Effects Of Phloretin Research Articles

Phloretin inhibits biofilm formation by affecting quorum sensing under different temperature

Biofilm is a critical factor for L. monocytogenes to persist in food processing. Phloretin is a natural product that is potent to antibacterial. In order to investigate the effects of phloretin on L. monocytogenes biofilm, we used sub-MIC phloretin to treat L. monocytogenes at 37 °C and 4 °C. The results show that L. monocytogenes biofilm maximum inhibitory rate was up to 60% when 20 μg/mL phloretin was used. The capacity of biofilm adhesion and aggregation of L. monocytogenes were declined. Meanwhile, biofilm protective capacity was impaired. The biofilm thickness of the treated group was decreased by 2 μm approximately at 20 μg/mL phloretin treatment. We also studied the mechanisms of biofilm inhibition by phloretin. The quorum sensing related genes were inhibited by phloretin. The relative transcript level of the agr system genes in quorum sensing was reduced by 50% with 20 μg/mL phloretin treatment. These findings suggest that phloretin has the potential to be developed as a natural antibacterial or biofilm inhibitor to control contamination in the food industry.

Phloretin Protects Macrophages from E. coli-Induced Inflammation through the TLR4 Signaling Pathway.

Macrophages are the cells of the first-line defense system, which protect the body from foreign invaders such as bacteria. However, Gram-negative bacteria have always been the major challenge for macrophages due to the presence of lipopolysaccharides on their outer cell membrane. In the present study, we evaluated the effect of phloretin, a flavonoid commonly found in apple, on the protection of macrophages from Escherichia coli infection. RAW 264.7 cells infected with standard E. coli, or virulent E. coli K1 strain were treated with phloretin in a dose-dependent manner to examine its efficacy in protection of macrophages. Our results revealed that phloretin treatment reduced the production of nitric oxide (NO) and generation of reactive oxygen species along with reducing the secretion of proinflammatory cytokines induced by the E. coli and E. coli K1 strains in a concentration-dependent manner. Additionally, treatment of phloretin downregulated the expression of E. coli-induced major inflammatory markers i.e. cyclooxygenase-2 (COX-2) and hemeoxygenase-1 (HO-1), in a concentration dependent manner. Moreover, the TLR4-mediated NF-κB pathway was activated in E. coli-infected macrophages but was potentially downregulated by phloretin at the transcriptional and translational levels. Collectively, our data suggest that phloretin treatment protects macrophages from infection of virulent E. coli K1 strain by downregulating the TLR4-mediated signaling pathway and inhibiting NO and cytokine production, eventually protecting macrophages from E. coli-induced inflammation.

Phloretin ameliorates dextran sulfate sodium-induced ulcerative colitis in mice by regulating the gut microbiota

Phloretin, extracted from the pericarp and velamen of apples or pears, is a dihydrochalcone flavonoid with anti-bacterial and anti-inflammatory activities. It has been reported that phloretin has anti-inflammatory effects in ulcerative colitis (UC) mice. However, the role of the gut microbiota in the phloretin anti-UC process remains unclear. In this study, we observed that the anti-UC effect of phloretin was affected by co-housing, probably because of the transmissible nature of the gut micobiota. Through fecal micobiota transplantation (FMT), the effects of the gut microbiota on the anti-UC of phloretin were further confirmed. UC was induced in mice by administrating 3% dextran sulfate sodium (DSS) in drinking water for 7 days. Phloretin (60 mg/kg) was administered by gavage every day during the experiment. Fecal microbes (109 CFU/mL) from phloretin-treated UC mice were administered by gavage to non-phloretin-treated UC mice for 7 days. The results showed that FMT, like phloretin, ameliorated UC by improving disease symptoms and colon inflammation, balancing inflammatory cytokines, maintaining intestinal barrier integrity, restoring systemic immune function, inhibiting NF-κB and NLRP3 inflammasome activation and ameliorating the oxidant stress. Both FMT and phloretin treatment increased the levels of Bacteroidetes, Alistipes and Lactobacillus and decreased those of Firmicutes, Oscillibacter and Ruminiclostridium_6. Correlation analysis between gut microbes and micro-environmental factors revealed that Alistipes abundance was negatively correlated with DAI, pathological score, and TNF-α, IL-6 and IL-1β levels, and Alistipes was more abundant in phloretin or FMT treated UC mice. Oscillibacter abundance was significantly positively correlated with IL-6 and IL-1β levels and pathological score, and Oscillibacter was increased in UC mice. Furthermore, network analysis of the dominant genera revealed that Alistipes abundance was negatively related to Oscillibacter abundance. In conclusion, this study suggests that the anti-UC effects of phloretin are achieved through regulation of the gut microbiota and phloretin has the potential to be developed as a promising agent for the treatment of UC.

