Isorhapontigenin from Traditionally used Iris Domestica Reduces the Inammatory Response by Suppressing NF-κB and MAPK Signaling in LPS-Activated RAW 264.7 Macrophages


 BackgroundIsorhapontigenin, a resveratrol analogue, isolated from Iris domestica can induce apoptosis in tumor cells. Here, we designed to explore whether isorhapontigenin reduced inflammatory response in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells.MethodsIsorhapontigenin treated with RAW 264.7 cells, and then with LPS to stimulate inflammatory response. Proinflammatory cytokine expressions were measured using ELISA, and protein expressions were detected using western blots. ResultsIsorhapontigenin significantly inhibited the proinflammatory cytokine expressions. Isorhapontigenin also decreased cyclooxygenase-2 and inducible nitric oxide synthase productions and promoted heme oxygenase-1 expression in LPS-stimulated RAW264.7 cells. Isorhapontigenin could significantly inhibit NF-κB subunit p65 protein localization to the nucleus and reduced MAPK signal pathway activation. Isorhapontigenin also decreased reactive oxygen species production. ConclusionThus, isorhapontigenin has potential anti-inflammation and anti-oxidative stress that inhibits inflammatory mediators and cytokines expressions through suppressing the MAPK and NF-κB pathways.


Abstract
Background Isorhapontigenin, a resveratrol analogue, isolated from Iris domestica can induce apoptosis in tumor cells. Here, we designed to explore whether isorhapontigenin reduced in ammatory response in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells.

Methods
Isorhapontigenin treated with RAW 264.7 cells, and then with LPS to stimulate in ammatory response.
Proin ammatory cytokine expressions were measured using ELISA, and protein expressions were detected using western blots.

Results
Isorhapontigenin signi cantly inhibited the proin ammatory cytokine expressions. Isorhapontigenin also decreased cyclooxygenase-2 and inducible nitric oxide synthase productions and promoted heme oxygenase-1 expression in LPS-stimulated RAW264.7 cells. Isorhapontigenin could signi cantly inhibit NF-κB subunit p65 protein localization to the nucleus and reduced MAPK signal pathway activation.
Isorhapontigenin also decreased reactive oxygen species production.

Conclusion
Thus, isorhapontigenin has potential anti-in ammation and anti-oxidative stress that inhibits in ammatory mediators and cytokines expressions through suppressing the MAPK and NF-κB pathways.

Background
In ammation can provide a dangerous signal, and in amed cells or tissues can release in ammatory cytokines or in ammatory mediators to maintain or assist the immune system against microbial invasion [1]. However, excessive release of in ammatory cytokines may lead to dysplasia or continued chronic in ammatory diseases, such as obesity, non-alcoholic steatohepatitis, Alzheimer's disease, atherosclerosis, asthma, and cancer [2]. Therefore, regulating acute and chronic in ammation is of great importance to reducing tissue damage or relieving many chronic diseases. Macrophages are immune cells that are mainly activated by microbial infection or stimulatory molecules [3]. Macrophage activation during microbial infection suppresses the spread of microbes and also stimulates the in ammatory response of macrophages to regulate immune response activation [4]. In gram-negative bacterial infections, lipopolysaccharide (LPS) bind to macrophages, activating toll-like receptor 4 (TLR4) and inducing in ammatory signal pathway expression [5]. Activated macrophages also release excessive in ammatory mediators, chemokines, and cytokines to increase immune cell activation and immune cell function in infected tissues [6].
The nuclear transcription factor κB (NF-κB) is an important signaling pathway in macrophage-caused in ammatory response [7]. NF-κB is composed of two heterodimeric subunits, p65 and p50. When cells are not receiving a proin ammatory stimulus, p50 and p65 are captured by inhibitor of NF-κB (IκB), which suppresses NF-κB activation. When LPS binds to macrophages, it activates TLR4. This induces IκB phosphorylation, releasing p65 and p50 into the nucleus to switch on the gene expressions of in ammatory cytokines and other in ammatory mediators [8]. In addition, LPS-stimulation of macrophages also induces mitogen-activated protein kinase (MAPK) pathway activation, assisting in the expression of the NF-κB pathway and causing more severe in ammatory responses [9]. Therefore, regulating in ammatory signal pathway expression will improve the in ammatory response of macrophages.
Hemeoxygenase-1 (HO-1) is an antioxidant protein, and can be induced by various stresses that protects cells against oxidative stress [10]. Hence, HO-1 can protect oxidative stress and maintain cellular redox homeostasis [11]. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor, and LPS-or in ammatory cytokine-stimulated macrophages will increase Nrf2 translocation into the nucleus to regulate HO-1 expression to protect cells from oxidative damage [12].
Isorhapontigenin is a derivative of resveratrol found in some Chinese herbal medicines [13]. Previous studies pointed out that isorhapontigenin has anti-cancer, anti-oxidant and cardioprotective functions [14][15][16]. Another study found that isorhapontigenin can also inhibit airway epithelial cell in ammatory responses and reduce IL-6 expression in IL-1β-activated A549 cells [17]. Thus, isorhapontigenin is a natural product as an anti-in ammatory agent. However, the molecular mechanism of isorhapontigenin's action on in ammatory macrophages remains unclear. Here, we would evaluate the anti-in ammatory molecular mechanism of isorhapontigenin and investigate in ammatory signaling expressions in RAW264.7 cells stimulated by LPS.

