Anti-inflammatory effect of oligostilbenoids from Vitis heyneana in LPS-stimulated RAW 264.7 macrophages via suppressing the NF-κB activation

Background Vitis heyneana is widely distributed in the north of Vietnam, it has been used in Vietnamese traditional medicine as an agent for treatment of arthritis, bronchitis, carbuncles and inflammatory conditions, and menstrual irregularities. However, this plant has not been investigated in phytochemical constituents and biological effects, especially in the anti-inflammatory property. Results Bioassay-guided fractionation of the EtOAc soluble fraction from the aerial part of Vitis heyneana resulted in the isolation of a series of oligostilbenoids as piceid (1), 2-r-viniferin (2), betulifol A (3), vitisinol C (4), (-)-trans-ε-viniferin (5), α-viniferin (6), shoreaketon (7), amurensin B (8), vitisinol B (9), and cis-vitisin B (10). Compound 5 showed the most potent inhibitory activities by suppressing LPS-induced COX-2 expression and PGE2 production. This compound exhibited significantly reduced LPS-induced nitric oxide (NO) release in a dose-dependent manner. These effects are accompanied with the inhibition of transcription factor NF-κB activation. Conclusion The results suggested that trans-ε-viniferin exerts anti-inflammatory effects via suppression the NF-κB activation in RAW 264.7 cells.


Background
The genus Vitis (family Vitaceae) is in the major group with more than 66 species identified and is distributed throughout the world [1]. In Vietnam, 6 species of Vitis genus have been reported until now, including V. larbusca L., V. heyneana Roem, & Schult., V. retordii Roman du Caill. Ex Planch. V. vinifera L., V. balansana Planch., V. flexuosa Thunb [2]. Typical constituents in Vitis genus have been reported to be oligomers of resveratrol named stilbenoids [3][4][5][6][7]. To date, over 1000 stilbenoids have been identified in various plant families such as Celastraceae, Cyperaceae, Fabaceae, Iritaceae, Moraceae, Paeoniaceae, and Vitaceae. The Vitaceae family includes about 900 species within 14-17 genera, primarily in tropical climate [8,9]. Of these genera, only five, the Ampelopsis, Cissus, Cyphostemma, Parthenocissus, and Vitis genera reported the presence of stilbenoids. However, chemical constituents of the species in Vitis genus have been studied the most. Stilbenoids possessed various pharmacological activities such as antioxidative, anti-inflammatory, and antimicrobial activities, as well as having cardioprotective, hepatoprotective, and neuroprotective effects [10][11][12][13][14]. Although approximately 100 stilbenoid monomers, dimers, and oligomers have been found in all Vitis species, research on the chemical compositions and biological activities of Vitis genus remains lacking [15]. Our screening antiinflammatory effect of an ethanol extract of 4 Vitis species including V. heyneana Roem. & Schult. (VH), Vitis vinifera L. (VV); Vitis balansana Planch. (VB), and Vitis labrusca L. (VL) collected in Vietnam via suppression of LPS-induced COX-2 expression found that 96% of ethanol extract of VH exhibited the most activity [16]. Besides, V. heyneana is widely distributed in northern Vietnam, for example in Cao Bang, Lang Son, and Lao Cai provinces. The stems and roots of this species are traditionally used for the treatment of arthritis, bronchitis, carbuncles and inflammatory conditions, and menstrual irregularities in Vietnamese indigenous peoples [17]. So far, few studies have been done to investigate the chemical constituents of VH. Currently, only a few studies have referred to the presence of stilbenoids in VH [17][18][19]. However, evaluation of anti-inflammatory activities of the VH species have not been studied yet. This study reports the anti-inflammatory effects of VH extracts and its isolated oligostilbenoids via suppressing LPS-induced COX-2 expression, PGE2 and NO productions, and NF-κB activation in RAW 264.7 macrophages.

Screening inhibitory activities
To study the cytotoxic effects of extract and its fractions on cell viability, the RAW 264.7 cells were incubated and treated a concentration of all materials (50 µg/mL). The results showed that all the extract and fractions that induced cell toxicities were negligible at the above concentrations [16]. In order to examine candidate extract/ fractions inhibiting COX-2 in RAW 264.7 cells, ethanol V. heyneana extract (VH) and its fractions (n-hexane-VHH, ethyl acetate-VHE and water-VHW) were tested on COX-2 mRNA expression level by qPCR. As shown in Fig. 1, the inhibitory effect of COX-2 mRNA was mostly potently suppressed by VHE and VH.

