Carthamus tinctorius Suppresses LPS-Induced Anti-Inﬂammatory Responses by Inhibiting the MAPKs/NF-κ B Signaling Pathway in HaCaT Cells

: This study aimed to elucidate the anti-inﬂammatory activity of C. tinctorius leaves by measuring inﬂammatory parameters such as nitric oxide (NO) production and mRNA expression of iNOS, interleukin-6 (IL-6), and IL-1 β in lipopolysaccharide (LPS)-induced HaCaT cells. Further, the effect of C. tinctorius ethanol extract on the MAPKs/NF-κ B signaling pathway was examined in HaCaT cells. The phytochemical proﬁle of the ethanol extract of C. tinctorius leaves was determined using UPLC-QTOF-MS/MS. The results indicated that the ethanol extract of C. tinctorius effectively attenuated LPS-induced secretion of NO, IL-6, and IL-1 β in HaCaT cells. Further, LPS-stimulated mRNA and protein expressions of iNOS were decreased by pre-treatment with C. tinctorius ethanol extract at the transcriptional level in HaCaT cells. Moreover, the ethanol extract of C. tinctorius suppressed NF-κ B signaling in LPS-induced HaCaT cells. This suppression was mediated by MAPKs/NF-κ B signaling, inhibiting the phosphorylation of p38 and p65 in HaCaT cells. However, there is no signiﬁcant effect on the phosphorylation of JNK by the ethanol extract. The QTOF-MS/MS analysis revealed the identiﬁcation of 27 components in the ethanol extract of C. tinctorius leaves. The data demonstrate that the ethanol extract of C. tinctorius leaves protects the LPS-induced HaCaT cells by inhibiting the expression of iNOS, IL-6, and IL-1 β and suppressing the phosphorylation of the p38, p65, p-JNK via inactivation of MAPKs/NF-κ B signaling pathway. These results demonstrate that C. tinctorius leaves may serve as a potential candidate to prevent inﬂammation-related diseases.


Introduction
Inflammation is an essential part of the defense mechanism of the host activated by the invasion of pathogens, damaged cells, and toxic compounds. Although inflammation plays an important role in the healing process, it is important pathogenesis of various chronic diseases, such as cardiovascular, diabetes, arthritis, and cancer [1]. In particular, the overproduction of nitric oxide (NO) is involved in inflammatory conditions, so it is regarded as an important pro-inflammatory mediator. NO synthases (NOS) enzymes are primarily responsible for the production of NO in mammalian cells [2,3]. NOS consists of three members which are neuronal NOS, endothelial NOS, and inducible NOS (iNOS). In these, iNOS is highly involved in the production of NO [4,5]. The inflammatory response is distinguished by producing several pro-inflammatory cytokines, including IL-1β, IL-6, TNF-α, etc. [6]. Overproduction of IL-1β upregulates adhesion molecule expression in endothelial cells to activate the translocation of leukocytes, which is related to hyperalgesia and fever [7,8].

Enzyme-Linked Immunosorbent Assay (ELISA)
HaCaT cells were pretreated with ethanol extract of C. tinctorius at 12.5-100 µg/mL for 1 h and then treated with LPS at 1 µg/mL concentration for 24 h. The amount of IL-6 and IL-1β was measured from the culture supernatants of HaCaT cells using ELISA kits (Invitrogen, Carlsbad, CA, USA) [29].

Phytochemical Analysis of C. tinctorius by UPLC-QTOF-MS/MS
The LC/MS systems consisted of a Waters Acquity UPLC I-Class system (Waters Co., Milford, MA, USA) with Waters Xeo G2 QTOF MS (Waters MS Technologies, Manchester, UK). An amount of 1 mg of different fractions (hexane, dichloromethane, and ethyl acetate) obtained from the ethanol extract of C. tinctorius was dissolved in 10 mL of 70% ethanol for LC-MS analysis. Then, 2 µL of diluted solution of C. tinctorius was injected into a Waters ACQUITY UPLC BEH C18 column (50 × 2.1 mm, 1.7 µm. The column conditions and the phytochemical characterization were followed based on the method described by Shin et al. [34]. The compounds in the ethanol extract of C. tinctorius were identified by UNIFI 1.8 (Waters, Milford, CT, USA). The traditional Chinese medicine library and the in-house library were used for the tentative identification of chemical components.