Preparation of total and nuclear protein
Cells treated with isorhapontigenin for 1 h. Next, cells stimulated with or without 100 ng/mL LPS for 30 min to detect protein phosphorylation, or for 24 h to investigate total protein expression. Brie y, cells collected and lysed using protein lysis buffer containing phosphatase inhibitor cocktail (Thermo Scienti c). Furthermore, nuclear proteins isolated using NE-PER cell fractionation kits (Thermo Scienti c).

ROS expression
Cells treated with isorhapontigenin for 1 h, then cells stimulated with LPS for 24 h. Cells washed and added with DCFH-DA as described previously [18]. ROS expressions observed using uorescence microscopy (Olympus). Cells also detected ROS levels using the multimode plate readers (BioTek synergy HT). Furthermore, MAPK inhibitors (Enzo Life Sciences) were treated with or without 10 μM isorhapontigenin to detect ROS expression.
Immuno uorescence staining Cells were used to seed microscope slides in culture plates and incubated with isorhapontigenin for 1 h, then LPS stimulated cells for 24 h. The slides treated with a speci c P65 antibody (1:100), followed by a uorescent secondary antibody. Finally, the slides treated with DAPI solution for nuclear staining and presented images using uorescence microscope (Olympus).

Statistics
Experimental results performed at least three independent experiments. Data are presented as the mean ± SD. Statistical analysis of experimental data performed using one-way ANOVA followed by Dunnett's test and post hoc analysis. Statistical signi cance de ned as p < 0.05.

Results
Effects of isorhapontigenin on cell viability Isorhapontigenin did not effectively affect cell viability at concentrations ≤40 μM (Fig. 1a). Hence, cellular experiments used concentrations of isorhapontigenin from 0-20 μM.

Isorhapontigenin attentuates IκBα phosphorylation and NF-κB activation
The results demonstrated that isorhapontigenin signi cantly reduced IκB-α degradation and phosphorylation compared to LPS-incubated RAW 264.7 cells (Fig. 3). Furthermore, p65 was mostly cytoplasmic in LPS unstimulated cells, while its level in nucleus increased after treatment with LPS.
Interestingly, western blotting of cytosolic and nuclear fractions demonstrated that the nuclear translocation of p65 decreased in isorhapontigenin-treated compared with untreated LPS-induced cells (Fig. 4a-c), and comparison of uorescent staining of p65 in cells under these conditions con rmed the signi cant suppression of nuclear translocation of p65 by isorhapontigenin (Fig. 4d).

Isorhapontigenin regulated the phosphorylation of MAPK pathway
Isorhapontigenin effectively decreased the phosphorylation of ERK1/2, JNK, and p38 ( Fig. 5a-b). Therefore, we used MAPK inhibitors to investigate how isorhapontigenin attenuates proin ammatory cytokine expression. The resulted demonstrated that isorhapontigenin enhanced the ability of MAPK inhibitors to suppress IL-6 and TNF-α expression, respectively (Fig. 5c-d).
Isorhapontigenin increased Nrf2 and HO-1 production We found that isorhapontigenin signi cantly promoted HO-1 levels in cytoplasm and increased Nrf2 expressions in nucleus compared with LPS-stimulated macrophages (Fig. 6a-c). In cells treated with DCFH-DA, uorescence microscopy revealed that isorhapontigenin could decrease intracellular ROS expressions (Fig. 6d). RAW 264.7 cells also incubated with DCFH-DA solution, and detected ROS expressions using Multi-Mode microplate reader. The result demonstrated that isorhapontigenin effectively reduced ROS expressions in LPS-stimulated RAW 264.7 cells (Fig. 6e). Furthermore, when isorhapontigenin added with MAPK inhibitors, ROS productions effectively decreased more effectively compared to isorhapontigenin treated with LPS-activated RAW 264.7 cells (Fig. 6f).