Phloretin ameliorates hyperuricemia-induced chronic renal dysfunction through inhibiting NLRP3 inflammasome and uric acid reabsorption

BackgroundHyperuricemia (HUA) is an important risk factor for renal diseases and contributes to renal fibrosis. It has been proved that phloretin has antioxidant and anti-inflammatory properties and could inhibit uric acid (UA) uptake in vitro. However, whether phloretin has a renal protective role in vivo remains unknown. PurposeThis study aims to evaluate the therapeutic effect of phloretin on HUA-induced renal injury in mice and to reveal its underlying mechanism. MethodsMice were induced hyperuricemic by oral gavage of adenine/potassium oxonate. The effects of phloretin on renal function, fibrosis, oxidative stress, inflammation, and UA metabolism in HUA mice were evaluated. The effect of phloretin on NLRP3 pathway was analyzed in human renal tubular cell lines (HK-2). ResultsHUA mice showed renal dysfunction with increased renal fibrosis, inflammation and mitochondrial stress. By contrast, phloretin reduced the level of serum blood urea nitrogen (BUN), urinary albumin to creatinine ratio (UACR), tubular necrosis, extracellular matrix (ECM) deposition and interstitial fibroblasts in HUA mice. The renal infiltration of inflammatory cells, cytokines such as NOD-like receptor family pyrin domain containing 3 (NLRP3) and interleukin-1β (IL-1β) release, mitochondrial reactive oxygen species (ROS) and morphological lesions in HUA mice also decreased. Furthermore, phloretin partly inhibited renal glucose transporter 9 (GLUT9) and promoted urinary UA excretion in HUA mice. In vitro, phloretin suppressed the NLPR3 pathway under LPS or UA stimulation in HK-2 cells. ConclusionsPhloretin could effectively attenuate UA-induced renal injury via co-inhibiting NLRP3 and UA reabsorption, and thus it might be a potential therapy to hyperuricemia-related renal diseases.

Mechanisms of Regulation of Amyloid-Induced Permeability of Model Lipid Membranes by Polyphenols

This work is devoted to the study of the processes of formation and functioning of ion channels by amyloidogenic peptides, pathological aggregation and accumulation of which is a cause of neurodegenerative disorders. The effect of the plant polyphenols phloretin, butein, resveratrol, isoliquiritigenin, 4'-hydroxychalcone, and cardamonine on the pore-forming activity of β-amyloid peptide fragment 25–35 in bilayer lipid membranes from palmitoyl-phosphocholine was studied. It was demonstrated that the introduction of phloretin, butein or isoliquiritigenin in membrane-bathing solutions to a concentration of 20 µM leads to the increase of macroscopic transmembrane currents induced by peptide. At the same time, cardamonine, 4'-hydroxychalcone, and resveratrol have no effect on the activity of β-amyloid peptide fragment 25–35. The comparison of the results of studying the effect of tested polyphenols on electric and elastic properties of model membranes and pore-forming ability of β-amyloid peptide fragment 25–35 allowed it to concluded that there is no connection between the potentiating effect of phloretin, butein, or isoliquiritigenin and changes in the physicochemical properties of lipid bilayers. Results obtained by means of a confocal fluorescent microscopy indicate that the domain organization of the lipid bilayer may play a role in the pore-forming activity of amyloidogenic peptide. The results of electrophysiological measurements obtained for α-synuclein (another protein forming ion-permeable pores) do not contradict the hypothesis of binding of polyphenols, hydroxylated in the 7 position of the A cycle and in the 4'-position of the B cycle, with an open propane fragment with β-layers formed by amyloid peptides.

