Natural compound phloretin restores periodontal immune homeostasis via HIF-1α-regulated PI3K/Akt and glycolysis in macrophages.

Periodontitis is a chronic inflammatory disease that affects about 45 %-50 % of adults worldwide, but the efficacy of current clinical therapies is unsatisfactory due to the complicated periodontal immune microenvironment. Thus, developing drugs that can regulate innate immune cells (e.g., macrophages) is a potent strategy to treat periodontitis. Here, we report that phloretin, a food plant-derived natural compound, is sufficient to alleviate periodontitis through immune regulation. In vivo, phloretin treatment could significantly reduce alveolar bone resorption and periodontal inflammation in mouse periodontitis models. In vitro, phloretin could suppress proinflammatory (M1-like) polarization and cytokine release in macrophages induced by LPS. Mechanistically, the immune regulatory role of phloretin in macrophages may be due to its metabolic regulation effect. Phloretin might restore the balance of M1/M2 macrophage transition in periodontitis by inhibiting HIF-1α-mediated glycolysis and PI3k/Akt pathways, thereby reducing the proinflammatory effect and immune disorder caused by over-activated M1 macrophages. Together, this study highlights that natural compound, such as phloretin, can restore periodontal immune homeostasis by metabolic regulation of macrophages, which may provide novel insight into the treatment of periodontitis.

Amorphous solid dispersion augments the bioavailability of phloretin and its therapeutic efficacy via targeting mTOR/SREBP-1c axis in NAFLD mice.

The escalating incidences of non-alcoholic fatty liver disease (NAFLD) and associated metabolic disorders are global health concerns. Phloretin (Ph) is a natural phenolic compound, that exhibits a wide array of pharmacological actions including its efficacy towards NAFLD. However, poor solubility and bioavailability of phloretin limits its clinical translation. Here, to address this concern we developed an amorphous solid dispersion of phloretin (Ph-SD) using Soluplus® as a polymer matrix. We further performed solid-state characterization through SEM, P-XRD, FT-IR, and TGA/DSC analysis. Phloretin content, encapsulation efficiency, and dissolution profile of the developed formulation were evaluated through reverse phase HPLC. Finally, the oral bioavailability of Ph-SD and its potential application in the treatment of experimental NAFLD mice was investigated. Results demonstrated that the developed formulation (Ph-PD) augments the dissolution profile and oral bioavailability of the native phloretin (Ph). In NAFLD mice, histopathological studies revealed the preventive effect of Ph-SD on degenerative changes, lipid accumulation, and inflammation in the liver. Ph-SD also improved the serum lipid profile, ALT, and AST levels and lowered the interleukin-6 and tumor necrosis factor-α levels in the liver. Further, Ph-SD reduced fibrotic changes in the liver tissues and attenuates NAFLD progression by blocking the mTOR/SREBP-1c pathway. In a nutshell, the results of our study strongly suggest that Ph-SD has the potential to be a therapeutic candidate in the treatment of NAFLD and can be carried forward for further clinical studies.

Concomitant inhibition of TLR-4 and SGLT2 by phloretin and empagliflozin prevents diabetes-associated ischemic acute kidney injury.

