Two methoxy derivatives of resveratrol, 3,3′,4,5′-tetramethoxy-trans-stilbene and 3,4′,5-trimethoxy-trans-stilbene, suppress lipopolysaccharide-induced inflammation through inactivation of MAPK and NF-κB pathways in RAW 264.7 cells

Background 3,3′,4,5′-tetramethoxy-trans-stilbene (3,3′,4,5′-TMS) and 3,4′,5-trimethoxy-trans-stilbene (3,4′,5-TMS) are two methoxy derivatives of resveratrol. Previous researches have proved that resveratrol and its analogues have anti-inflammatory effect through suppressing mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signaling pathways. This study aims to study whether 3,3′,4,5′-TMS and 3,4′,5-TMS alleviate inflammation and the underlying mechanism. Methods RAW 264.7 macrophage cells were treated with lipopolysaccharide (LPS) to induce inflammation and pretreated with 3,3′,4,5′-TMS or 3,4′,5-TMS. Cell viability was measured with the 3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Nitric oxide (NO) release was detected by Griess reagent. The secretions of pro-inflammatory cytokines were assessed by ELISA kits. Protein expressions of signaling molecules were determined by Western blotting. Reactive oxygen species (ROS) production was detected by fluorescence staining and malondialdehyde (MDA) assay. Results 3,3′,4,5′-TMS and 3,4′,5-TMS suppressed LPS-induced NO release and pro-inflammatory cytokines (IL-6 and TNF-α) secretions in a dose-dependent manner in RAW 264.7 cells. 3,3′,4,5′-TMS and 3,4′,5-TMS significantly down-regulated the LPS-induced expressions of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), and partially suppressed the activation of MAPK (phosphorylation of p38, JNK, ERK), and NF-κB (phosphorylation of IKKα/β, p65 and IκBα) signaling pathways; where phosphorylation of ERK and p65 was mildly but not significantly decreased by 3,3′,4,5′-TMS. LPS-induced NF-κB/p65 nuclear translocation was inhibited by both 3,3′,4,5′-TMS and 3,4′,5-TMS. Moreover, both resveratrol derivatives decreased the ROS levels. Conclusions 3,3′,4,5′-TMS and 3,4′,5-TMS significantly suppress LPS-induced inflammation in RAW 264.7 cells through inhibition of MAPK and NF-κB signaling pathways and also provide anti-oxidative effect. This study reveals potential therapeutic applications of 3,3′,4,5′-TMS and 3,4′,5-TMS for inflammatory diseases.


Background
Inflammation, widely defined as a nonspecific response to tissue injury or infection, is a complicated and pervasive form of defense and is employed by both innate and adaptive immune systems to combat pathogenic intruders [1]. Macrophage plays a critical role in the initiation, maintenance, and resolution of inflammation and it can be activated by many signals including cytokines [interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), and granulocyte-monocyte colony stimulating factor (GM-CSF)], bacterial lipopolysaccharide (LPS), extracellular matrix proteins, and other chemical mediators [2]. When inflammation is triggered by a pathogen, resident macrophages produce various pro-inflammatory mediators such as nitric oxide (NO) from inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), reactive oxygen species (ROS), TNF-α, and interleukin (IL)-6 [2,3].
To date, only few studies explore the anti-inflammatory effect of 3,4′,5-TMS and even no studies working on 3,3′,4,5′-TMS. In the present study, we used LPS-induced RAW 264.7 macrophages as an in vitro model to investigate the anti-inflammatory effects of 3,3′,4,5′-TMS and 3,4′,5-TMS and the underlying mechanism. The potency was also compared.

Cell culture
RAW 264.7, a mouse macrophage cell line from the American Type Culture Collection (ATCC, Manassas, VA, USA), was cultured in high-glucose DMEM supplemented with 10% FBS plus 100 U/mL penicillin and 100 µg/mL streptomycin at 37 ℃ in a humidified incubator with 5% CO 2 .

Determination of nitric oxide (NO) release
The RAW 264.7 cells were seeded in 24-well plates (2 × 10 5 cells/well) and allowed to adhere overnight at 37 ℃ with 5% CO 2 . The cells were pretreated with 3,3′,4,5′-TMS (10 and 50 µM) or 3,4′,5-TMS (10 and 50 µM) for 4 h and then co-treated with LPS (1 µg/mL) for another 12 h. The release of NO was determined by measuring the accumulated nitrite in the culture medium with Griess reagent according to the manufacturer's instructions. The absorbance was detected at 548 nm using the microplate reader.