Phloretin Suppresses Bone Morphogenetic Protein-2-Induced Osteoblastogenesis and Mineralization via Inhibition of Phosphatidylinositol 3-kinases/Akt Pathway.

Phloretin has pleiotropic effects, including glucose transporter (GLUT) inhibition. We previously showed that phloretin promoted adipogenesis of bone marrow stromal cell (BMSC) line ST2 independently of GLUT1 inhibition. This study investigated the effect of phloretin on osteoblastogenesis of ST2 cells and osteoblastic MC3T3-E1 cells. Treatment with 10 to 100 µM phloretin suppressed mineralization and expression of osteoblast differentiation markers, such as alkaline phosphatase (ALP), osteocalcin (OCN), type 1 collagen, runt-related transcription factor 2 (Runx2), and osterix (Osx), while increased adipogenic markers, peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), fatty acid-binding protein 4, and adiponectin. Phloretin also inhibited mineralization and decreased osteoblast differentiation markers of MC3T3-E1 cells. Phloretin suppressed phosphorylation of Akt in ST2 cells. In addition, treatment with a phosphatidylinositol 3-kinase (PI3K)/Akt inhibitor, LY294002, suppressed the mineralization and the expression of osteoblast differentiation markers other than ALP. GLUT1 silencing by siRNA did not affect mineralization, although it decreased the expression of OCN and increased the expression of ALP, Runx2, and Osx. The effects of GLUT1 silencing on osteoblast differentiation markers and mineralization were inconsistent with those of phloretin. Taken together, these findings suggest that phloretin suppressed osteoblastogenesis of ST2 and MC3T3-E1 cells by inhibiting the PI3K/Akt pathway, suggesting that the effects of phloretin may not be associated with glucose uptake inhibition.

Phloretin suppresses metastasis by targeting protease and inhibits cancer stemness and angiogenesis in human cervical cancer cells

BackgroundPhloretin, a dihydrochalcone flavonoid, possesses anti-inflammatory activity and inhibits the growth of various cancers. However, the flavonoid's effect on cervical cancer metastasis and angiogenesis remains unknown. PurposeIn this study, we provide molecular evidence associated with the antimetastatic and antiangiogenic effects of phloretin. MethodsIn this study, the anti-invasive effect of phloretin (0–60 μM) in cervical cancer cells was evaluated using the Matrigel invasion assay, gelatin zymography, cell-matrix adhesion assay, wound healing assay, and Western blotting. Antiangiogenic potential of phloretin (0–100 μM) was assessed by the Matrigel tube formation assay. The in vivo antitumor effect of phloretin (10 or 20 mg/kg) was fed by oral gavage and determined using subcutaneous inoculation and tail vein injection in immunodeficient nude mice. ResultsPhloretin (60 μM) showed marked suppression of invasion and migration through downregulation of matrix metalloproteinase (MMP)-2, MMP-3, and cathepsin S in human SiHa cervical cancer cells. Phloretin (60 μM) reversed the epithelial-mesenchymal transition induced by transforming growth factor-β1 and downregulated mesenchymal markers, such as fibronectin, vimentin, and RhoA. Phloretin (100 μM) treatment significantly inhibited the aldehyde dehydrogenase 1 activity of SiHa cells, reduced the self-renewal properties and stemness signatures of CD44 and Sox-2 in sphere-forming cervical cancer-derived tumor-initiating cells, and inhibited the invasion, MMP-2 activity, and tube formation capacity of human umbilical vein endothelial cells. The ability of phloretin (20 mg/kg) to suppress lung metastasis and tumor growth in SiHa cells was evidenced by tail vein injection and subcutaneous inoculation in a tumor xenograft model. ConclusionIn summary, the findings indicate that phloretin inhibits the metastatic and angiogenic abilities and cancer stemness of SiHa cells, thereby suggesting that this flavonoid is a promising therapeutic agent for the treatment of human cervical cancer cells.