Toll-like receptor-4 (TLR4) and sodium-glucose co-transporter 2 (SGLT2) signaling is involved in the pathogenesis of diabetes-associated kidney diseases. The purpose of this study was to explore the role and effect of phloretin, a TLR4 inhibitor, as an adjuvant therapy to empagliflozin, an SGLT2 inhibitor, in ischemic acute kidney injury (AKI) under diabetic conditions. To achieve this, firstly we induced type 1 diabetes using streptozotocin (55 mg per kg per intraperitoneally (i.p.)) followed by performing bilateral ischemia-reperfusion kidney injury to induce AKI in male Wistar rats. Treatment with phloretin (50 and 100 mg per kg per orally) and empagliflozin (10 mgper kg per orally) alone or in combination was administered to the diabetic rats for 4 days and 1 h before surgery. Moreover, a hypoxia-reperfusion injury was induced using sodium azide in NRK52E cells under a hyperglycemic environment to mimic the in vivo model. The cells were treated with phloretin (50 µM) and empagliflozin (100 nM) for 24 h. For biochemical analysis, plasma and urine samples were used. The kidney tissues were used to perform immunoblotting, histopathology, and immunohistochemistry. Other experiments like immunofluorescence, cell viability assay, and flow cytometry analysis were performed using the in vitro samples. The study outcomes revealed that compared to monotherapy, combination therapy of phloretin and empagliflozin was significantly effective. Phloretin and empagliflozin target the HMGB1/TLR4/MyD88/IK-ß/α/NF-κB pathway to reduce inflammation and apoptosis, in addition to their antihyperglycemic effect. Thus, phloretin, a natural dietary supplement, as an adjuvant therapy to empagliflozin can be helpful to reduce empagliflozin-associated side effects, by reducing its clinical dose and increasing its therapeutic efficacy in AKI-diabetes comorbidity.

Phloretin induces G2/M arrest and apoptosis by suppressing the ß-catenin signaling pathway in colorectal carcinoma cells.

Colorectal carcinoma (CRC) is the third most prevalent cancer, causing a significant mortality worldwide. Present available therapies are surgery, chemotherapy including radiotherapy, and these are known to be associated with heavy side effects. Therefore, nutritional intervention in the form of natural polyphenols has been well recognised to prevent CRC. Phloretin, a known dihydrochalcone is present in apple, pear and strawberry. This has been proven to induce apoptosis in cancer cells and also exhibited anti-inflammatory activity, thus can be explored as a potential anticancer nutraceutical agent. This study demonstrated phloretin's significant in vitro anticancer activity against CRC. Phloretin suppressed cell proliferation, colony forming ability and cellular migration in human colorectal cancer HCT-116 and SW-480 cells. Results also revealed that phloretin generated reactive oxygen species (ROS) which provoked depolarization of mitochondrial membrane potential (MMP) and further contributed to cytotoxicity in colon cancer cells. Phloretin also modulated the cell cycle regulators including cyclins and cyclin-dependent kinases (CDKs) and halted cell cycle at G2/M phase. Moreover, it also induced apoptosis by regulating the expression of Bax and BCl-2. The Wnt/ß-catenin signaling is inactivated by phloretin by targeting the downstream oncogenes namely CyclinD1, c-Myc and Survivin which are involved in the proliferation and apoptosis of colon cancer cells. In our study we showed that lithium chloride (LiCl) induced the expression of ß-catenin and its target genes and the co-treatment of phloretin circumvent its effect and downregulated the Wnt/ß-catenin signaling. In conclusion, our results strongly suggested that phloretin can be utilized as a nutraceutical anticancer agent for combating CRC.

Therapeutic Potential and Pharmaceutical Development of a Multitargeted Flavonoid Phloretin.

Phloretin is a flavonoid of the dihydrogen chalcone class, present abundantly in apples and strawberries. The beneficial effects of phloretin are mainly associated with its potent antioxidant properties. Phloretin modulates several signaling pathways and molecular mechanisms to exhibit therapeutic benefits against various diseases including cancers, diabetes, liver injury, kidney injury, encephalomyelitis, ulcerative colitis, asthma, arthritis, and cognitive impairment. It ameliorates the complications associated with diabetes such as cardiomyopathy, hypertension, depression, memory impairment, delayed wound healing, and peripheral neuropathy. It is effective against various microbial infections including Salmonella typhimurium, Listeria monocytogenes, Mycobacterium tuberculosis, Escherichia coli, Candida albicans and methicillin-resistant Staphylococcus aureus. Considering the therapeutic benefits, it generated interest for the pharmaceutical development. However, poor oral bioavailability is the major drawback. Therefore, efforts have been undertaken to enhance its bioavailability by modifying physicochemical properties and molecular structure, and developing nanoformulations. In the present review, we discussed the pharmacological actions, underlying mechanisms and molecular targets of phloretin. Moreover, the review provides insights into physicochemical and pharmacokinetic characteristics, and approaches to promote the pharmaceutical development of phloretin for its therapeutic applications in the future. Although convincing experimental data are reported, human studies are not available. In order to ascertain its safety, further preclinical studies are needed to encourage its pharmaceutical and clinical development.