Enzyme-linked immunosorbent assay (ELISA)
RAW 264.7 cells (6 × 10 5 cells/well in 6-well plates) were pretreated with different concentrations (10 and 50 µM) of 3,3′,4,5′-TMS or 3,4′,5-TMS for 4 h, followed by the coincubation of LPS (1 µg/mL) for 12 h. The culture media were collected after the drug treatment and the releases of IL-6 and TNF-α from the cells were detected using immunoassay kits (Mlbio, Shanghai, China) according to the manufacturer's protocol. The absorbance values at 450 nm were determined by the SpectraMax M5 microplate reader.

Western blotting analysis
RAW 264.7 cells (6 × 10 5 cells/well in 6-well plates) were pretreated with of 3,3′,4,5′-TMS or 3,4′,5-TMS at 10 and 50 µM for 4 h, followed by the addition of LPS (1 µg/mL) for 12 h. The cells were harvested on ice and lysed with RIPA solution containing 1% Protease Inhibitor Cocktail (Beyotime Biotechnology, Shanghai, China) and 1% phenylmethanesulfonyl fluoride (PMSF). The cell lysates were centrifuged at 15,000 rpm for 30 min at 4 ℃ to collect supernatants. The total protein content was determined by BCA assay (Beyotime Biotechnology, Shanghai, China). Protein samples were separated by 8 or 10% SDS-PAGE gels and the blots were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membrane was blocked by 5% skimmed milk or 1% BSA in Tween-20 phosphate-buffered saline (TBST) buffer, followed by incubation with appropriate primary antibodies overnight at 4 ℃ and with the corresponding secondary antibodies for another 1 h at room temperature. Finally, the specific protein bands were visualized with an American ECL ™ Advanced Western Blotting Detection Kit (GE Healthcare Life Sciences, Uppsala, Sweden) and scanned by ChemiDoc ™ MP Imaging System (BIO-RAD, USA).

Immunofluorescence assay
RAW 264.7 cells (3 × 10 5 cells/confocal dish) were pretreated with 3,3′,4,5′-TMS or 3,4′,5-TMS (50 µM) for 4 h, followed by the co-incubation of LPS (1 µg/mL) for 12 h. The cells were then washed by PBS for 10 min and fixed with 4% PFA at room temperature for 20 min. The fixed cells were permeabilized with 0.1% Triton X-100 for 10 min and blocked with 3% BSA for 1 h at room temperature. Then the cells were incubated with the primary NF-κB p65 antibody (1:500) overnight at 4 ℃. Finally, the cells were washed by PBS for 10 min and were incubated with Alexa Fluor 488-labeled secondary antibody (1:100) for 1 h at 37 ℃. After washed, the cells nuclei were dyed by DAPI. The fluorescence images were captured under the Leica-DMi8 Inverted fluorescence microscope (Leica, Wetzlar, Germany).

MDA assay
RAW 264.7 cells (6 × 10 5 cells/well in 6-well plates) were pretreated with of 3,3′,4,5′-TMS or 3,4′,5-TMS at 10 and 50 µM for 4 h, followed by the addition of LPS (1 µg/mL) for 12 h. The cells were harvested on ice and lysed with RIPA solution containing 1% Protease Inhibitor Cocktail and 1% PMSF. The cell lysates were centrifuged at 15,000 rpm for 30 min at 4 ℃ to collect supernatants. The total protein content was determined by BCA assay. The MDA content was then detected with assay kit according to the manufacturer's instructions. All the results were normalized by the total protein content.