Statistical Analysis
GraphPad Prism Version 8.0 (GraphPad, La Jolla, CA, USA) software was used for data analyses. The values are expressed as the mean of three independent determinations ± SEM. Statistical differences were assessed using the Student-Newman-Keuls test for multiple comparisons after being analyzed with a one-way ANOVA. The p values < 0.05 were considered statistically significant.

The Effect of Ethanol Extract of C. tinctorius on the Viability of HaCaT Cells
Previously, several studies reported atopic dermatitis-related inflammation in macrophages and keratinocytes [35][36][37]. Therefore, we investigated whether ethanol extract of C. tinctorius exhibits LPS-induced anti-inflammatory effects on HaCaT cells. For this purpose, the cytotoxicity of the ethanol extract of C. tinctorius was determined by MTT assays after incubating HaCaT cells with different concentrations of the ethanol extract of C. tinctorius (0-1000 µg/mL) for 24 h. After the treatment, there was no significant effect observed in HaCaT with up to 100 µg/mL concentration of the ethanol extract of C. ticntorius (cell viability > 90%) ( Figure 1). Hence, we selected the concentration of C. tinctorius ethanol extract up to 100 µg/mL for further experiment.

Effect of C. tinctorius Ethanol Extract on Nitric Oxide Production in LPS-Stimulated HaCaT Cells
It is well known that LPS stimulation is responsible for the release of different inflammatory mediators, especially NO. NO is an important mediator that regulates the molecules with different biological functions including pathological processes in normal physiological conditions. However, excessive production of NO induces inflammation in abnormal physiological conditions [38,39]. Therefore, the inhibitory effect of C. tinctorius ethanol extract on NO production in LPS-induced HaCaT cells was evaluated ( Figure 2). The NO production was significantly increased by accumulating a higher level of nitrite in HaCaT cells (40.97 µM). To investigate the effect of C. tinctorius extract on NO production, HaCaT cells were simultaneously treated with 1 µg/mL LPS and various concentrations of extracts (12.5-100 µg/mL) separately for 24 h. When compared to the untreated control, HaCaT cells pre-treated with ethanol extract of C. tinctorius significantly (p < 0.001) reduced the nitrite concentration in the medium to 4.02 µM at the concentration of 100 µg/mL ( Figure 2). cules with different biological functions including pa iological conditions. However, excessive production normal physiological conditions [38,39]. Therefore, th anol extract on NO production in LPS-induced HaCa NO production was significantly increased by accum HaCaT cells (40.97 µM). To investigate the effect of C.
HaCaT cells were simultaneously treated with 1 µg/ of extracts (12.5-100 µg/mL) separately for 24 h. Whe HaCaT cells pre-treated with ethanol extract of C. t duced the nitrite concentration in the medium to 4 µg/mL ( Figure 2).

The Effect of Ethanol Extract of C. tinctorius on mRNA and Protein Expressions of iNOS in LPS-Stimulated HaCaT Cells
To determine the inhibitory action of C. tinctorius ethanol extract on NO production from HaCaT cells, we calculated the mRNA expression level of iNOS by RT-PCR and protein expression level by Western blot analysis. In response to LPS, the iNOS mRNA levels were significantly increased in HaCaT cells. However, the pre-treatment with ethanol extract of C. tinctorius drastically suppressed the mRNA expression of iNOS in HaCaT cells ( Figure 3a). Western blot analysis also demonstrated that LPS-treated HaCaT cells markedly increased the protein expression of iNOS. However, pre-treatment with ethanol extract of C. tinctorius showed strong inhibitory activity against iNOS protein expression HaCaT cells (Figure 3b). tein expression level by Western blot analysis. In response to LPS, the iNOS mRNA levels were significantly increased in HaCaT cells. However, the pre-treatment with ethanol extract of C. tinctorius drastically suppressed the mRNA expression of iNOS in HaCaT cells ( Figure 3a). Western blot analysis also demonstrated that LPS-treated HaCaT cells markedly increased the protein expression of iNOS. However, pre-treatment with ethanol extract of C. tinctorius showed strong inhibitory activity against iNOS protein expression HaCaT cells (Figure 3b).