Discussion
Isorhapontigenin is a tetrahydroxylated stilbenoid isolated from I. domestica, G. cleistostachyum, and wine grapes [19]. Isorhapontigenin has been known to have anticancer, anti-in ammatory, antioxidant effects [14,20,21]. Isorhapontigenin was found that could inhibited cartilage matrix damage and chondrocyte in ammation in rats, suppressing COX-2 and iNOS expression in IL-1β-induced chondrocytes [17]. However, anti-in ammatory molecular mechanism of isorhapontigenin on immune cells is not yet fully clear. In this current study, isorhapontigenin signi cantly reduced pro-in ammatory cytokine productions by LPS-stimulated macrophages. In addition, in LPS-stimulated macrophages, isorhapontigenin also reduced iNOS and COX-2 productions, and increased HO-1 and Nrf2 protein expression. We also found that isorhapontigenin signi cantly attenuated the nuclear translocation of p65 and decreased MAPK phosphorylation in in ammatory signaling pathways, and improved the nuclear translocation of Nrf2 to promote its antioxidant effect in LPS-induced macrophages.
When infecting bacteria invade human tissues or organs, innate immune system detect and devour them, preventing bacterial spread, and reducing the likelihood of diseases such as sepsis and bacteremia [5].
The LPS binds to the TLR4 receptor of macrophages, activated in ammatory signaling and released in ammatory mediators and cytokines to promote the ability of immune cells to resist bacterial infection [22]. LPS-stimulated macrophages can induce excessive iNOS expression, and iNOS can convert Larginine to L-citrulline and produce NO [23]. These NO molecules in turn activate in ammatory molecular messages to inform in ammatory cells and tissues of danger, interfering with the normal physiological functions of tissues [24]. Previous research found that isorhapontigenin could reduce NO and iNOS expression in IL-1β-induced rat chondrocytes [17]. In this study, isorhapontigenin signi cantly inhibited iNOS expression in LPS-stimulated macrophages, and thus could reduce the expression of in ammatory signaling pathways caused by excessive NO. In addition, LPS-stimulated macrophages can also strongly increase COX-2 expression [25]. COX-2 metabolizes arachidonic acid to produce PGE 2 , which increases pain in in amed tissue and causes vasodilation, allowing increased immune-cell migration into the tissue [26]. COX-2 inhibitors as anti-in ammatory and analgesic drugs can decrease PGE 2 production by inhibiting COX-2 expression, thus reducing in ammation [26]. Isorhapontigenin was reported to reduce COX-2 and PGE 2 expression and reduce IL-1β-induced rat cartilage explant damage [17]. Our experiments also found that isorhapontigenin inhibits COX-2 and PGE 2 expressions in LPS-activated macrophages.
Hence, isorhapontigenin has the ability to reduce secretions of macrophage in ammatory mediator and thus reduce the excessive activation of immune cells.
During bacterial infection, activated macrophages release in ammatory cytokines, which cause more immune cell activation and severe in ammation [27]. LPS-stimulated macrophages can express high levels of TNF-α to increase the permeability of vascular endothelial cells and promote their expression of cell adhesion molecules. This allows more neutrophils or monocytes to in ltrate the infected tissue to promote immunity and attack microorganisms [28]. However, bacterial infection induces excessive activation of macrophages, which release excessive amounts of TNF-α, causing fever, lowered blood pressure, and even organ necrosis, sepsis, and death [22,29]. Therefore, reducing the uncontrolled activation of macrophages would reduce the excessive production of TNF-α and reduce the risk of death from organ failure and shock [30]. In this study, 5-20 μM isorhapontigenin inhibited TNF-α production in LPS-induced macrophages. Isorhapontigenin is an analog of resveratrol. Previously, scholars found that 1-25 μM resveratrol can reduce the secretion of TNF-α by LPS-stimulated macrophages [31]. Therefore, we thought that isorhapontigenin should also have a good anti-in ammatory effect. In amed macrophages also release a large amount of IL-6. IL-6 is considered to be an important factor in the expression of C-reactive protein, which initiates acute in ammation [32]. In some patients with chronic obstructive pulmonary disease, the lungs contain large amounts of C-reactive protein and IL-6, which stimulate vasodilation and aggravate in ammation [33]. In addition, LPS-induced macrophages secrete IL-1β to stimulate the hypothalamus to increase body temperature and promote the innate immune response against bacterial infection [34]. IL-1β can also induce lung neutrophil in ltration and increase lung in ammation in mice with LPS-induced lung injury [35]. This study also found that 10-20 μM isorhapontigenin inhibited the production of IL-1β and IL-6 by in ammatory macrophages. Therefore, isorhapontigenin may be a natural anti-in ammatory compound that can reduce in ammation during bacterial infections.
NF-κB can be translocated to the nucleus where it induces the expression of in ammation-related genes, including those encoding proin ammatory cytokines, iNOS, and COX-2 [7]. LPS binds to the CD14 molecule on macrophages, activating TLR4, which triggers signaling through the NF-κB pathway [36,37]. In inactivated macrophages, IκB (composed of IκB-α and IκB-β subunits) inhibits NF-κB activity, restricting it to the cytoplasm. LPS stimulates IκB phosphorylation and release, allowing NF-κB to enter the nucleus to drive the expression of in ammation-related gene expression [36]. In this current study, isorhapontigenin inhibited IκB phosphorylation and suppressed IκB degradation. Therefore, in in ammatory macrophages treated with isorhapontigenin, most NF-κB remained in the cytoplasm, attenuating the expression of in ammation-related genes. To further con rm that isorhapontigenin can reduce NF-κB translocation into the nucleus of in amed macrophages, we used immuno uorescent staining to demonstrate that isorhapontigenin treatment reduces p65 protein expression in the nucleus of LPS-stimulated macrophages. Recent studies demonstrated that the NF-κB pathway is important in regulating the increased expression of COX-2 and iNOS in activated macrophages [38]. Previous studies have found that MAPK signaling also promotes NF-κB activity in these calls. We found that isorhapontigenin treatment of LPS-stimulated macrophages signi cantly reduced JNK, ERK 1/2, and p38 phosphorylation. Additionally, MAPK inhibitors co-cultured with isorhapontigenin and found that isorhapontigenin addition decreased proin ammatory cytokine expression. Thus, our results show that isorhapontigenin reduces the expression of in ammatory mediators and cytokines by inhibiting the NF-κB and MAPK pathways.

