Numerous studies on blueberry treatment of murine cell lines use TLR4-activated models, through the addition of the bacterial cell wall component LPS at doses varying from 0.01 to 10 μg/mL . This produces a strong inflammatory response through, but not limited to, the expression of IL-6, TNF-α, or NO . The capacity of blueberry components to reduce proinflammatory marker gene expression and secretion inLPS-induced murine cell models has been shown in several studies, although the anti-inflammatory effect seems to be carried out by an array of compounds rather than an individual blueberry phenolic fraction. Diverse blueberry products, including blueberry pomace, whole blueberry extracts, polyphenol-rich extracts, or specific phenolic fractions lowered the production of cytokines by the cells in a dose-dependent manner. Further details on the treatments and doses used in the studies are reported in Table 1. For instance, blueberry phytochemicals reduced the gene expression and secretion of proinflammatory cytokines induced by LPS, particularly IL-6 , IL-1β , and TNF-αin RAW 264.7 cells and BMDMs compared with the LPS-induced control. However, it is not clear which fraction or specific phenolic compound exerts a more potent effect, especially since their activities seem to be cytokine specific. For example, Esposito et al. reported a better ability of a proanthocyanidin fraction to reduce IL-1β compared with the polyphenol extract, anthocyanin, or phenolic acid fractions. However, IL-6 gene expression was not suppressed by that same proanthocyandin fraction, and monocyte chemoattractant protein -1 expression , a chemokine that regulates monocyte infiltration ,drainage planter pot was inhibited by the blueberry anthocyanins but not by the other phenolic fractions. Although the large variation between the blueberry treatment doses used across different reports hinders direct comparisons between the studies, most have shown a dose-dependent regulation of cytokines .

Cheng et al. , however, reported 40–60% inhibition of IL-1β gene expression with ≤200 μg/mL blueberry extract, but no effect when the treatment dose was increased to 400 μg/mL . This observation was tentatively explained by the increased phagocytic activity of the macrophages in the presence of a high polyphenol concentration. iNOS is an enzyme responsible for the synthesis of NO, a mediator secreted by neutrophils and macrophages to induce vasodilation, mediate the immune response, or regulate apoptosis . After stimulation of RAW 264.7 cells by LPS, blueberry extracts inhibited iNOS gene expression and NO production , with extracts from anthocyanin-rich cultivars and blueberry proanthocyanidin fractions being particularly effective . COX-2 also plays a central role in the induction of inflammation. It is involved in the formation of prostaglandins, including prostaglandin E2 responsible for the induction of pain . Blueberry extracts have consistently reduced COX-2 gene expression in RAW 264.7 cells , although Mueller et al. and Grace et al. did not report a significant reduction of iNOS and COX-2 gene expression by whole blueberry or polyphenol-rich extracts. Although most studies have focused on blueberry polyphenols, Gu et al. reported the anti-inflammatory effect of the volatile extracts of several berries compared with their phenolic counterpart. Volatile and phenolic blueberry extracts showedsimilar inhibitory effects on inflammatory cytokines, NO, and COX-2 production, even though the volatile fraction was tested at a lower concentration than the phenolic fraction. This provides initial evidence that the volatile fraction of blueberries also contains molecules with anti-inflammation properties, but this remains to be validated. The anti-inflammatory activities of blueberry phytochemicals have also been demonstrated in cell lines derived from humans , including the U-937 and THP-1 monocytelike cells, that can be differentiated into macrophages after stimulation with phorbol-12-myristate-13-acetate , and human primary peripheral blood mononuclear cells . In the context of blueberry studies, a variety of compounds have been used to induce inflammation, including Fusobacterium nucleatum bacteria , LPS , or cytokines .

