK, and p38, which was accompanied with an increase in the AP-1 expression. However, these significantly decreased following melatonin treatment. These observations were also consistent with those of the TXNIP expression assays.

Melatonin Attenuates mRNA Expression of Inflammatory Cytokines in SiONPs-Stimulated Cells
The TNF-α and IL-6 mRNA expression were markedly higher in the SiONPs-stimulated cells than in non-stimulated cells (Figure 6a,b, respectively). However, melatonin treatment reduced this increase in a concentration-dependent manner. Additionally, the elevated IL-1β mRNA expression in the SiONPs markedly decreased following treatment with melatonin ( Figure 6c).

Melatonin-Mediated TXNIP Inhibition Reduces the MAPKs Phosphorylation and AP-1 Expression in SiONPs-Stimulated Cells
SiONPs treatment markedly elevated TXNIP expression (Figure 7a-c). However, melatonin treatment suppressed TXNIP expression in SiONPs-stimulated cells when compared with that in non-stimulated cells, which was accompanied with increases in JNK, ERK, and p38 MAPK phosphorylation. However, this reduced following the addition of melatonin, consistent with the TXNIP expression trend (Figure 7a,b). Furthermore,

Melatonin Attenuates mRNA Expression of Inflammatory Cytokines in SiONPs-Stimulated Cells
The TNF-α and IL-6 mRNA expression were markedly higher in the SiONPs-stimulated cells than in non-stimulated cells (Figure 6a,b, respectively). However, melatonin treatment reduced this increase in a concentration-dependent manner. Additionally, the elevated IL-1β mRNA expression in the SiONPs markedly decreased following treatment with melatonin (Figure 6c).

Melatonin-Mediated TXNIP Inhibition Reduces the MAPKs Phosphorylation and AP-1 Expression in SiONPs-Stimulated Cells
SiONPs treatment markedly elevated TXNIP expression (Figure 7a-c). However, melatonin treatment suppressed TXNIP expression in SiONPs-stimulated cells when compared with that in non-stimulated cells, which was accompanied with increases in JNK, ERK, and p38 MAPK phosphorylation. However, this reduced following the addition of melatonin, consistent with the TXNIP expression trend (Figure 7a,b). Furthermore, SiONPs treatment noticeably elevated AP-1 expression, which was declined following the melatonin treatment (Figure 7a-c).
TXNIP or MAPK expression was not affected by the control siRNA, whereas their expression was downregulated by the TXNIP siRNA (Figure 8a,b). Furthermore, melatonin and TXNIP siRNA together demonstrated an even more pronounced reduction in TXNIP expression and the MAPKs phosphorylation in SiONPs-stimulated cells than TXNIP siRNA treatment alone.
SiONPs treatment noticeably elevated AP-1 expression, which was declined following the melatonin treatment (Figure 7a-c).

Discussion
SiONPs have various industrial applications owing to their many advantages, but their increasing use has proven detrimental to the health of workers dealing with them [2,3]. Furthermore, SiONPs are a major constituent of Asian sand dust; thus, they are the underlying cause of increasing susceptibility to bacteria or viral infections in populations exposed to Asian sand dust [9]. In recent studies, SiONPs were shown to induce changes in the immune function of exposed cell; in particular, exposure to these particles has been linked to excessive inflammation in the respiratory tract [4,6,7,24]. Conversely, melatonin has exhibited protective effects against asthma and COPD owing to its pharmacological properties [17,20,21,25]. Based on literature, we investigated the activity of melatonin on SiONPs-induced pulmonary inflammation using both experimental animal models and cell lines. In this study, melatonin effectively attenuated SiONPs-induced pulmonary inflammation and this anti-inflammatory effect was linked to the downregulation of the TXNIP/MAPK/AP-1 pathway.
Excessive inflammatory responses result in the development and exacerbation of lung injuries due to the production of inflammatory cytokines [26,27]. SiONPs increase inflammatory cell infiltration and cytokine expression levels by activating inflammatory signalling, resulting in lung injury [7,16]. Therefore, a reduction in the inflammatory responses induced by SiONPs may offer a potential therapeutic strategy for preventing and treating SiONPs-related lung damage. The therapeutic effects of melatonin against inflammatory responses have been reported by several experimental studies, which suggest that melatonin is effective in preventing lung damage associated with enhanced inflammatory responses [20,21,[28][29][30]. Several studies have shown that melatonin reduces inflammatory mediators produced in response to various stimuli including radiation, carbon tetrachloride, and allergens, thereby preventing lung injury [28,29,31]. Moreover, melatonin treatment alleviates the pulmonary inflammation associated with cigarette smoke-induced COPD by suppressing MAPK phosphorylation [21]. In this study, SiONPs induced the inhibition of several inflammatory mediators as previously reported [15,16]. However, treatment with melatonin reduced the inflammatory index, including those of inflammatory cells and cytokines, in both the SiONPs-exposed mice and SiONPs-stimulated cells. These changes were accompanied by histological evidence of a decrease in inflammatory

