Biological and computational evaluation of resveratrol inhibitors against Alzheimer’s disease

It has been reported that beta amyloid induces production of radical oxygen species and oxidative stress in neuronal cells, which in turn upregulates β-secretase (BACE-1) expression and beta amyloid levels, thereby propagating oxidative stress and increasing neuronal injury. A series of resveratrol derivatives, known to be inhibitors of oxidative stress-induced neuronal cell death (oxytosis) were biologically evaluated against BACE-1 using homogeneous time-resolved fluorescence (TRF) assay. Correlation between oxytosis inhibitory and BACE-1 inhibitory activity of resveratrol derivatives was statistically significant, supporting the notion that BACE-1 may act as pivotal mediator of neuronal cell oxytosis. Four of the biologically evaluated resveratrol analogs demonstrated considerably higher activity than resveratrol in either assay. The discovery of some “hits” led us to initiate detailed docking studies associated with Molecular Dynamics in order to provide a plausible explanation for the experimental results and understand their molecular basis of action.     

Activation of Sirt1 by resveratrol inhibits TNF-α induced inflammation in fibroblasts

Inflammation is one of main mechanisms of autoimmune disorders and a common feature of most diseases. Appropriate suppression of inflammation is a key resolution to treat the diseases. Sirtuin1 (Sirt1) has been shown to play a role in regulation of inflammation. Resveratrol, a potent Sirt1 activator, has anti-inflammation property. However, the detailed mechanism is not fully understood. In this study, we investigated the anti-inflammation role of Sirt1 in NIH/3T3 fibroblast cell line. Upregulation of matrix metalloproteinases 9 (MMP-9), interleukin-1beta (IL-1β), IL-6 and inducible nitric oxide synthase (iNOS) were induced by tumor necrosis factor alpha (TNF-α) in 3T3 cells and resveratrol suppressed overexpression of these pro-inflammatory molecules in a dose-dependent manner. Knockdown of Sirt1 by RNA interference caused 3T3 cells susceptible to TNF-α stimulation and diminished anti-inflammatory effect of resveratrol. We also explored potential anti-inflammatory mechanisms of resveratrol. Resveratrol reduced NF-κB subunit RelA/p65 acetylation, which is notably Sirt1 dependent. Resveratrol also attenuated phosphorylation of mammalian target of rapamycin (mTOR) and S6 ribosomal protein (S6RP) while ameliorating inflammation. Our data demonstrate that resveratrol inhibits TNF-α-induced inflammation via Sirt1. It suggests that Sirt1 is an efficient target for regulation of inflammation. This study provides insight on treatment of inflammation-related diseases.     

4,4′-Dihydroxy-trans-stilbene,a resveratrol analogue,exhibited enhanced antioxidant activity and cytotoxicity

Resveratrol (3,5,4′-trans-trihydroxystibene) is a natural phytoalexin present in grapes and red wine, which possesses a variety of biological activities including antioxidant activity. In order to find more active antioxidant with resveratrol as the lead compound we synthesized 4,4′-dihydroxy-trans-stilbene (4,4′-DHS). The antioxidant activities of resveratrol and 4,4′-DHS were evaluated by the reaction kinetics with galvinoxyl radical or Cu(II) ions, and the inhibition effects against free-radical-induced peroxidation of human erythrocyte ghosts. It was found that 4,4′-DHS exhibits remarkably higher antioxidant activity than resveratrol. The oxidative products of resveratrol and 4,4′-DHS in the presence of Cu(II) in acetonitrile were identified as the dihydrofuran dimers by spectroscopic method, and the antioxidant mechanism for 4,4′-DHS was proposed. In addition, 4,4′-DHS exhibits remarkably higher cytotoxicity against human promyelocytic leukemia (HL-60) cells than resveratrol.     