Phloretin loaded chitosan nanoparticles enhance the antioxidants and apoptotic mechanisms in DMBA induced experimental carcinogenesis

The main aim of this study was to investigate the effects of phloretin loaded chitosan nanoparticles (PhCsNPs) on 7,12-dimethylbenz[a]anthracene (DMBA) induced experimental cancer in hamsters. Oral squamous cell carcinoma (OSCC) was induced in male golden Syrian hamsters by painting with 0.5% DMBA three times a week for 14 weeks. Varying concentration of PhCsNPs (5, 10, and 20 mg/kg b.wt.) was orally administered on alternative days to evaluate the optimum dose. The experiment design was terminated at the end of the 14th week. The development of OSCC was confirmed by histopathological and biochemical analysis (lipid peroxidation, antioxidant profile, and detoxification enzymes) in plasma, erythrocyte, buccal, and liver tissues. Significant increases in oxidation and lipid peroxidation were noticed in DMBA-painted hamsters. Oral administration of PhCsNPs in various doses on alternate days reversed the deleterious effects induced by DMBA. In addition, immunoblot analyses of PhCsNPs treatment enhanced the release of Bcl-2 associated X protein (Bax), cytochrome c, caspase-3, 9 and suppressed the B-cell lymphoma 2 (Bcl-2) expression, which the use of PhCsNPs for mitochondrial-mediated apoptosis. These findings suggest biofabricated PhCsNPs may act as a potent antioxidant and anti-carcinogenic in DMBA induced oral cancer in experimental animals.

Phloretin ameliorates hyperuricemia‐induced chronic renal dysfunction through inhibiting NLRP3 inflammasome and uric acid reabsorption

ObjectiveHyperuricemia is an important risk factor for renal diseases and contributes to renal fibrosis, and it's a big challenge for clinic. Phloretin, a plant‐derived dihydrochalcone, has shown antioxidant/anti‐inflammatory properties, and could inhibit uric acid (UA) uptake in vitro, but its role on renal injury remains unknown. In this study, we aimed to investigate the therapeutic potential of phloretin on hyperuricemia‐induced renal dysfunction and underlying mechanism in mice.MethodsMice were induced hyperuricemic by oral gavage of adenine/potassium oxonate mixture, and then treated with phloretin. The effects of phloretin on renal function, inflammation and interstitial fibrosis were evaluated in hyperuricemic mice. The changes of renal mitochondrial oxidative stress and UA metabolism were also analyzed.ResultsHyperuricemic mice showed renal dysfunction with elevated renal inflammation and fibrosis. By contrast, phloretin significantly improved renal function and morphologic lesions, as indicated by reduced levels of serum blood urea nitrogen (BUN), creatinine and urinary albumin to creatinine ratio (UACR), as well as decreased levels of kidney tubular necrosis, interstitium extracellular matrix (ECM) deposition and interstitial fibroblasts activation in the kidneys of hyperuricemic mice. Furthermore, phloretin significantly suppressed renal mitochondrial reactive oxygen species (ROS) production and NOD‐like receptor family pyrin domain containing 3 (NLRP3) inflammasome signaling, and inhibited glucose transporter 9 (GLUT9) and UA reabsorption in hyperuricemic mice.ConclusionPhloretin can ameliorate hyperuricemia‐induced chronic renal injury through co‐inhibiting NLRP3 pathway and UA reabsorption, and thus it might be a promising therapy to hyperuricemia‐related renal diseases.Support or Funding InformationThis work was supported by grants from National Natural Science Foundation of China (31200754, 81571808, 31871001) and China Postdoctoral Science Foundation (2012M511931).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Effect of Phloretin on the Thermotropic Behavior of Membrane Forming Lipids

The Effect of Phloretin on the Thermotropic Behavior of Membrane Forming Lipids

Biochemical Basis of Anti-Cancer-Effects of Phloretin-A Natural Dihydrochalcone.