Discussion
SiONPs have various industrial applications owing to their many advantages, but their increasing use has proven detrimental to the health of workers dealing with them [2,3]. Furthermore, SiONPs are a major constituent of Asian sand dust; thus, they are the underlying cause of increasing susceptibility to bacteria or viral infections in populations exposed to Asian sand dust [9]. In recent studies, SiONPs were shown to induce changes in the immune function of exposed cell; in particular, exposure to these particles has been linked to excessive inflammation in the respiratory tract [4,6,7,24]. Conversely, melatonin has exhibited protective effects against asthma and COPD owing to its pharmacological properties [17,20,21,25]. Based on literature, we investigated the activity of melatonin on SiONPs-induced pulmonary inflammation using both experimental animal models and cell lines. In this study, melatonin effectively attenuated SiONPs-induced pulmonary inflammation and this anti-inflammatory effect was linked to the downregulation of the TXNIP/MAPK/AP-1 pathway.
Excessive inflammatory responses result in the development and exacerbation of lung injuries due to the production of inflammatory cytokines [26,27]. SiONPs increase inflammatory cell infiltration and cytokine expression levels by activating inflammatory signalling, resulting in lung injury [7,16]. Therefore, a reduction in the inflammatory responses induced by SiONPs may offer a potential therapeutic strategy for preventing and treating SiONPs-related lung damage. The therapeutic effects of melatonin against inflammatory responses have been reported by several experimental studies, which suggest that melatonin is effective in preventing lung damage associated with enhanced inflammatory responses [20,21,[28][29][30]. Several studies have shown that melatonin reduces inflammatory mediators produced in response to various stimuli including radiation, carbon tetrachloride, and allergens, thereby preventing lung injury [28,29,31]. Moreover, melatonin treatment alleviates the pulmonary inflammation associated with cigarette smoke-induced COPD by suppressing MAPK phosphorylation [21]. In this study, SiONPs induced the inhibition of several inflammatory mediators as previously reported [15,16]. However, treatment with melatonin reduced the inflammatory index, including those of inflammatory cells and cytokines, in both the SiONPs-exposed mice and SiONPs-stimulated cells. These changes were accompanied by histological evidence of a decrease in inflammatory cell infiltration in the respiratory tract. Overall, these results indicate that melatonin can act as a therapeutic agent and prevent lung damage caused by exposure to SiONPs.
TXNIP has been implicated in the development of many biological reactions, including inflammatory responses [10]. TXNIP overexpression in response to various stimuli induces the phosphorylation of various MAPKs including p38 and JNK, whereas silencing of TXNIP suppresses the activation of MAPK signalling [32,33]. This increase in MAPK phosphorylation increases AP-1 activation, leading to an increase in inflammatory mediators and consequently making TXNIP an important regulator of the inflammatory responses [34,35]. Exposure to SiONPs induces airway inflammation in experimental animals, and these increases were associated with an elevation in TXNIP expression levels [15]. However, silibinin, an antioxidant and anti-inflammatory agent, suppressed the pulmonary inflammation caused by SiONP exposure by inhibiting TXNIP expression [16]. Thus, TXNIP inhibitors may effectively suppress the lung inflammation associated with SiONP exposure. In this study, exposure to SiONPs caused pulmonary inflammation and activated the TXNIP/MAPK/AP-1 pathway in mice. However, in both the experimental animal models and H292 cell lines, melatonin reduced the activation of TXNIP/MAPK/AP1 signalling associated with SiONP exposure, resulting in a decline in the production of inflammatory mediators and the eventual reduction of lung inflammation, thus indicating that melatonin acts as a TXNIP inhibitor and can effectively inhibit the lung inflammation associated with SiONPs.

Conclusions
Our data show that melatonin suppresses the SiONP-induced inflammatory responses in both the mouse lung tissues and H292 cells. These effects were closely associated with the downregulation of the TXNIP/MAPK/AP-1 pathway, allowing us to establish that melatonin effectively inhibits SiONP-induced lung inflammation by downregulating TXNIP expression.