The inhibition of GLUT1-induced glycolysis in macrophage by phloretin participates in the protection during acute lung injury.

The increased level of glycolysis in macrophage aggravates lipopolysaccharide (LPS)-induced acute lung injury (ALI). Glucose transporter 1 (GLUT1) serves as a ubiquitously expressed glucose transporter, which could activate inflammatory response by mediating glycolysis. Phloretin (PHL), an apple polyphenol, is also an inhibitor of GLUT1, possessing potent anti-inflammatory effects in various diseases. However, the potential role of PHL in ALI remains unclear till now. This study aims to investigate the impacts of PHL on ALI as well as its possible mechanisms. A mouse ALI model was established via intratracheal injection of LPS. LPS-induced primary macrophages were used to mimic in vitro ALI. Mice were pretreated with low or high dosage of PHL for 7 days via intragastric administration once a day before LPS injection. The results showed that PHL pretreatment significantly prevented LPS-induced lung pathological injury and inflammatory response. Meantime, PHL pretreatment also decreased the level of glycolysis in macrophage during ALI. In terms of mechanism, PHL inhibited the mRNA and protein expression of GLUT1. In vitro experiments further showed GLUT1 overexpression in macrophage by infection with lentivirus could abolish the inhibition of inflammation and glycolysis mediated by PHL, suggesting that GLUT1 was essential for the protection of PHL. Taken together, PHL pretreatment may protect against LPS-induced ALI by inhibiting glycolysis in macrophage in a GLUT1-dependent manner, which may be a candidate against ALI in the future.

Phloretin attenuation of hepatic steatosis via an improvement of mitochondrial dysfunction by activating AMPK-dependent signaling pathways in C57BL/6J mice and HepG2 cells.

Phloretin, a dihydrochalcone, widely exists in the fruits of apple trees and crabapple trees (Malus prunifolia) with multiple biological activities. Presently, we studied the function of phloretin on the attenuation of hepatic steatosis and further explored the underlying mechanisms both in vitro and in vivo. Male C57BL/6J mice were fed a normal diet or high fat diet (HFD) with or without phloretin (100 mg kg-1) for 12 weeks. HepG2 cells were induced by 200 µM palmitic acid (PA) and co-incubated with phloretin (50 µM) for 24 h. The results showed that phloretin treatment significantly decreased the accumulation of lipids in the liver of the HFD-fed C57BL/6J mice and PA-induced HepG2 cells. Also, phloretin effectively ameliorated hepatic steatosis via promoting fatty acid ß-oxidation (FAO). This biological activity of phloretin was closely related to its capacity to improve mitochondrial dysfunction, including the promotion of mitochondrial biosynthesis and inhibition of mitochondrial swelling through the AMPK-dependent SIRT1/PGC-1α and SIRT3/CypD signaling pathways, respectively. These results demonstrate that phloretin effectively improves mitochondrial function and ameliorates HFD-induced hepatic steatosis through an AMPK-dependent signaling pathway.

Phloretin suppresses neuroinflammation by autophagy-mediated Nrf2 activation in macrophages.