The Effect of Ethanol Extract of C. tinctorius on Pro-inflammatory Cytokines in LPS-Stimulated HaCaT Cells
We also examined the inhibitory effects of C. tinctorius on the release of IL-6 and IL-1β in HaCaT cells induced by LPS. HaCaT cells were pretreated with different concentrations of C. tinctorius ethanol extract for 1 h and then stimulated with LPS (1 µg/mL) separately for 24 h. The mRNA expression level of IL-6 and IL-1β was determined in HaCaT cells by RT-PCR analysis. C. tinctorius ethanol extract significantly inhibited the mRNA expression levels of IL-6 and IL-1β in LPS-induced HaCaT cells ( Figure 4) compared to that of the control.

The Effect of Ethanol Extract of C. tinctorius on Pro-Inflammatory Cytokines in LPS-Stimulated HaCaT Cells
We also examined the inhibitory effects of C. tinctorius on the release of IL-6 and IL-1β from HaCaT cells, we calculated the mRNA expression level of iNOS by RT-PCR and protein expression level by Western blot analysis. In response to LPS, the iNOS mRNA levels were significantly increased in HaCaT cells. However, the pre-treatment with ethanol extract of C. tinctorius drastically suppressed the mRNA expression of iNOS in HaCaT cells (Figure 3a). Western blot analysis also demonstrated that LPS-treated HaCaT cells markedly increased the protein expression of iNOS. However, pre-treatment with ethanol extract of C. tinctorius showed strong inhibitory activity against iNOS protein expression HaCaT cells (Figure 3b).

The Effect of Ethanol Extract of C. tinctorius on Pro-inflammatory Cytokines in LPS-Stimulated HaCaT Cells
We also examined the inhibitory effects of C. tinctorius on the release of IL-6 and IL-

The Effect of C. tinctorius Ethanol Extract on MAPKs/NF-κB Activation in LPS-Stimulated HaCaT Cells
We investigated whether C. tinctorius ethanol extract inhibited the phosphorylation of p65 (a subunit of NF-κB) in HaCaT cells. The LPS treatment increased the phosphory-

The Effect of C. tinctorius Ethanol Extract on MAPKs/NF-κB Activation in LPS-Stimulated HaCaT Cells
We investigated whether C. tinctorius ethanol extract inhibited the phosphorylation of p65 (a subunit of NF-κB) in HaCaT cells. The LPS treatment increased the phosphorylation of p65 in HaCaT cells, but C. tinctorius ethanol extract significantly attenuated the phosphorylation of P65 in LPS-stimulated HaCaT cells (Figure 5a). Subsequently, we identified which kinase was involved in the regulation of MAPKs/NF-κB activity in HaCaT cells. The results revealed that the phosphorylation of p38, ERK, and JNK (Figure 5b-   1% ethanol alone, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. LPS alone.

The Effect of C. tinctorius Ethanol Extract on MAPKs/NF-κB Activation in LPS-Stimulated HaCaT Cells
We investigated whether C. tinctorius ethanol extract inhibited the phosphorylation of p65 (a subunit of NF-κB) in HaCaT cells. The LPS treatment increased the phosphorylation of p65 in HaCaT cells, but C. tinctorius ethanol extract significantly attenuated the phosphorylation of P65 in LPS-stimulated HaCaT cells (Figure 5a). Subsequently, we identified which kinase was involved in the regulation of MAPKs/NF-κB activity in Ha-CaT cells. The results revealed that the phosphorylation of p38, ERK, and JNK (Figure 5b

UPLC-QTOF-MS/MS Analysis of C. tinctorius Leaves
The total phenolic content of the ethanol extract of C. tinctorius leaves was 18.27 ± 0.13 mg GAE/g. QTOF-MS/MS analysis was performed to detect the chemical profile in the ethanol extract of the leaves of C. tinctorius. For better separation of components, the ethanol extract was fractionated first with hexane, dichloromethane, and ethyl acetate successively to produce fractions of different polarities. Figure 6 shows the base peak inten-

UPLC-QTOF-MS/MS Analysis of C. tinctorius Leaves
The total phenolic content of the ethanol extract of C. tinctorius leaves was 18.27 ± 0.13 mg GAE/g. QTOF-MS/MS analysis was performed to detect the chemical profile in the ethanol extract of the leaves of C. tinctorius. For better separation of components, the ethanol extract was fractionated first with hexane, dichloromethane, and ethyl acetate successively to produce fractions of different polarities. Figure 6 shows the base peak intensity (BPI) chromatogram of different components from C. tinctorius leaves. The results revealed the presence of 27 different chemical components in the leaves of C. tinctorius ( Table 2). The major components in the leaves of C. tinctorius are including protocatechuic acid (C 7 H 6 O 4 ) caffeic acid (C 9 H 8 O 4 ), coumaroylquinic acid (C 16 Table 2.   Table 2.

