NLRP3; NF-κB

Advanced glycation end products (AGEs) could interact with the receptor for AGE (RAGE) as a sterile danger signal to induce inflammation. 4′-methoxyresveratrol (4′MR), a polyphenol derived from Dipterocarpaceae, has not been studied for its anti-inflammation effects. In the present study, we sought to explore the protective role of 4′MR in AGEs-induced inflammatory model using RAW264.7 macrophages. 4′MR significantly inhibited gene expression of pro-inflammatory cytokines and chemokines, such as interleukin 1β (IL-1β), interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α) and monocyte chemoattractant protein-1 (MCP-1), as well as two typical pro-inflammatory enzymes, inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX2). Besides, 4′MR significantly decreased oxidative stress, demonstrated by levels of ROS production, protein carbonyl and advanced oxidation protein product via down-regulation of NADPH oxidase. Further analysis showed that 4′MR attenuated the RAGE overexpression induced by MGO-BSA. It also blocked the downstream signal of AGE-RAGE, particularly, MAPKs including p38 and JNK, and subsequently reduced NF-κB activation. Additionally, 4′MR significantly abated the activation of NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome including NLRP3 and cleaved caspase-1 and reduced the secretion of mature IL-1β. Taken together, our results suggest that the anti-inflammatory effect of 4′MR is mainly through suppressing RAGE-mediated MAPK/NF-κB signaling pathway and NLRP3 inflammasome activation. 4′MR could be a novel therapeutic agent for inflammation-related diseases.[1]

Intracellular ROS Production Measurement[1]
The effect of 4'-O-Methylresveratrol on ROS production was determined by a method according to our previous study. For quantification of intracellular ROS level, RAW264.7 macrophages were plated in a 96-well plate for 24 h, and then further stimulated with 200 µg/mL BSA or MB with or without 10 µM of 4'-O-Methylresveratrol for 24 h. After the cultivation, supernatant was aspirated and cells were washed by warm PBS twice. Then cells were incubated with 25 µM 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) in DF10 for 1 h at 37 °C. After that, cells were washed with warm PBS twice. Finally, the fluorescence intensity of DCFH-DA-activated cells was determined by emission at 485 nm and excitation at 528 nm employing a microplate reader after adding 200 µL HBSS to each well. Results were expressed as a percentage of non-glycated BSA.

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Protein Carbonyl and AOPP Levels Measurement[1]
After treatment with 200 µg/mL BSA or MB with or without 10 µM 4'-O-Methylresveratrol for 24 h, lysates from the cell culture were prepared using a homogenizer. The concentration of total protein was measured by a BCA protein assay kit. Protein carbonyl expressed as nmol per mg total protein measured by a Protein Carbonyl Assay Kit according to the manufacturer’s protocol. The content of advanced oxidation protein products (AOPP) was determined as described by our previous study. In brief, the standard curve was draw by 0, 10, 20, 40, 80, and 100 µmol/L chloramine-T and 300 µL of protein lysates were used for sample detection. Seventy-five microliters of 1.16 M KI and 150 µL of acetic acid were added into the tubes which served as reactants with chloramine-T or samples. After that, their absorbance was measured by microplate reader at 340 nm immediately. The AOPP quantity was calculated with respect to chloramine-T and results showed in nmol of chloramine-T equivalent per mg total protein.

RNA Isolation and qPCR Analysis[1]
RAW264.7 macrophages (106 cells/well) were cultured in DF10 supplemented with 200 µg/mL BSA or MB with or without 10 µM 4'-O-Methylresveratrol for 24 h, the total RNAs were extracted with a standard TRIzol method according to the manufacturer’s instructions. The concentrations of extracted RNAs were measured by a spectrophotometer. The cDNA synthesis was performed with reverse transcription by a PrimeScript RT reagent Kit according to the manufacturer’s instructions. A real-time PCR quantitation of target genes was performed using QuantiFast SYBR-Green RT-PCR kits. 18S expression was used as an internal control. The sequences of forward and revere primers used are shown in Appendix A (Table A1). The measurements were analyzed using the ΔCT method with LightCycler 96 in the qPCR apparatus.