Apple is a rich source of bioactive phytochemicals that help improve health by preventing and/or curing many disease processes, including cancer. One of the apple polyphenols is phloretin [2′,4′,6′-Trihydroxy-3-(4-hydroxyphenyl)-propiophenone], which has been widely investigated for its antioxidant, anti-inflammatory and anti-cancer activities in a wide array of preclinical studies. The efficacy of phloretin in suppressing xenograft tumor growth in athymic nude mice implanted with a variety of human cancer cells, and the ability of the compound to interfere with cancer cells signaling, have made it a promising candidate for anti-cancer drug development. Mechanistically, phloretin has been reported to arrest the growth of tumor cells by blocking cyclins and cyclin-dependent kinases and induce apoptosis by activating mitochondria-mediated cell death. The blockade of the glycolytic pathway via downregulation of GLUT2 mRNA and proteins, and the inhibition of tumor cells migration, also corroborates the anti-cancer effects of phloretin. This review sheds light on the molecular targets of phloretin as a potential anti-cancer and anti-inflammatory natural agent.

The modulatory role of phloretin in Aβ25-35 induced sporadic Alzheimer's disease in rat model.

Alzheimer's disease (AD) is the leading neurodegenerative disorder with extracellular senile plaques and neurofibrillary tangles as the major hallmarks. The objective was to evaluate the effect of phloretin in a chronic model of sporadic AD by injecting aggregated form of Aβ25-35 peptide sequence intracerebroventricularly (icv) in Wistar rats. To achieve this, male Wistar rats were injected with aggregated Aβ25-35 peptide icv, followed by 21days phloretin (2.5mg/kg, 5mg/kg) administration after recovery period. Barnes maze and elevated plus maze along with the biochemical estimation of antioxidant enzymes activities were conducted. The hippocampus region of the rat brains were stained with Congo red and Nissl stain. TNF-α was estimated in the brain homogenates using the ELISA assay. In this study, phloretin improved the spatial memory formation and retention in Barnes maze test. Additionally, phloretin alleviated the antioxidant defense biomarkers and thereby reduced oxidative stress, decreased TNF-α-mediated neuroinflammation. Furthermore, phloretin treatment showed decreased amyloid beta accumulation in the CA1 region and less number of pyknotic nuclei in the dentate gyrus of the Aβ25-35-injected rat brains. The above experimental findings evinced the promising role of phloretin in Aβ25-35-injected rats and which further envisage its potential to be explored in the treatment of AD.

Decrease of microbial community diversity, biogenic amines formation, and lipid oxidation by phloretin in Atlantic salmon fillets

Phloretin is a naturally occurring flavonoid that has antimicrobial and antioxidant activities. The objective of this study is to examine the feasibility of using phloretin as a preservative for salmon fillets. Salmon samples were dipped in phloretin solution (2 mg/mL and 4 mg/mL), stored at 4 °C for three days. The effect of phloretin on the quality of salmon fillets was investigated during storage time. The results showed that phloretin reduced microbial species diversity and the relative abundance of bacterial community composition in genus level in salmon fillets. Specific spoilage organisms (Pseudomonas, Photobacterium, and Shewanella), Acinetobacter, Flavobacterium, and Chryseobacterium were identified as dominant bacteria in salmon fillets during storage time. Besides, phloretin inhibited the growth of test microorganisms, in particular, Gram-positive bacteria including Listeria monocytogenes, Staphylococcus aureus, and Bacillus subtilis. When dipped into 4 mg/mL of phloretin, phloretin showed growth inhibitory effects against Pseudomonas and Photobacterium by 1.6 and 0.7 logarithmic, respectively; and total viable bacteria counts were decreased. Notably, the formation of biogenic amines was retarded by 18.84 times and lipid oxidation measured by thiobarbituric acid-reactive substances (TBARS) was reduced by 3.23 times compared to control samples. Overall, phloretin is promising as a natural preservative to improve seafood safety.