Screening inhibitory activities of oligostilbenoids by suppressing LPS-induced COX-2 expression and PGE2 production in RAW 264.7 macrophages
The effects of all isolated compounds at concentration of 10 µM, and a reference agent, meloxicam (20 µM) were further examined on LPS-induced COX-2 protein, mRNA expression and LPS-induced PGE2 production in RAW 246.7 cells by reported method with slight modification. As the results in Fig. 3, western blotting and real time-PCR assays revealed that, among the active metabolites, trans-ε-viniferin (5; 10 µM) potently suppressed LPS-induced COX-2 expression in RAW 246.7 cells (Fig. 3a, b). Furthermore, ELISA assay confirmed that 5 most inhibited the level of LPS-induced PGE2 production in RAW 264.7 cells (Fig. 3c).

Effects of 5 on COX-2, iNOS expression and PGE2 production in RAW 264.7 macrophages
Considering that the 5 was the most active metabolite, the effects of this compound was investigated on LPSinduced PGE2 production in RAW 264.7 cells using ELISA method with slight modification. As the results in Fig. 4a show, stimulation of cells resulted in the increase of PGE2 production compared with unstimulated vehicle cells. In the administration of 5 (1-30 µM), the PGE2 productions were remarkably reduced in a concentration dependent manner (p < 0.05). We further investigated whether the inhibition effect by the same range of concentration of 5 was related to the gene expression,   3'
These results were also confirmed by dose dependent manner.

Effect of 5 on NO productions and NF-κB activation
As shown in Fig. 5a, 5 (Code VH07, 1-30 µM) significantly inhibited LPS-induced NO production in a dose dependent manner. Especially, at the highest concentration (30 µM), this compound could decrease the amount of NO production more than 2-fold compared to unstimulated vehicle. Since NF-κB was identified as an important transcription factor that controls several pro-inflammatory mediations, we investigated the NF-κB transcription activity by performing luciferase reporter gene assay and the results are shown in Fig. 5b. Compound 5 (1-30 µg/mL) dose dependent reduced LPSinduced NF-κB transitivity (p < 0.05).

Discussion
Vitis sp. is widely distributed and has been used as the raw material for juice and wine all over the world. For the pharmaceutical application, it has been reported that the roots, stems and leaves possessed anti-inflammatory, antioxidants, and anti-tumour activities, and contains a number of stilbenoid and resveratrol oligomers. Previously, resveratrol (3,4′,5-trihydroxystilbene) isolated mostly from Vitis sp. as a main metabolite with high concentration, was shown to play an important role in human health with extremely extensive bioactivities such as anti-bacterial, anti-thrombotic, anti-oxidation, anti-inflammatory, reduce hypertension, and especially anti-cancer [9][10][11][12][13]. In addition, several studies focused on resveratrol oligomers that are characterized by the polymerization of several resveratrol units [9][10][11][12][13]. In this study, we demonstrated the potential involvement of the NF-κB pathway in the anti-inflammation of metabolites from V. heyneana. Among the active components, 5 was found to be the most anti-inflammatory activity  in suppression of NO, PGE2, and COX-2 production in LPS-stimulated RAW 264.7 macrophage cells. We also found that, this oligostilbenoid inhibited LPS-induced NF-κB activation. RAW 264.7 cells are the murine macrophage cells line that plays an important testable model for anti-inflammatory agent nowadays. When the macrophage cells were activated by LPS, a number of cytokines were released and recordable. Among them, NO is an inflammation mediator, in which, NO produced by iNOS coursed toxicity to cells and directly concern to the pathogenesis of inflammation process. COX-1 and COX-2 are the isozymes that convert arachidonic acid to prostaglandin, however, COX-2 responses mainly to produce a huge amount of PGEs in macrophage cells. Inhibition of the above productions may be the effective method for the treatment of several types of inflammation. Of our experiments, this is the first report confirming that oligostibene from V. heyneana suppresses the LPS-induced inflammatory response by activating NF-κB in vitro.
(-)-Trans-ε-viniferin was first isolated from V. vinifera and classified as a model for its bio-synthesis from resveratrol. Similar to resveratrol, this dimer was shown to have several biological properties such as anti-oxidant, antidepressant, and anti-adipogenesis [27][28][29][30]. This compound showed cytotoxicity in several cancer cell lines as C6, Hep G2, HeLa, and MCF-7n and exerted the anti-proliferative and pro-apoptotic effect in U266, RPMI8226, Jurkat, K562 and U937 and other cancer cell lines [4,31]. (-)-Trans-ε-viniferin and its derivative compounds slightly reduced cell proliferation on human adenocarcinoma colon cells and could constitute new putative anti-cancer agents on colon carcinoma [32]. (-)-Transε-viniferin significantly attenuated mutant Htt-induced depletion of SIRT3 and protected cells from mutant Htt. This compound also decreased levels of reactive oxygen species and prevented loss of mitochondrial membrane potential in cells expressing mutant Htt [33]. The other form, α-viniferin, also down-regulated the LPS-induced expression of pro-inflammatory genes such as iNOS and COX-2 by suppressing the activity of NF-κB via dephosphorylation of Akt/PI3K. This compound suppressed NO and PGE2 production in the late stage of inflammation through induction of heme oxygenase-1 (HO-1), and the expression of pro-inflammatory genes iNOS and COX-2 in the early stage of inflammation by inhibiting the Akt/ PI3K-dependent NF-κB activation in BV2 microglial cells [34]. The V. thunbergii extract that was rich in (-)-transɛ-viniferin significantly inhibited PGE2 production in LPS-induced PHCs cells without exhibiting significant cytotoxicity [35].
Even though (-)-trans-ɛ-viniferin and its isomers have been shown to have several anti-inflammatory effects, the molecular mechanism underlying the anti-inflammatory in LPS-induced RAW 264.7 has not been completely elucidated thus far. The results of our research indicate that among the active components, treating the RAW 264.7 macrophage cells with several concentrations of (-)-trans-ɛ-viniferin (5) could inhibit LPS-induced NO, PGE2, iNOS, COX-2 productions in a dose dependent manner. With the highest concentration at 30 µM, this oligostibene significantly reduced the above productions more than 50% as compared to the LPS-treated cell alone.