Probing the binding sites of resveratrol,genistein, and curcumin with milk β-lactoglobulin

We determined the binding sites of curcumin (cur), resveratrol (res), and genistein (gen) with milk β-lactoglobulin (β-LG) at physiological conditions. Fourier transform infrared spectroscopy, circular dichroism, and fluorescence spectroscopic methods as well as molecular modeling were used to determine the binding of polyphenol–protein complexes. Structural analysis showed that polyphenols bind β-LG via both hydrophilic and hydrophobic contacts with overall binding constants of K_{curcumin–β-LG}?=?4.4 (±?.4)?×?10⁴ M^?1, K_{resveratrol–β-LG}?=?4.2 (±?.2)?×?10⁴ M^?1, and K_{genistein–β-LG}?=?1.2 (±?.2)?×?10⁴?M^?1. The number of polyphenol molecules bound per protein (n) was 1 (cur), 1.1 (res), and 1 (gen). Molecular modeling showed the participation of several amino acid residues in polyphenol–protein complexation with the free binding energy of ?12.67 (curcumin–β-LG), ?12.60 (resveratrol–β-LG), and ?10.68?kcal/mol (genistein–β-LG). The order of binding was cur?>?res?>?gen. Alteration of the protein conformation was observed in the presence of polyphenol with a major reduction of β-sheet and an increase in turn structure, causing a partial protein structural destabilization. β-LG might act as a carrier to transport polyphenol in vitro.     

TRAF6 is functional in inhibition of TLR4-mediated NF-κB activation by resveratrol

Resveratrol was suggested to inhibit Toll-like receptor (TLR)4-mediated activation of nuclear factor-κB (NF-κB) and Toll/interleukin-1 receptor domain-containing adaptor inducing interferon-β (TRIF)–(TANK)-binding kinase 1, but the myeloid differentiation primary response gene 88–tumor necrosis factor receptor-associated factor 6 (TRAF6) pathway is not involved in this effect. However, involvement of TRAF6 in this process is still elusive since cross talk between TRIF and TRAF6 has been reported in lipopolysaccharide (LPS)-induced signaling. Using RAW 264.7 macrophages, we determined the effect of resveratrol on LPS-induced TRAF6 expression, ubiquitination as well as activation of mitogen-activated protein (MAP) kinases and Akt in order to elucidate its involvement in TLR4 signaling. LPS-induced transient elevation in TRAF6 mRNA and protein expressions is suppressed by resveratrol. LPS induces the ubiquitination of TRAF6, which has been reported to be essential for Akt activation and for transforming growth factor-β activated kinase-1–NAP kinase kinase 6 (MKK6)-mediated p38 and c-Jun N-terminal kinase (JNK) activation. We found that resveratrol diminishes the effect of LPS on TRAF6 ubiquitination and activation of JNK and p38 MAP kinases, while it has no effect on the activation of extracellular-signal-regulated kinase (ERK)1/2. The effect of resveratrol on MAP kinase inhibition is significant since TRAF6 activation was reported to induce activation of JNK and p38 MAP kinase while not affecting ERK1/2. Moreover, Akt was identified previously as a direct target of TRAF6, and we found that, similarly to MAPKs, phosphorylation pattern of Akt followed the activation of TRAF6, and it was inhibited by resveratrol at all time points. Here, we provide the first evidence that resveratrol, by suppressing LPS-induced TRAF6 expression and ubiquitination, attenuates the LPS-induced TLR4–TRAF6, MAP kinase and Akt pathways that can be significant in its anti-inflammatory effects.     

3,4,4′-Trihydroxy-trans-stilbene,an analogue of resveratrol,is a potent antioxidant and cytotoxic agent

Abstract

Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is a naturally occurring polyphenol widely distributed in food and dietary plants. This phytochemical has been intensively studied as an efficient antioxidant and anticancer agent, and a variety of substituted stilbenes have been developed in order to improve the potency of resveratrol. In this work, we described the synthesis of 3,4,4 -trihydroxy-trans-stilbene (3,4,4′-THS), an analogue of resveratrol, and studied its antioxidant and cytotoxic activity in vitro. 3,4,4 -THS was much more efficient than resveratrol in protecting against free radical-induced lipid peroxidation, photo-sensitized DNA oxidative damage, and free radical-induced hemolysis of human red blood cells. More potent growth inhibition in cultured human leukemia cells (HL-60) was also observed for 3,4,4 -THS. The relationship between the antioxidant efficiency and cytotoxic activity was discussed, with the emphasis on inhibition of the free radical enzyme ribonucleotide reductase by antioxidants. The result that this subtle structure modification of resveratrol drastically improves its bioactivity provides important strategy to develop novel resveratrol-based molecules.     