Anticancer Activity of Phloretin Against Human Gastric Cancer Cell Lines Involves Apoptosis, Cell Cycle Arrest, and Inhibition of Cell Invasion and JNK Signalling Pathway.

BackgroundGastric cancer is one of most commonly diagnosed cancers and causes significant mortality worldwide. In this study, the antiproliferative and anticancer effects of Phloretin were evaluated against gastric cancer cell lines.Material/MethodsMTT assay was used to assess the proliferation rate of gastric cancer cells. DAPI and annexin V/PI were used for detection of apoptotic cell death. Cell invasion was investigated by Transwell assays and the expression of the proteins was estimated by immunoblotting.ResultsThe results revealed that Phloretin exerts anticancer effects on all the gastric cancer cell lines used in this study. However, the anticancer effects were more significant (p<0.05) on the AGS cell line. Further, the effect of Phloretin on the viability of normal GES-1 cells was minimal. Apoptosis assays showed that Phloretin triggers apoptotic cell death in AGS gastric cancer cells. Phloretin could also cause the arrest of the AGS gastric cancer cells in the G2/M phase of the cell cycle and suppress their ability to migrate. Western blotting analysis revealed that Phloretin significantly decreased the expression of p-JNK and p-38. However, comparatively lower effects were observed on the expression of JNK and P38.ConclusionsWe showed that Phloretin is an important molecule for the treatment of gastric cancer.

The effect of phloretin on synaptic proteins and adult hippocampal neurogenesis in Aβ (1-42)-injected male Wistar rats.

Considering the deleterious effect of Aβ1-42, a study was designed to evaluate the effect of phloretin on altered synaptic proteins and adult hippocampal neurogenesis in Aβ1-42-injected Wistar rats. The rats were pretreated with 5 mg/kg p.o dose of phloretin and donepezil (positive control) for 28 days, followed by intrahippocampal injections of aggregated Aβ1-42. After termination, perfused brains were isolated and subjected to Western blot and immunohistochemistry (IHC) analysis. The Western blot revealed that Aβ1-42-injected rats had significantly low levels of synaptophysin as compared to sham control. Phloretin pretreatment significantly protected the presynaptic protein synaptophysin against the effects of Aβ1-42. There were no significant changes in the levels of PSD95 between different groups. The IHC findings showed that Aβ1-42 significantly reduced the Ki67 and DCX in the dentate gyrus as compared to sham control. However, phloretin significantly improved the number of Ki67- and DCX-positive neurons in the dentate gyrus region as compared to Aβ1-42 group. This study demonstrated the protective effect of phloretin on synaptophysin and adult neuronal proliferating cells in Aβ1-42-injected rats. The encouraging findings highlight the potential of phloretin as a dietary supplement targeting key therapeutic mechanisms in neurodegenerative disorders such as AD.

A novel electrochemical biosensor for antioxidant evaluation of phloretin based on cell-alginate/ʟ-cysteine/gold nanoparticle-modified glassy carbon electrode

Antioxidant evaluation of bioactive compounds is limited, since many methods lack a real physiological environment that can be used conveniently and intuitively. In this study, a simple, label-free and effective electrochemical biosensor method has been developed to evaluate the antioxidant effect of phloretin (Ph) by 3D cell modification on a glassy carbon electrode (GCE). In response to this, A549 cells were immobilized onto a self-assembled ʟ-cysteine/gold nanoparticle (AuNPs/ʟ-Cys)-modified GCE surface by a simple drop casting after encapsulated in alginate. The electrochemical impedance spectroscopy (EIS) results showed that the impedance value (Ret) increased with the concentration of H2O2 in the range of 0–60 μmol/L with the correlation of 0.990 which acted as an oxidative stress model inducer. However, the EIS value decreased with the co-incubation of Ph ranging from 10 to 100 μmol/L, showing a dose-dependent manner and time effect, indicating that the variation of Ret was responded to the antioxidant effect. The response impedance of the biosensor is linear to Ph concentrations from 20 μmol/L to 100 μmol/L with the detection limit (LOD) as 1.96 μmol/L. A significant correlation was observed between reactive oxygen species (ROS) values and Ret values following the concentrations of Ph, thus demonstrating the good biological relevance of cell-based electrochemical method. The strategy has been used to evaluate Ph antioxidant capacity in real cells with satisfactory results, indicating the feasibility of biosensor analysis for antioxidant evaluation.