Discussion
Carthamus tinctorius L. is utilized to treat numerous ailments, including inflammationmediated diseases in Asian countries. Inflammation is an essential part of our body's defense mechanism that can be activated by various factors. However, the mechanisms of the anti-inflammatory activity of C. tinctorius leaves have not been evaluated. In this study, the LPS-stimulated anti-inflammatory activity of C. tinctorius ethanol extract in HaCaT cells was demonstrated. LPS can generate many pro-inflammatory cytokines, including NO. The excessive production of NO results in many inflammatory diseases and it is an important inflammatory mediator [48][49][50]. In the cytotoxicity study, C. tinctorius ethanol extract did not produce any effect on the viability of HaCaT cells at 100 µg/mL concentration (Figure 1). We evaluated the inhibitory effect of C. tinctorius on the NO production of HaCaT cells stimulated with LPS. The results demonstrated that C. tinctorius ethanol extract effectively inhibited the LPS-stimulated NO production in HaCaT cells ( Figure 2) and downregulated mRNA and protein expressions of iNOS. The LPS-induced HaCaT cells produce a rapid inflammatory reaction that can release pro-inflammatory cytokines (IL-6 and IL1β) and inflammatory mediators (iNOS) [51].
Attracting circulatory immune function cells, especially, neutrophils to fight infection is beneficial [52]. Yet, the excessive inflammatory reaction can lead to injury of tissues and organs. Hence, during the inflammatory response, the production of inflammatory mediators and pro-inflammatory cytokines should be strictly controlled [53,54]. We revealed that C. tinctorius ethanol extract inhibited the mRNA and protein expressions of iNOS in HaCaT cells (Figure 3). In addition, we determined the inhibitory effects of C. tinctorius on the release of IL-6 and IL-1β in LPS-induced HaCaT cells. We found that C. tinctorius ethanol extract also inhibited IL-6 and IL-1β mRNA expression in HaCaT cells. A previous study reported that C. tinctorius has a significant effect on the downregulation of iNOS and IL-1β in LPS-activated RAW 264.7 cells [55]. NF-κB is a regulatory transcription factor and is mainly involved in cellular responses to stimuli for the expression of TNFα, IL-1β, IL-6, iNOS, and COX-2 [50].
Dimers of NF-κB (p50/p65) are released and phosphorylated by LPS stimulation, immediately enter the nucleus, and bind exclusively with DNA sequences for promoting the expression of target genes [17,56]. Therefore, the inhibitory effect of C. tinctorius ethanol extracts on LPS-stimulated NF-κB was evaluated. This study found that phosphorylation of p65 was significantly increased by LPS stimulation for 1 h, which means increased NF-κB activation. However, C. tinctorius ethanol extract reduced the phosphorylation of p65 in HaCaT cells, revealing a notable inhibitory effect on NF-κB activity. Consequently, C. tinctorius ethanol extract inhibited the inflammatory mediator and pro-inflammatory cytokines expression in LPS-induced HaCaT cells by downregulating the NF-κB. A previous study reported that C. tinctorius aqueous extract proved to suppress bleomycin-stimulated mRNA levels of IL-1β, TNF-α, and TGF-β1 in lung homogenates. Furthermore, it inhibits the increased activity of NF-κB and p38 MAPK phosphorylation in lung tissues [57]. C. tinctorius methanol extract showed anti-inflammatory activity through increased HO-1 induction via Nrf-2 signals by inhibiting LPS-induced expression of iNOS and COX-2 and TNFα-mediated VCAM-1 upregulation in RAW 264.7 cells [58].
A recent study proved that C. tinctorius honey extract inhibited the NO production, suppressed iNOS, IL-1β, TNF-α, and MCP-1 expressions, decreased the phosphorylation of IκBα, inhibited the NF-κB-p65 protein, in LPS-induced RAW 264.7 cells through the activation of Nrf-2/HO-1 signaling pathway [59]. Moschamine isolated from C. tinctorius significantly suppressed mRNA and protein expression of COX-2, mPGES-1, iNOS, IL-6, and IL-1β by downregulating STAT1/3 activation in RAW 264.7 cells induced with LPS [60]. The florets of C. tinctorius contain a major active constituent called safflower yellow. This constituent inhibited inflammation in TNF-α-induced