NF-κB inhibitory action of resveratrol: A probable mechanism of neuroprotection in experimental diabetic neuropathy

Resveratrol has shown array of biological actions, and is under clinical development for various disease conditions. The etiology of diabetic neuropathy revolves around oxidative stress, AGE formation, lipid peroxidation etc. All these stimulate inflammatory processes and NF-κB cascade is considered as one of the major players of inflammatory response. Activation of NF-κB results in elevated levels of inflammatory mediators. COX-2 and TNF-α activity have also been correlated with inflammatory damage in the pathophysiology of diabetic neuropathy (DN). Therefore we investigated the effect of resveratrol on NF-κB inflammatory cascade, COX-2, TNF-α and IL-6 levels in experimental DN.We found that resveratrol protected against various functional and behavioral deficits in diabetic neuropathy in line with our earlier published reports. In this study we found that the resveratrol treatment decreased the expression of p65 and IκB-α in treated rats. Treatment also ameliorated the elevated levels of TNF-α, IL-6 and COX-2. Resveratrol treatment produced significant decrease in nerve MDA levels in treated animals which may also be contributing to reduction in neuro-inflammation. This study confirms the NF-κB inhibitory activity and anti-inflammatory activity of resveratrol which may contribute to neuroprotection in diabetic neuropathy apart from its antioxidant effect.     

Protective effects of resveratrol on postmenopausal osteoporosis： regulation of SIRT1-NF-κB signaling pathway

Postmenopausal osteoporosis severely jeopardizes human health. Seeking for therapeutic drugs without side effects is of great necessity. Our study was designed to investigate whether resveratrol, an agonist of SIRT1, could have favor- able effect on osteoporosis and to explore the underlying mechanisms. Rat osteoporosis model （ovariectomy group, OVX） was established by bilateral ovariectomy. Three dif- ferent doses of resveratrol were used： 5 mg/kg/d （low- dosed, RESCD）, 25 mg/kg/d （medium-dosed, RESMD）, and 45 mg/kg/d （high-dosed, RESUD）. Results showed that RESLD did not show any significant effect on OVX altera- tions, while RESMD and RESXD significantly elevated the decreased bone mineral density induced by osteoporosis （RESMD 0.205±0.023, RESm） 0.214 ± 0.053 vs. OVX 0.165 ± 0.050 g/cm2 respectively; P 〈 0.05）. Serum markers alkaline phosphatase （ALP） and osteocalcin were moderately restored by resveratrol. Moreover, resveratrol improved bone structure in OVX rats, demonstrated by hematoxylin-eosin staining and micro-computed tomo- graphic results. In vitro results revealed that resveratrol promoted osteoblast differentiation of bone marrow mesen- chymal stromal cells, evidenced by the increase of ALP generation and mRNA expression of collagen 1 （P 〈 0.05; RESMD, RESm） vs. control group）. SIRT1 gene silencing by siRNA transfection blocked these beneficial effects of resveratrol （P〈0.05; RES ＋ SIRT1^KD vs RES^HD）. Western blot results showed that resveratrol activated SIRT1 and subsequently suppressed the activity of NF-κB with decreased expression level of p-IκBa and NF-κB p65 （P〈 0.05）. Our findings verified the effects of specific dosed resveratrol on postmenopausal osteoporosis through osteoblast differentiation via SIRT1-NF-κB signaling pathway. This study suggested the therapeutic potential of resveratroi against osteoporosis and stressed the importance of effective doses.     

Synthesis of 4′-ester analogs of resveratrol and their evaluation in malignant melanoma and pancreatic cell lines

4′-Ester analogs of the disease preventative agent resveratrol were synthesized and evaluated for their potential as anti-melanoma and pancreatic cancer agents. A decarbonylative Heck coupling was used to assemble the protected stilbene core structure. The 4′-acetate and the palmitoate analogs demonstrated selective activity with DM443 and DM738 cells over normal NHDF cells.     