Phloretin as a Potent Natural TLR2/1 Inhibitor Suppresses TLR2-Induced Inflammation.

Toll-like receptor 2 (TLR2) responses are involved in various inflammatory immune disorders. Phloretin is a naturally occurring dietary flavonoid that is abundant in fruit. Here, we investigated whether the anti-inflammatory activity of phloretin is mediated through TLR2 pathways, and whether phloretin acts as an inhibitor of TLR2/1 heterodimerization using the TLR2/1 agonist Pam3CSK4. We tested the effects of phloretin on tumor necrosis factor (TNF)-α production induced by various TLRs using known TLR-specific agonists. Phloretin significantly inhibited Pam3CSK4-induced TRL2/1 signaling in Raw264.7 cells compared to TLR signaling induced by the other agonists tested. Therefore, we further tested the effects of phloretin in human embryonic kidney (HEK) 293-hTLR2 cells induced by Pam3CSK4, and confirmed that phloretin has comparable inhibition of TLR2/1 heterodimerization to that induced by the known TLR2 inhibitor CU-CPT22. Moreover, phloretin reduced the secretion of the inflammatory cytokines TNF-α and interleukin (IL)-8 in Pam3CSK4-induced HEK293-hTLR2 cells, whereas it did not significantly reduce these cytokines under Pam2CSK4-induced activation. Western blot results showed that phloretin significantly suppressed Pam3CSK4-induced TLR2 and NF-κB p65 expression. The molecular interactions between phloretin and TLR2 were investigated using bio-layer interferometry and in silico docking. Phloretin bound to TLR2 with micromolar binding affinity, and we proposed a binding model of phloretin at the TLR2–TLR1 interface. Overall, we confirmed that phloretin inhibits the heterodimerization of TLR2/1, highlighting TLR2 signaling as a therapeutic target for treating TLR2-mediated inflammatory immune diseases.

Phloretin Promotes Adipogenesis via Mitogen-Activated Protein Kinase Pathways in Mouse Marrow Stromal ST2 Cells.

Phloretin, a glucose transporter (GLUT) inhibitor, has pleiotropic effects. The present study examined the effects of phloretin on the commitment of marrow stromal cells to adipocytes, using the mouse marrow stromal cell line ST2. Oil red O staining showed that treatment with phloretin 10–100 µM promoted lipid accumulation. Real-time PCR showed that phloretin significantly increased the expression of adipogenic markers, including PPARγ, C/EBPα, fatty acid synthase, fatty acid-binding protein 4, and adiponectin. Western blotting showed that phloretin inhibited ERK1/2 and JNK but activated p38 MAPK. Treatment with a MAPK/ERK kinase inhibitor and a JNK inhibitor enhanced adipogenesis, similar to phloretin. In contrast, a p38 MAPK inhibitor suppressed phloretin-induced adipogenesis. Although phloretin phosphorylated AMP-activated protein kinase (AMPK), co-incubation with an AMPK inhibitor did not block phloretin-induced adipogenesis. The 2-deoxyglucose colorimetric assay showed that phloretin and siRNA silencing of GLUT1 decreased glucose uptake. However, unlike phloretin treatment, GLUT1 silencing inhibited adipogenesis. In addition, phloretin enhanced adipogenesis in GLUT1 knocked-down cells. Taken together, phloretin induced adipogenesis of marrow stromal cells by inhibiting ERK1/2 and JNK and by activating p38 MAPK. The adipogenic effects of phloretin were independent of glucose uptake inhibition. Phloretin may affect energy metabolism by influencing adipogenesis and adiponectin expression.