Plant materials
The aerial parts of V. heyneana were collected from Lao Cai province (north of Vietnam) in September 2016 and botanically identified by Assoc. Prof. Dr. Nguyen The Cuong, Institute of Ecology and Biological Resources. A voucher specimen (TL07) has been deposited at the Herbarium of IEBR and Department of Phytochemistry, Hanoi, Vietnam.

Western blot analysis
RAW 264.7 cells were seeded in 6 well-plate and treated with extractions or compounds from V. heyneana. Cells were collected and washed with cold phosphate-buffered saline (PBS). The collected cells were lysed on ice for 30 min in 100 μL lysis buffer [120 mM NaCl, 40 mM Tris (pH 8), 0.1% NP40 (Nonidet P-40)] and centrifuged at 12,000 rpm for 30 min. BCA protein assay kit (Pierce, Rockford, IL) was used to measure protein concentrations as described in a previous report [36]. Finally, bands were detected by ECL kit (Pierce West Femto), and images were acquired using the Odyssey Fc Imager (LI-COR Biosciences).

Measurement of nitrite
RAW 264.7 cells (5 × 10 5 cells) were pre-incubated at 37 °C for 12 h in serum-free medium and NO production was monitored by measuring nitrite levels in culture media using Griess reagent (1% sulfanilamide, 0.1% N-1-naphthylenediamine dihydrochloride, and 2.5% phosphoric acid). Absorbance was measured at 540 nm after incubating for 10 min.

Reporter gene assay
One µg of the plasmid NF-κB or 50 ng of pRL Renilla was transfected into the cells using LipofectAMINE2000 (Invitrogen Corp., Carlsbad, CA) using the Dual-Luciferase Reporter Assay Systems (Promega, Madison, WI, USA). After 6 h, the transfection medium was replaced with the DMEM without serum and the cells were further incubated for 18 h. The firefly and hRenilla luciferase activity was detected using a multilaber counter. Ratio of activity was determined by normalizing the promoterdriven luciferase activity versus hRenilla luciferase.

Enzyme-linked immunosorbent assay (ELISA)
Prostaglandin E2 (PGE2) concentrations in DMEM media were measured by ELISA kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer's protocols.

Extraction and isolation
The aerial part of V. heyneana (5.0 kg) was extracted with EtOH 96% (3 h × 3L) at 50 °C. The combined extracts were filtered and evaporated under pressure to give green residue (235.5 g) which was suspended in water and partitioned with organic solvent to get n-hexane (26.

Statistics
Values are presented as mean ± SE unless otherwise stated. P-values were calculated by Student's t test or one-way analysis of variance followed by Bonferroni post hoc testing using GraphPad Prism 5 (GraphPad Software Inc.). P < 0.05 was considered statistically significant. Data are expressed as mean ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Conclusion
This the first time that a Vietnamese V. heyneana extract and its phytochemical constituents have been reported to possess an anti-inflammatory activity. The results demonstrated that one of the most active compounds, (-)-trans-ɛ-viniferin, decreased NO, PGE2, iNOS, and COX-2 productions in RAW 264.7 macrophage cells after LPS stimulation. This anti-inflammatory activity may mediate by the NF-κB activation mechanism in the RAW 264.7 cells. However, to imply that this Vitis sp. and its components may be useful in the prevention or treatment of inflammatory diseases may be premature as further studies and confirmation of these results are required.