BACKGROUND: Macrophages play a dual role in neuroinflammatory disorders such as multiple sclerosis (MS). They are involved in lesion onset and progression but can also promote the resolution of inflammation and repair of damaged tissue. In this study, we investigate if and how phloretin, a flavonoid abundantly present in apples and strawberries, lowers the inflammatory phenotype of macrophages and suppresses neuroinflammation. METHODS: Transcriptional changes in mouse bone marrow-derived macrophages upon phloretin exposure were assessed by bulk RNA sequencing. Underlying pathways related to inflammation, oxidative stress response and autophagy were validated by quantitative PCR, fluorescent and absorbance assays, nuclear factor erythroid 2-related factor 2 (Nrf2) knockout mice, western blot, and immunofluorescence. The experimental autoimmune encephalomyelitis (EAE) model was used to study the impact of phloretin on neuroinflammation in vivo and confirm underlying mechanisms. RESULTS: We show that phloretin reduces the inflammatory phenotype of macrophages and markedly suppresses neuroinflammation in EAE. Phloretin mediates its effect by activating the Nrf2 signaling pathway. Nrf2 activation was attributed to 5' AMP-activated protein kinase (AMPK)-dependent activation of autophagy and subsequent kelch-like ECH-associated protein 1 (Keap1) degradation. CONCLUSIONS: This study opens future perspectives for phloretin as a therapeutic strategy for neuroinflammatory disorders such as MS. TRIAL REGISTRATION: Not applicable.

Targeting Colorectal Cancer with Conjugates of a Glucose Transporter Inhibitor and 5-Fluorouracil.

Overexpression of glucose transporters (GLUTs) in colorectal cancer cells is associated with 5-fluorouracil (1, 5-FU) resistance and poor clinical outcomes. We designed and synthesized a novel GLUT-targeting drug conjugate, triggered by glutathione in the tumor microenvironment, that releases 5-FU and GLUTs inhibitor (phlorizin (2) and phloretin (3)). Using an orthotopic colorectal cancer mice model, we showed that the conjugate exhibited better antitumor efficacy than 5-FU, with much lower exposure of 5-FU during treatment and without significant side effects. Our study establishes a GLUT-targeting theranostic incorporating a disulfide linker between the targeting module and cytotoxic payload as a potential antitumor therapy.

Decrypting the Molecular Mechanistic Pathways Delineating the Chemotherapeutic Potential of Ruthenium-Phloretin Complex in Colon Carcinoma Correlated with the Oxidative Status and Increased Apoptotic Events.

To explore fresh strategies in colorectal cancer (CRC) chemotherapy, we evaluated the capability of the ruthenium-phloretin complex in exterminating colon cancer by effectively addressing multiple apoptotic mechanisms on HT-29 cancer cells together with an animal model of colorectal cancer activated by 1,2-dimethylhydrazine and dextran sulfate sodium. Our current approach offers tangible evidence of the application of the ruthenium-phloretin complex in future chemotherapy. The complex triggers intrinsic apoptosis triggered by p53 and modulates the Akt/mTOR pathway along with other inflammatory biomarkers. The ruthenium-phloretin complex has been synthesized and successfully characterized by numerous spectroscopic methodologies accompanied by DPPH, FRAP, and ABTS assays assessing its antioxidant potential. Studies conducted in human cell lines revealed that the complex improved levels of p53 and caspase-3 while diminishing the activities of VEGF and mTOR, triggers apoptosis, and induces fragmentation of DNA in the HT-29 cells. Toxicity studies were conducted to identify the therapeutic doses of the novel complex in animal models. The outcomes of the in vivo report suggest that the complex was beneficial in repressing multiplicity of aberrant crypt foci as well as hyperplastic lesions and also promoted increased levels of CAT, SOD, and glutathione. In addition, the ruthenium-phloretin complex was able to control cell proliferation and boosted apoptotic outbursts in cancer cells associated with the increase in cellular response towards Bax while diminishing responses towards Bcl-2, NF-κB, and MMP-9. Our observations from the experiments deliver testament that the ruthenium-phloretin complex has the potential to act as a promising chemotherapeutic agent in colorectal cancer because it can affect the growth of ACF and hyperplastic abrasions in the colon tissues by evoking cell death.