Soluble polyphenols: Synthesis and bioavailability of 3,4′,5-tri(α-d-glucose-3-O-succinyl) resveratrol

We report the development of a chemical modification method of general applicability to polyphenols, which increases solubility to influence absorption. Glucosyl groups were added to the resveratrol kernel via a succinate linker, yielding 3,4′,5-tri-(α-d-glucose-3-O-succinyl) resveratrol. The construct was only slowly hydrolyzed in acid and at pH 6.8, but it was destroyed by blood esterases in less than 1 h. In rats its administration resulted in a blood concentration versus time curve shifted to longer times in comparison to resveratrol, a useful modulation of pharmacokinetics. The area-under-curve parameter and the metabolite mix were similar to those of resveratrol. The method may be advantageously employed to solubilize other polyphenols and to make them more palatable.     

Synthetic resveratrol aliphatic acid inhibits TLR2-mediated apoptosis and an involvement of Akt/GSK3β pathway

As resveratrol derivatives, resveratrol aliphatic acids were synthesized in our laboratory. Previously, we reported the improved pharmaceutical properties of the compounds compared to resveratrol, including better solubility in water and much tighter binding with human serum albumin. Here, we investigate the role of resveratrol aliphatic acids in Toll-like receptor 2 (TLR2)-mediated apoptosis. We showed that resveratrol aliphatic acid (R6A) significantly inhibits the expression of TLR2. In addition, overexpression of TLR2 in HEK293 cells caused a significant decrease in apoptosis after R6A treatment. Moreover, inhibition of TLR2 by R6A decreases serum deprivation-reduced the levels of phosphorylated Akt and phosphorylated glycogen synthase kinase 3β (GSK3β). Our study thus demonstrates that the resveratrol aliphatic acid inhibits cell apoptosis through TLR2 by the involvement of Akt/GSK3β pathway.     

Chronic estradiol-17β exposure increases superoxide production in the rostral ventrolateral medulla and causes hypertension: reversal by resveratrol

Women are exposed to estrogen in several forms, such as oral contraceptive pills and hormone replacement therapy. Although estrogen was believed to be cardioprotective, lately, its beneficial effects are being questioned. Recent studies indicate that oxidative stress in the rostral ventrolateral medulla (RVLM) may play a role in the development of hypertension. Therefore, we hypothesized that chronic exposure to low levels of estradiol-17β (E(2)) leads to hypertension in adult-cycling female Sprague Dawley (SD) rats potentially through generation of superoxide in the RVLM. To test this hypothesis, young adult (3 or 4 mo old) female SD rats were either sham-implanted or implanted (subcutaneously) with slow-release E(2) pellets (20 ng/day) for 90 days. A group of control and E(2)-treated animals were fed lab chow or chow containing resveratrol (0.84 g/kg of chow), an antioxidant. Rats were implanted with telemeters to continuously monitor blood pressure (BP) and heart rate (HR). At the end of treatment, the RVLM was isolated for measurements of superoxide. E(2) treatment significantly increased mean arterial pressure (mmHg) and HR (beats/min) compared with sham rats (119.6 ± 0.8 vs. 105.1 ± 0.7 mmHg and 371.7 ± 1.5 vs. 354.4 ± 1.3 beats/min, respectively; P < 0.0001). Diastolic and systolic BP were significantly increased in E(2)-treated rats compared with control animals. Superoxide levels in the RVLM increased significantly in the E(2)-treated group (0.833 ± 0.11 nmol/min·mg) compared with control (0.532 ± 0.04 nmol/min·mg; P < 0.05). Treatment with resveratrol reversed the E(2)-induced increases in BP and superoxide levels in the RVLM. In conclusion, these findings support the hypothesis that chronic exposure to low levels of E(2) induces hypertension and increases superoxide levels in the RVLM and that this effect can be reversed by resveratrol treatment.     