Statistical analysis
All data of experiments are showed as mean ± standard error of mean (SEM) of n independent experiments. Variance between two groups was analyzed using one-way analysis of variance (ANOVA) by GraphPad Prism software (GraphPad Software, United States). P < 0.05 is considered to be statistically significant.
As shown in Fig. 2e, morphological changes in RAW 264.7 cells were obvious. LPS-stimulated RAW 264.7 cells differentiated into an irregular shape with pseudopodia and accelerated spreading as compared to the normal state in the control group. When treated by 3,3' ,4,5'-TMS and 3,4' ,5-TMS in addition, the cells showed less pseudopodia formation and cell spreading.
Inflammation is involved in the development of various deadly illness such as arthritis, arteriosclerosis, cancer, liver diseases, neurological disorder, and renal disorders [20]. Macrophages function to control and clear infections, remove dead cells and derbies, and promote wound healing and tissue repair; nevertheless, prolonged and excessive activation of macrophages contributes to tissue damage and pathology in inflammatory diseases [21]. Upon stimulation of LPS in macrophages, upregulated expression of iNOS results in the burst of NO generation and contributes to the development of inflammation. Cyclooxygenase (COX) with COX-2 as the inducible isoform catalyzes the conversion from arachidonic acid (AA) to prostaglandin E2 (PGE2) which is also a pivotal indicator of inflammation [22]. Cytokines play important roles in regulating an inflammation process and various cells express different cytokines. IL-6 and TNF-α are the most common cytokines expressed in macrophages [23]. The present study showed that 3,3′,4,5′-TMS and 3,4′,5-TMS, two methoxy derivatives of resveratrol, alleviated inflammation through suppressing the secretions of NO, IL-6 and TNF-α and expressions of iNOS and COX-2 in LPS-induced RAW 264.7 cells. These results were in consistent with the previous study reporting that 3,4′,5-TMS possesses anti-inflammatory effect [19]. However, that earlier study only excludes the role of heme oxygenase-1 (HO-1) and the underlying molecular mechanisms remain to be explored. Of note, we were the first to suggest the anti-inflammatory activity of 3,3′,4,5′-TMS.
Our results strongly supported the protective effects of 3,3′,4,5′-TMS and 3,4′,5-TMS against inflammation in LPS-treated macrophages. Next, we examined the underlying mechanism. It has been well established that LPS activates TLR4 and triggers the downstream MAPKs and NF-κB signaling pathways, modulating inflammatory responses in RAW 264.7 cells [24]. JNK, p38, and ERK are kinase modules of MAPK family and participate in many pathological processes such as inflammation, cell apoptosis, gene transcription, and differentiation [5,25,26]. Normally, NF-κB is bound with IκB so that the complex cannot translocate into the nucleus from cytoplasm and maintain in an inactive form. Once the cells being exposed to extracellular stimuli like LPS, rapid phosphorylation by IKK, ubiquitination, and proteolytic degradation will happen to IκB, releasing NF-κB to translocate to the nucleus [27]. As a result, the transcription of a mass of pro-inflammatory cytokines and mediators including IL-6, TNF-α, and COX-2 was triggered [7]. Here we found that 3,3′,4,5′-TMS reduced the phosphorylation levels of JNK and p38 in MAPK pathway as well as the phosphorylation levels of IKKα/β and IκBα in NF-κB pathway. Similarly, 3,4′,5-TMS reduced the phosphorylation of all these proteins: JNK, p38, ERK, IKKα/β, IκBα, and p65. Besides, both 3,3′,4,5′-TMS and 3,4′,5-TMS exerted inhibition effect on the nuclear translocation of NF-κB p65. These results indicated that the anti-inflammatory effects of these two resveratrol derivatives are at least partially mediated through inhibiting MAPK and NF-κB activation. Contradiction about the impact of LPS on the expression of IκBα was found in the previous studies. Some studies showed that LPS treatment effectively suppressed the expression of IκBα [28,29]; whilst other studies showed that LPS increased the phosphorylation of IκBα without affecting the expression of total protein [30,31]. This different result might be caused by the difference in treatment time and/or source of antibodies. Our present results showed that LPS treatment induced the phosphorylation of IκBα which was reversed by 3,3′,4,5′-TMS and 3,4′,5-TMS but  Extensive evidence supported the diverse beneficial effects of resveratrol against various diseases such as cancer, neurological disorders, diabetes, cardiovascular diseases and so on [32]. Regarding the remarkable therapeutic potentials of resveratrol, studies on the derivatives of resveratrol with more potent protective effects have raised attention. Resveratrol is widely known to activate Sirtuin 1 (SIRT1). SIRT1, a NAD+-dependent class III histone deacetylase, has been proved to take part in many pathophysiological processes including antiinflammation via modulating specific proinflammatory mediators [33][34][35]. Furthermore, the deacetylation of NF-κB regulated by SIRT1 can suppress the expression of downstream signaling pathways, thus alleviate inflammation induced by LPS [22,36,37]. Resveratrol is reported to regulate MAPK and NF-κB signaling pathways for its immunomodulating functions [38]. Our recent study confirmed that resveratrol upregulated SIRT1 expression to ameliorate vascular dysfunction associated with diabetes and obesity [39]. Both 3,3′,4,5′-TMS and 3,4′,5-TMS are methoxy derivatives of resveratrol, sharing the similar structure with resveratrol, exhibit the same anti-inflammatory properties via suppressing MAPK and NF-κB signaling pathways. Importantly, 3,3′,4,5′-TMS and 3,4′,5-TMS showed differential effects that they did not upregulate SIRT1 expression.

Conclusions
In summary, our data revealed that 3,3′,4,5′-TMS and 3,4′,5-TMS suppress inflammatory responses in LPSinduced RAW 264.7 cells through the inhibition of MAPK and NF-κB pathways. Further in vivo studies on these two methoxy derivatives of resveratrol are warranted to develop them into therapeutic applications for inflammatory disorders considering that 3,3′,4,5′-TMS and 3,4′,5-TMS have great potential for treatment of inflammation.