rat chondrocytes by regulating the NF-κB/SIRT1/AMPK pathways and endoplasmic reticulum stress [61]. Another study found that aqueous extracts from the petals of C. tinctorius and its main constituent safflower yellow prevented NO production and PGE2 in LPS-stimulated RAW264.7 cells by downregulating iNOS and COX-2 expressions [62]. Polyacetylene glucosides isolated from florets of C. tinctorius significantly inhibited NO production in LPS-stimulated RAW264.7 cells [27]. Hydroxysafflor yellow A from C. tinctorius L showed an anti-inflammatory effect in LPSinduced microglia activation by activating a nicotinamide adenine dinucleotide-dependent enzyme, SIRT1 [63]. In addition, dichloromethane extract and its water-ethanolic part of Carthamus lanatus aerial parts exhibited anti-inflammatory activity in activated human neutrophils [64].
The MAPK family, including p38, JNK, and ERK, play an essential role in the transcriptional regulation of the LPS-stimulated expression of iNOS [65]. MAPKs are the upstream activator of NF-κB via blocking the transcriptional activation of NF-κB by MAPK inhibitors [66,67]. In this study, p38, JNK, and ERK phosphorylation dramatically increased LPS-stimulated HaCaT cells, suggesting that LPS upregulated the MAPK pathway in Ha-CaT cells. C. tinctorius ethanol extract inhibited p38 and ERK phosphorylation in HaCaT cells. A previous study reported that schizandrin A showed a significant protective effect of LPS-induced inflammation in HaCaT cells by suppressing apoptosis, promoting cell viability, inhibiting the expression of IL-1β, IL-6, and TNF-α, downregulating the miR-127 expression, inactivating the p38MAPK/ERK and JNK pathways [68]. Liu et al. [69] demonstrated that sinomenine retards LPS-induced phosphorylation of p65, IκBα, and p38MAPK in HaCaT cells by downregulating the long non-coding RNA colon cancerrelated transcript-1. Tanshinol showed a protective effect on LPS-induced HaCaT cells by downregulating the miR-122 and inhibiting the JNK and NF-κB pathways [70]. This study suggested that the anti-inflammatory effect of C. tinctorius ethanol extract was associated with the regulation of MAPKs/NF-κB signaling pathways in LPS-stimulated HaCaT cells.
In this study, 27 different components were identified from the dried leaves of C. tinctorius by UPLC-QTOF. Previous studies reported that derivatives of quinic acid, coumaric acid, quercetin, and kaempferol attenuated immune responses through various mechanisms [71][72][73][74]. In particular, kaempferol and quercetin effectively downregulated the activation of important transcription factors for iNOS (NF-κB and STAT-1) in LPS-stimulated macrophages [71]. In LPS-induced RAW 264.7 cells, ferulic acid and its derivatives inhibited the production of NO through the suppression of iNOS expression and decreasing the production of prostaglandin E2 and TNF-α [75]. Quinic acid derivatives from Uncaria tomentosa also inhibited NF-κB activation in TNF-α-stimulated type II alveolar epithelial-like, A549 cells [72]. In another study, Zhao et al. [76] reported that p-coumaric acid inhibited inflammatory cytokines production in LPS-stimulated RAW 264.7 cells by blocking NF-kB and MAPK signaling pathways. In HaCaT cells, caffeic acid and ferulic acid inhibited UVA-stimulated matrix metalloproteinase-1 by regulating antioxidant defense systems [77].
Flavonoids, kaempferol-3-O-rutinoside, and kaempferol-3-O-glucoside isolated from flowers of C. tinctorius showed remarkable anti-nociceptive and anti-inflammatory activities [78]. Therefore, the presence of various bioactive metabolites in the ethanol extract of C. tinctorius leaves might be ascribed to its anti-inflammatory potential.

Conclusions
The results demonstrated that C. tinctorius ethanol extract inhibited LPS-stimulated NO, IL-1β, and IL-6 production as well as protein and mRNA expressions of iNOS in HaCaT cells. Further, the mechanistic study revealed that the anti-inflammatory effect of C. tinctorius ethanol extract is mediated via MAPKs/NF-κB by inhibiting the phosphorylation of p65, p38, and ERK in HaCaT cells. It could be concluded that the ethanol extract of C. tinctorius leaves can be utilized for the development of an anti-inflammatory agent.