ε-Viniferin is more effective than its monomer resveratrol in improving the functions of vascular endothelial cells and the heart

The present study compared the effects of resveratrol and its dimer ε-viniferin on vascular endothelial cells (VECs) functions, and on the blood pressure and cardiac mass of spontaneously hypertensive rats (SHRs). Treatment of VECs with these compounds enhanced cell proliferation via nitric oxide generation and protected the cells from oxidative stress by suppressing increases in intracellular oxygen species. ε-Viniferin was more potent than resveratrol in most of these effects. ε-Viniferin, but not resveratrol inhibited angiotensin-converting enzyme activity in vitro. Three weeks of ε-viniferin treatment (5 mg/kg) reduced the systolic blood pressure and improved the whole cardiac mass and left ventricle mass indexes in SHRs. In contrast, resveratrol administration (2.5 mg/kg) failed to lower the blood pressure and significantly improve these mass indexes. These data suggest that ε-viniferin as well as resveratrol may be involved in protecting the functions of VECs and the heart.     

Dynamic mobilization of PGC-1α mediates mitochondrial biogenesis for the protection of RGC-5 cells by resveratrol during serum deprivation

Mitochondrial dysfunction contributing to the pathogenesis of glaucomatous neurodegeneration has stimulated considerable interest recently. In this study, we explored the role of peroxisome proliferator activated receptor-γ co-activator 1α (PGC-1α) in resveratrol-triggered mitochondrial biogenesis for preventing apoptosis in a retinal ganglion cell line RGC-5. Our results showed that serum deprivation induced cell apoptosis in a time-dependent manner. Applying resveratrol maintained the normal mitochondrial membrane potential, decreased the levels of both total and cleaved caspase-3, and inhibited the release of cytochrome c, which subsequently enhanced cell survival. Moreover, resveratrol stimulated mitochondrial biogenesis by increasing the absolute quantity of mitochondria as well as their DNA copies. Treatment with resveratrol promoted the protein expression of SIRT1, but not PGC-1α; instead, resveratrol facilitated PGC-1α translocation from the cytoplasm to the nucleus and up-regulated NRF1 and TFAM, which were blocked by nicotinamide. Collectively, we demonstrate that the SIRT1-dependent PGC-1α subcellular translocation following resveratrol application potentially attenuates serum deprivation-elicited RGC-5 cell death, thereby raising the possibility of mitigating glaucomatous retinopathy by enhancement of mitochondrial biogenesis.     

Comparative studies of the antioxidant effects of a naturally occurring resveratrol analogue – <Emphasis Type="Italic">trans</Emphasis>-3,3′,5,5′-tetrahydroxy-4′-methoxystilbene and resveratrol – against oxidation and nitration of biomolecules in blood platelets

The action of two phenolic compounds isolated from the bark of Yucca schidigera: trans-3,3',5,5'-tetrahydroxy-4'-methoxystilbene and its analogue -- resveratrol (trans-3,4',5-trihydroxystilbene, present also in grapes and wine) on oxidative/nitrative stress induced by peroxynitrite (ONOO(-), which is strong physiological oxidant and inflammatory mediator) in human blood platelets was compared. The trans-3,3',5,5'-tetrahydroxy-4'-methoxystilbene, like resveratrol, significantly inhibited protein carbonylation and nitration (measured by enzyme-linked immunosorbent assay method) in the blood platelets treated with peroxynitrite (0.1 mM) and markedly reduced an oxidation of thiol groups of proteins (estimated with 5,5'-dithio-bis(2-nitro-benzoic acid)] or glutathione (measured by high performance liquid chromatography method) in these cells. The trans-3,3',5,5'-tetrahydroxy-4'-methoxystilbene, like resveratrol, also caused a distinct reduction of platelet lipid peroxidation induced by peroxynitrite. The obtained results indicate that in vitro trans-3,3',5,5'-tetrahydroxy-4'-methoxystilbene and resveratrol have very similar protective effects against peroxynitrite-induced oxidative/nitrative damage to the human platelet proteins and lipids. Moreover, trans-3,3',5,5'-tetrahydroxy-4'-methoxystilbene proved to be even more potent than resveratrol in antioxidative tests. We conclude that the novel tested phenolic compound -- trans-3,3',5,5'-tetrahydroxy-4'-methoxystilbene isolated from Y. schidigera bark possessing Generally Recognized As Safe label given by the Food and Drug Administration and allows their human dietary use -- seems to be a promising candidate for future evaluations of its antioxidative activity and may be a good candidate for scavenging peroxynitrite.