Blueberry extracts exerted an inhibitory effect toward cytokine secretion and matrix metalloproteinase -8 and 9 production , in cells triggered by bacteria or LPS. Blueberry extracts decreased TNF-α gene expression induced by LPS in THP-1 monocytes and U937 macrophages , but on the contrary, increased its expression in THP- 1 differentiated macrophages . The regulatory effect reported in most studies was associated with a decrease in NF-κB translocation in THP-1 cells . In PBMCs and THP-1 cells alternatively induced with either IFN-γ or TNF- α, the effects of a blueberry treatment were less robust . Cytokine secretion and adhesion molecule gene expression were inhibited by a blueberry extract in IFN- γ -induced PBMCs, but the same blueberry extract further increased the proinflammatory marker secretion when the cells were induced with TNF-α . These observations were tentatively explained by looking at the pathways activated by the different cytokines : the signal transducers and activators of transcription pathway activation was inhibited by coincubation of IFN-γ with the berry treatment, but NF-κB was enhanced by the addition of TNF-α combined with a blueberry extract . A follow-up study demonstrated that the IFN-γ receptor 2, responsible for transducing the signal conveyed by the proinflammatory cytokines, was inhibited by the blueberry anthocyanins . These observations on cell models induced with non-LPS ligands suggest that the immunomodulatory effects of blueberry compounds are context and pathway specific. In summary, blueberry phenolic and polyphenolic extracts have been shown to dampen inflammation in RAW 264.7, U-937, BMDMs, and PBMCs challenged with inflammation inducers, through the reduction of proinflammatory cytokine gene expression and secretion, and inhibition of NF-κB translocation to the nucleus. No specific fraction emerges as being more potent, suggesting a general effect of multiple phytomolecules rather than a single compound. More studies are warranted to better define molecular targets of blueberry-derived molecules and to assess the involvement of TLR-dependent and -independent pathways.ROS and free radicals are natural by-products of enzymatic reactions produced during metabolism .

When controlled, ROS production is used for signaling in metabolic processes . Environmental factors, lifestyle, and pathologies contribute to an unbalanced state, where ROS production overwhelms the defense capacity of the cells and induces oxidative stress . This state leads to protein and nucleic oxidation, and lipid peroxidation, which can impair enzymatic processes, induce breakage of DNA strands, and may lead to cell death . Endogenous antioxidant defense mechanisms exist in the body to limit the production and deleterious effects of ROS. Superoxide dismutase , found in the membrane or cytosolic fractions of cells, converts superoxide radicals to H2O2 and O2 . Glutathione peroxidase , via the oxidation of glutathione S-transferase, reduces lipid peroxide and converts H2O2 to H2O . Enzymes, including DNA glycosylases, repair damaged DNA . Oxidation and inflammation are intricately related, as cytokines and chemokines secreted by inflammatory cells can trigger ROS production. In turn, ROS activate proinflammatory pathways, including NF- κB, and sustain the cycle of oxidative and inflammatory stress . These conditions favor the development of chronic pathologies such as cancer , cardiovascular , inflammatory , and neurodegenerative diseases . The effect of blueberry phytochemicals on oxidative stress has been evaluated using several cell models, including neurons , fibroblasts , hepatocytes , enterocytes , and epithelial cells . Despite the diversity in models used, studies overlap in terms of the endpoints measured, which focus on evaluation of the modulation in ROS production and lipid peroxidation, increases in antioxidant enzyme activities, and protection of DNA against oxidative damage . The induction of oxidative stress in a variety of cells was attenuated by treatment with blueberry extracts, principally through the decreased formation of ROS , but also increased scavenging activity , and/or reduction of lipid peroxidation . This effect, however,plant pot with drainage was only partly explained by the regulatory effect of the blueberry compounds on antioxidant enzymes, which were upregulated in neuronal cells treated with blueberry juice and Caco-2 cells incubated with a polyphenol-rich blueberry extract . Glutathione concentrations, however, remained unchanged in Caco-2 cells treated with an anthocyanin fraction . In addition, a blueberry pomace extract tested on Chinese hamster ovary epithelial cells and a human colon cancer cell line failed to stimulate the transcription of detoxification enzymes such as heme oxygenase and NADPH quinone oxidoreductase- 1 , both involved in the antioxidant/oxidant balance . In contrast with those observations made on blueberry parent compounds, phloroglucinol aldehyde increased thetranscription activity of nuclear factor erythroid 2-related factor 2 , which when induced by oxidative stress stimulates the transcription of HO-1 and NQO-1 .A consequence of oxidation is DNA damage and potentially increases in cell death. Blueberry extracts demonstrated a protection against DNA damage induced by hydroxide peroxide and tert-butylhydroperoxide , although in Caco-2 cells, only the blueberry phenolcarbonic acid fraction reduced DNA damage compared with whole blueberry extract, anthocyanin, and polymeric fractions . Protective effects in terms of DNA damage and cell death were attributed to blueberry anthocyanin fractions in liver cells and pulmonary epithelial cells exposed to light or ionizing radiation, through modulation of apoptosis and cell cycle regulatory gene expressions . However, no improvement of cell cycle perturbation induced by 2,2-azobisdihydrochloride was reported in intestinal epithelial cells IPEC-1 treated with blueberry anthocyanins . To summarize, blueberry extracts and phenolic fractions demonstrated protective effects against oxidative stress, which were mainly explained through reduction of ROS production and protection against DNA damage induced during oxidation. Additional studies comparing cell models and/or blueberry fractions using similar experimental parameters are necessary to demonstrate the effects of the treatment on antioxidant enzymes, and fully understand the antioxidant contribution of blueberry phytochemicals versus their metabolic by-products.