HO-1 protein expression is induced under various stresses. Stimulation of macrophages by in ammation
or oxidative molecules induces massive expression of HO-1 [39]. HO-1 metabolizes heme to produce carbon monoxide, biliverdin, and iron [10]. Resveratrol can stimulate macrophages to express a large amount of HO-1, inhibit the production of iNOS, COX-2, and TNF-α, and reduce the expression of ROS in LPS-induced macrophages [31]. Recent studies demonstrated that Nrf2 is an essential transcription factor for regulated antioxidant pathway [40]. Nrf2 translocation into the nucleus to regulate the expression of HO-1, which enhances the cell's antioxidant ability [12]. In addition, isorhapontigenin added together with MAPK inhibitors could signi cantly attenuate ROS productions in LPS-stimulated macrophages. Therefore, our results indicate that isorhapontigenin promotes the nuclear translocation of Nrf2 and induces HO-1 synthesis in macrophages, thus reducing the levels of ROS via blocked MAPK signal pathways in LPS-stimulated macrophages.

Conclusion
In conclusion, isorhapontigenin reduced proin ammatory cytokine and mediator expression by suppressing of the activation of the MAPK and NF-κB signaling pathways in LPS-stimulated macrophages. Isorhapontigenin could decreased ROS levels by upregulating the Nrf2/HO-1 pathway ( Figure 7). Hence, isorhapontigenin has anti-in ammatory and antioxidant activity and is a potential novel anti-in ammatory agent. The data that support the ndings of this study are available from the corresponding author upon reasonable request.

Competing interests
The authors have no con icts of interest to declare.

Funding
This study was supported in part by grants from the Chang Gung Memorial Hospital (CMRPF1F0083, CMRPF1G0181, CMRPF1H0023, and CMRPF1H0043) and the Chang Gung University of Science and Technology (ZRRPF3K0011 and ZRRPF3K0111-7), and the Ministry of Science and Technology in Taiwan (109-2320-B-255-006-MY3).