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Measurement of NO and IL-1β Levels[1]
RAW264.7 macrophages (106 cells/well) were cultured in DF10 supplemented with 200 µg/mL BSA or MB with or without 10 µM 4'-O-Methylresveratrol for 24 h, and supernatants were collected for nitric oxide (NO) and interleukin 1β (IL-1β) measurement. Levels of NO and IL-1β were determined by a mouse NO and IL-1β ELISA kit according to the manufacturer’s instructions.

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Protein Extraction and Western Blot Analysis[1]
RAW264.7 macrophages (106 cells/well) were cultured in DF10 supplemented with 200 µg/mL BSA or MB with or without 10 µM 4'-O-Methylresveratrol for 45 min after being starved for 4 h in six-well dishes. Then, cells were washed with pre-cooled PBS twice and lysed with 200 µL of RIPA lysis buffer on ice for 15 min. The lysates were subsequently transferred into 2 mL tubes for collecting supernatant by centrifugation (12,000× g, 10 min, 4 °C). The concentration of protein was determined by a BCA protein assay kit. A total of 30 µg protein was run per lane on SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked for 1 h at room temperature by 5% fat-free milk and incubated with primary antibodies (ERK1/2, p-ERK1/2, JNK, p-JNK, p38 MAPK, p-p38 MAPK, p65, p-p65, NLRP3, and cleaved caspase-1 were used at 1:1000; RAGE and β-actin were used at 1:3000) overnight at 4 °C accompanied by soft shaking. After washing 3 times per 5 min with PBST, the PVDF membranes were then incubation for 30 min by a HRP-conjugated secondary antibody (1:1000) at room temperature. Finally, immunoreactive bands were visualized with enhanced chemiluminescence (ECL) reagents and detected by ChemiDoc® MP Image Lab. The image Lab Software was used for quantifying band densities.

[1]. 4'-Methoxyresveratrol Alleviated AGE-Induced Inflammation via RAGE-Mediated NF-κB and NLRP3 Inflammasome Pathway. Molecules. 2018 Jun 14;23(6).

5-[2-(4-methoxyphenyl)ethenyl]benzene-1,3-diol is a stilbenoid.
(E)-5-(4-Methoxystyryl)benzene-1,3-diol has been reported in Alpinia hainanensis, Dracaena cochinchinensis, and other organisms with data available.

C15H14O3

242.26986

242.094

C, 74.36; H, 5.82; O, 19.81

33626-08-3

33626-08-3

6255462

White to off-white solid powder

1.252

446.5±14.0 °C at 760 mmHg

223.8±20.1 °C

0.0±1.1 mmHg at 25°C

1.692

3.62

2

3

3

18

259

0

COC1=CC=C(C=C1)C=CC1=CC(O)=CC(O)=C1

IHVRWFJGOIWMGC-NSCUHMNNSA-N

InChI=1S/C15H14O3/c1-18-15-6-4-11(5-7-15)2-3-12-8-13(16)10-14(17)9-12/h2-10,16-17H,1H3/b3-2+

5-[(E)-2-(4-methoxyphenyl)ethenyl]benzene-1,3-diol

4'-O-Methylresveratrol; 33626-08-3; (E)-5-(4-Methoxystyryl)benzene-1,3-diol; Desoxyrhapontigenin; RESVERATROL 4'-METHYL ETHER; 4'O-Methylresveratrol; 4-Methoxyresveratrol; Deoxyrhapontigenin; 3,5-Dihydroxy-4'-methoxystilbene;

2934.99.03.00

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 48~50 mg/mL (198.1~206.4 mM)
Ethanol: ~48 mg/mL (~198.1 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (10.32 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (10.32 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)