There is also a need to evaluate the potential effects of blueberry volatiles in this regard.Atherosclerosis, a chronic inflammatory disease of the arterial wall , is characterized by the buildup of plaques in arteries and is the most frequent underlying condition for the development of cardiovascular diseases . Human umbilical vein endothelial cells and human microvascular vein endothelial cells provide a model to study normal as well as oxidation and inflammation-related dysfunctions. Studies on the effect of blueberry compounds in endothelial cell models are detailed in Table 3. Risk factors including smoking, aging, hypercholesterolemia, and hyperglycemia promote the retention of lipids, particularly LDL prone to oxidation in the vascular wall, causing the activation of inflammatory processes . The treatment of endothelial cells exposed to oxidative stress triggers with blueberry anthocyanins demonstrated protective effects toward ROS secretion and lipid peroxidation . A similar observation was reported with cells treated with blueberry exosome-like nanoparticles ,an extracellular messenger vesicle presents in plants that contains proteins, lipids, mRNA, and microRNA . In addition to reducing ROS production, blueberry ELN also regulated gene expression involved in endothelial activation and leukocyte recruitment [MAPK1 and intercellular adhesion molecule ] and inflammation . The antioxidant effect of blueberry extract on endothelial cells is likely due to the activity of several phytochemicals, but the extent to which other compounds contribute to the effect remains unclear, particularly due to the low number of studies focusing on nonphenolic compounds. The secretion of chemokines and adhesion molecules by the endothelium is primarily regulated by TNF-α and Creactive protein , and leads to monocyte recruitment . After infiltration, monocytes differentiate into macrophages and phagocytose LDL . Macrophages that accumulate lipids eventually turn into foam cells, becoming surrounded by smooth-muscle cells and a collagen matrix, ultimately resulting in plaque formation . Blueberry anthocyanins reduced the adhesion of THP-1 monocytes to HUVEC endothelial cells with a better efficacy than the phenolic acid fraction derived from the same extract . The action of blueberry anthocyanins was further investigated and the individual compounds malvidin and cyanidin 3-glucoside, protocatechuic, and gallic acid reduced THP-1 adhesion . Blueberry extracts also decreased platelet- and endothelial-derived microvesicles through the inhibition of P2X7 transcription and Akt phosphorylation, both contributing to the release of extracellular vesicles associated with monocyte interaction with endothelial cells . Su et al. observed that blueberry extracts led to down regulation of noncoding miR-21, miR-146a, and miR125b, miRs typically increased in macrophages involved in plaque formation . In cardiovascular disease, endothelial cell migration and angiogenesis are reduced, leading to structural and functional alterations of the endothelium . Akt is a major signaling pathway in angiogenesis, regulating cell survival, cell cycle, and migration . Treatment with blueberry polyphenols increased angiogenesis in endothelial cells through the upregulation of the Akt pathway . However, abnormal angiogenesis promoted by vascular endothelial growth factor was counterbalanced by blueberry extract treatment through the inhibition of ERK and Akt phosphorylation . In addition, blueberry polyphenol extract did not modulate polyphospholipase C expression and phosphorylation, involved in angiogenesis, in HUVEC cells following induction with VEGF or LPS .