Phloretin flavonoid exhibits selective antiproliferative activity in doxorubicin-resistant gastric cancer cells by inducing autophagy, inhibiting cell migration and invasion, cell cycle arrest and targeting ERK1/2 MAP pathway.

PURPOSE: Studies have shown that Phloretin exerts anticancer effects on several types of cancer cells. Nonetheless, the anticancer effects of Phloretin have not been fully explored against the human gastric cancer cells. Therefore, this study was undertaken to evaluate the anticancer effects of Phloretin against the human gastric cancer cells. METHODS: Cell proliferation was evaluated by WST-1 assay while cell cycle analysis was carried out by flow cytometry. The effects on cell migration and invasion were evaluated by wound healing assay and transwell assays, respectively. Electron microscopy and western blot methods were used to study effects on autophagy and ERK1/2/MAPK signalling pathway. RESULTS: The results showed that Phloretin inhibited the proliferation rate of the human SNU-1 gastric cancer cells and showed an IC50 of 18 µM. However, Phloretin showed very high IC50 (80 µM) against the normal GES-1 normal gastric cells. Electron microscopy showed that Phloretin triggered autophagy in the SNU-1 gastric cancer cells which was accompanied by enhancement in the expression of LC3B II and Beclin 1. Cell cycle analysis showed that Phloretin caused accumulation of the SNU-1 cells in the G0/G1 phase of the cell cycle triggering G0/G1 cell cycle arrest. The G0/G1 arrest of SNU-1 cells was also associated with depletion of cyclin D1 and D2 expression. Wound healing and transwell assays showed that Phloretin suppressed the migration of the SNU-1 gastric cancer cells, suggestive of the anti-metastatic potential of this molecule. Finally, this molecule also blocked the ERK1/2/MAPK signalling pathway in SNU-1 cells in a concentration-dependent manner. CONCLUSIONS: Phloretin may prove beneficial as a promising drug candidate for gastric cancer treatment provided further studies are carried out on it, especially toxicological studies.

Anticancer activity of Phloretin against the human oral cancer cells is due to G0/G1 cell cycle arrest and ROS mediated cell death.

PURPOSE: Phloretin is one of the important polyphenolics abundantly present across the plant kingdom. Studies have reported the anticancer effects of Phloretin against different human cancer cells. Nonetheless, the anticancer effects of Phloretin have not been explored against the human oral cancer cells. Therefore this study was designed to investigate the anticancer effects of Phloretin against the human oral cancer cells. METHODS: CCK-8 assay was used for the determination of cell viability. Annexin V/propidium iodide (PI) staining and flow cytometry were used for necrosis detection and cell cycle analysis, respectively. Wound healing assay was used for cell migration analysis. Western blot analysis was used for protein expression analysis. RESULTS: The results showed that Phloretin suppressed the proliferation rate of the human SCC-1 oral cancer cells and showed an IC50 of 12.5 µM. Nonetheless, Phloretin had negligible effects on the proliferation rate of the EBTr normal oral cells. DAPI staining showed that Phloretin did not induce apoptosis and western blot showed that it had no apparent effects on the Bax and Bcl-2 expression. Nonetheless annexin V/PI staining showed that Phloretin caused cell death in SCC-1 oral cancer cells. Flow cytometric analysis showed that Phloretin caused increase in the reactive oxygen species (ROS) levels of the SCC-1 cells in a time and dose-dependent manner. Cell cycle analysis showed that Phloretin caused increase in the percentage of the SCC-1 cells in the G0/G1 phase of the cell cycle leading to G0/G1 cell cycle arrest. The G0/G1 arrest of SCC-1 cells was also associated with depletion of cyclin D1, CDK4 and CDK6 expression. Wound healing assay was also performed which showed that Phloretin suppressed the migration of the SCC-1 oral cancer cells, indicative of the anti-metastatic potential of Phloretin. CONCLUSION: Phloretin exhibits significant growth inhibitory effects on the human oral cancer cells and may prove beneficial in oral cancer treatment.