The protective effect of phloretin in osteoarthritis: an in vitro and in vivo study.

Osteoarthritis (OA) is a degenerative joint disease characterized by the degradation and inflammation of cartilage. Phloretin, a type of dihydrochalcone mainly found in apples and apple-derived products, has been reported to possess various potent biological effects such as antioxidant, anticancer, anti-inflammatory, and immunomodulatory. However, the anti-inflammatory effects of phloretin on OA have not been reported. This study was aimed at assessing the effects of phloretin on human OA chondrocytes. Human OA chondrocytes were pretreated with phloretin (10, 30, and 100 μM) for 2 h and subsequently stimulated with IL-1β for 24 h. The production of NO, PGE2, TNF-α, and IL-6 was determined using the Griess reagent and ELISAs. The mRNA expression of COX-2, iNOS, MMP-3, MMP-13, and ADAMTS-5 was measured by real-time PCR. Changes in the protein expression of COX-2, iNOS, MMPs, ADAMTS, aggrecan, collagen-II, NF-κB, and the PI3K/Akt signaling pathway were detected by western blotting. In this study, we found that phloretin significantly inhibited the IL-1β-induced production of NO, PGE2, TNF-α, and IL-6, the expression of COX-2, iNOS, MMP-3, MMP-13, and ADAMTS-5, and the degradation of aggrecan and collagen-II in human OA chondrocytes. Furthermore, phloretin dramatically suppressed the IL-1β-stimulated phosphorylation of PI3K/Akt and activation of NF-κB in human OA chondrocytes. In addition, treatment with phloretin not only prevented the destruction of cartilage and the thickening of subchondral bone but also relieved synovitis in a mouse model of OA. Moreover, immunohistochemical results showed that phloretin significantly decreased the expression of MMP-13 and increased the expression of collagen-II in OA in mice. In conclusion, these results suggest that phloretin may be a potential agent for the treatment of OA.

Phloretin attenuates mucus hypersecretion and airway inflammation induced by cigarette smoke

BackgroundsCigarette smoke (CS)-induced airway mucus hypersecretion and inflammation are the prominent features of chronic obstructive pulmonary disease (COPD). As an anti-inflammatory flavonoid, phloretin was found to be involved in various inflammatory disorders such as sepsis. In this study, the effects of phloretin on CS-induced airway mucin secretion and inflammation were investigated in vivo and in vitro. MethodsPhloretin dissolved in 1% DMSO was daily injected intraperitoneally to mice, which were then exposed to CS for four weeks. Mouse lung histologic changes were evaluated, the expression of mucin 5ac (MUC5AC) was measured, bronchoalveolar lavage fluid (BALF) total cells, neutrophils, and macrophages were counted. BALF and lung levels of tumor necrosis factor-alpha and interleukin-1 beta (IL-1β) were quantified. Moreover, the effects of phloretin on cigarette smoke extract (CSE)-induced expression of MUC5AC and IL-1β were investigated in NCI-H292 cells. Then, to explore the potential mechanisms, the signaling molecules including epidermal growth factor receptor (EGFR), extracellular signal-regulated kinase (ERK) and P38 were evaluated. ResultsPhloretin pretreatment dramatically suppressed the mucins secretion, inflammatory cell infiltration and inflammatory cytokine release in mouse lungs induced by CS, and it also suppressed CSE-induced expression of MUC5AC and IL-1β in NCI-H292 bronchial epithelial cells. Furthermore, western blot showed that phloretin attenuated the activation of EGFR, ERK and P38 both in vivo and in vitro. ConclusionsThis study highlights the protective effect of phloretin on CS-related airway mucus hypersecretion and inflammation, where EGFR, ERK and P38 might be involved. These findings suggest that phloretin could be a potential therapeutic drug for COPD.