TNF-α modulation by natural bioactive molecules in mouse RAW 264.7 macrophage cells.

BACKGROUND: The present study was designed to examine the anti-inflammatory effects of plant-derived products marketed for human health benefits. METHODS: The tumor necrotic factor-α (TNF-α) was used as a proinflammatory biomarker generated by mouse macrophage RAW 264.6 cells. The in vitro tested plant products include Saskatoon berry (SKB), quercetin, purified oat beta-glucan (OBG), curcumin, and turmeric. Quantification of TNF-α in cell culture supernatants was carried out using mouse TNF-α assay kit and the cell proliferation was determined by MTT (3-(4, 5-dimethylthiazolyl-2)-2,5- diphenyltetrazolium bromide) assay. The cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum and 100 U/mL penicillin and 100 µg/mL streptomycin. Bacterial lipopolysaccharide (LPS) at a concentration of 500 ng/mL was employed to stimulate the TNF-α production in mouse macrophage cells. RESULTS: Results showed that curcumin at 10 µM (3.7 µg/mL) level effectively attenuated the LPS-induced inflammatory response, and at 100 µM completely inhibited macrophage RAW cell growth (p<0.05). The aqueous turmeric extract caused inhibitory effect on TNF-α at 25, 50, 100, and 500 µg/mL. SKB inhibited TNF-α production at 50, 100, 500, and 1,000 µg/mL. On the other hand, at 10, 25, 500, and 1,000 µg/mL SKB promoted significant cell growth/proliferation. Quercetin at 10, 25, 50 and 100 µg/mL inhibited TNF-α, but at 500 and 1,000 µg/mL stimulated cell growth. OBG at 10, 25, and 50 µg/mL inhibited TNF-α, but in some cases OBG stimulated TNF-α At 1,000 and 10,000 µg/mL OBG proved to be extremely toxic or lethal to the macrophage cells. CONCLUSIONS: Overall, the plant products showed anti-inflammatory effects as well as cell proliferation or inhibition in the in vitro system used in this investigation. The underlying mechanisms of dualistic actions caused by plant-derived ingredients, viz., macrophage cellular growth stimulation or retardation, remain to be elucidated.

Evaluation of the antioxidant properties of tryptophan and its metabolites in in vitro assay.

BACKGROUND: Human milk contains a number of nutrients and bioactive ingredients which play an important role in the growth and development of infants. One important nutrient and bioactive ingredient of human milk is L-tryptophan. L-Tryptophan is an essential aromatic α-amino acid and is required in the diet of children and adult humans. As an essential amino acid, it is needed for protein synthesis and as a precursor of key biomolecules such as serotonin, melatonin, tryptamine, niacin, quinolinic acid and kynurenic acid, nicotinamide adenine dinucleotide. The aim of the study was to evaluate the antioxidant, anti-inflammatory and antiproliferative properties of tryptophan isolated from enzymatic hydrolysates from human milk and its metabolites on human glioma U251 cells and to evaluate the effects of human recombinant (hrIFNγ) on molecular ions of tryptophan and its metabolites in human glial U251 cells. METHODS: The cytotoxicity was determined by MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay. The antioxidant property was assessed by the oxygen radical scavenging capacity (ORAC) method. The anti-inflammatory effect was determined by the enzyme-linked immunosorbent assay (ELISA) against cytokines IL-6 and TNF-α. The effects of recombinant human (rhIFNγ) on molecular ions of tryptophan and its catabolites were evaluated by mass spectrometry. The tryptophan was isolated from milk peptides following enzymatic digestion, followed by separation by chromatographic and mass spectrometric methods. RESULTS: Tryptophan from human milk exhibited profoundly higher oxygen radical absorption capacity (7,986±468 µm Trolox equivalent (TE)/g) than that of whole human milk (80.4±13.3 µm TE/g). Tryptophan showed a moderate degree of anti-inflammatory activity against TNF-α and IL-6. rhIFNγ inhibited tryptophan metabolism. A low concentration of L-tryptophan (10-25 µg/mL) inhibited nearly 25% of cell growth. When U251 cells were treated with 25 µg/mL L-tryptophan and subsequently challenged with 30 ng/mL of human recombinant IFNγ, a significant inhibitory effect on cell growth was observed. Low concentrations of Xanthurenic acid, L-kynurenine, and 3-OH DL kynurenine were found to inhibit cell growth except melatonin and 3-OH anthranilic acid. Melatonin was a strong inducer of TNF-α in RAW cells, whereas 3-OH kynurenine at 25, 50 and 100 µg/mL inhibited IL-6 in RAW cells. No significant change was observed in the IL-8 profile in tryptophan-treated U251 cells except that L-kynurenine at 10 µg/mL produced significantly high level of an inflammatory cytokine IL-8. Melatonin, 3-OH, DL kynurenine at high concentrations (100 µg/mL) induced proliferation of U251 cells. Melatonin seemed to show synergistic effects with recombinant human IFNγ (rhINFγ) in promoting growth of human glioma cells. While treatment of U251 cells with tryptophan alone and subsequent treatment with rhIFNγ inhibited the growth of human cancer glioma cells, and conversely melatonin combined with rhIFNγ promoted growth of the U251 cells. CONCLUSIONS: The findings from this study suggest that human milk-derived tryptophan and its metabolites possess strong antioxidant properties. Such effects might play a significant role in regulating the cell proliferation and growth of human cancer cells in a concentration-dependent manner.

Cell transplantation in wounded mixed connective tissues.

Direct transplantation of multipotent precursor cells into the periodontium could provide a therapeutic approach for restoring periodontal tissues destroyed by periodontitis or trauma. To improve the understanding of cell migration, proliferation, and differentiation, we used a rodent model combining orthodontic tooth movement and transplantation of Lac-Z-positive murine-cultured periodontal ligament (PL) or femur-derived bone marrow precursor cells into a defined mandibular wound site, thus promoting tissue regeneration in wounded periodontium. Our results show that in orthodontically traumatized tissues, transplanted PL and bone marrow cells migrated systemically, contributing to the repopulation of sites with reduced cell/matrix density. The transplanted PL cells proliferated in adjacent alveolar bone marrow spaces, thus migrating to vascular tissues in the PL. The capillary walls in the PL serve as delivery sites for these cells and other marrow-derived hematopoietic cells, including monocytes. The transplanted marrow cells, extracted from femur of transgenic (TgR) mice exhibited similar behavior to those of transplanted PL cells, showing high proliferative activity in alveolar marrow as well as intensive repopulating capacity in wounded periodontium. On the other hand, the buccal skin fibroblasts failed to migrate and home effectively and thus the transplantation of these cells had no effect on periodontium regeneration. Based on these results, we conclude that the transplanted PL and bone marrow cells migrate systemically and following a cyclical process of growth and development and differentiate into PL fibroblasts, osteoblasts, and cementoblasts, thereby contributing to periodontal regeneration.