Beneficial effects of combination therapy of phloretin and metformin in streptozotocin-induced diabetic rats and improved insulin sensitivity in vitro.

The GLUT4 and PI3K/AKT signaling pathways are the key sensors of energy status and they regulate glucose and lipid metabolism. Phloretin activates the PI3K/AKT pathway by promoting GLUT4 translocation and expression, thereby improving glucose consumption and tolerance. As metformin can regulate glucose metabolism, we hypothesized that phloretin can amplify its gluco-regulatory effects. Male Sprague Dawley rats were fed with a high-fat and high-sugar diet for 8 weeks and injected with a low dose of streptozotocin to induce type 2 diabetes. The diabetic rats were randomized to receive phloretin (100 mg kg-1 d-1), metformin (250 mg kg-1 d-1), or phloretin + metformin via oral gavage for another 4 weeks. Random blood glucose, serum insulin, free fatty acid, total cholesterol, triglyceride, and low-density lipoprotein levels were detected in type 2 diabetic rats. Hematoxylin-eosin and Oil Red O staining were used to observe the pathological changes in the liver, pancreas, and adipose tissues of type 2 diabetic rats. The expression levels of IRS-1, PI3K, P-AKT, and GLUT4 in skeletal muscle were detected using western blotting. Phloretin plus metformin improved fasting blood glucose levels, glucose tolerance, and insulin sensitivity in type 2 diabetic rats. In addition, this combination reduced lipid accumulation, improved the pathological changes in the liver, pancreas, and adipose tissue, and increased IRS-1, PI3K, P-AKT, and GLUT4 expression in skeletal muscle and the liver of type 2 diabetic rats. Thus, phloretin can be used in a potential combination therapy with metformin for the prevention and rescue of type 2 diabetes.

Role of Apple Phytochemicals, Phloretin and Phloridzin, in Modulating Processes Related to Intestinal Inflammation.

Plant-derived food consumption has gained attention as potential intervention for the improvement of intestinal inflammatory diseases. Apple consumption has been shown to be effective at ameliorating intestinal inflammation symptoms. These beneficial effects have been related to (poly)phenols, including phloretin (Phlor) and its glycoside named phloridzin (Phldz). To deepen the modulatory effects of these molecules we studied: i) their influence on the synthesis of proinflammatory molecules (PGE2, IL-8, IL-6, MCP-1, and ICAM-1) in IL-1ß-treated myofibroblasts of the colon CCD-18Co cell line, and ii) the inhibitory potential of the formation of advanced glycation end products (AGEs). The results showed that Phlor (10-50 µM) decreased the synthesis of PGE2 and IL-8 and the formation of AGEs by different mechanisms. It is concluded that Phlor and Phldz, compounds found exclusively in apples, are positively associated with potential beneficial effects of apple consumption.

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.

Pharmacological Aspects and Potential Use of Phloretin: A Systemic Review.

Over the past two decades, many researchers have concluded that a diet rich in polyphenolic compounds plays an important therapeutic role in reducing the risk of cancer, cardiovascular disease, inflammation, diabetes, and other degenerative diseases. Polyphenolic compounds have been reported to be involved in neutralization of reactive oxygen species and charged radicals, and have anticarcinogenic effects, hepatoprotective effects, low-glycaemic response, and other benefits. The benefits of fruits and vegetables may be partly attributable to polyphenolic compounds, which have antioxidant and free radical scavenging properties. Fruits such as apples contain a variety of phytochemicals, including (+)-catechin and (-)-epicatechin, phlorizin, phloretin quercetin, cyanidin-3-Ogalactoside, chlorogenic acid, and p-coumaric acid, all of which are strong antioxidants. Phloretin, a natural phenolic compound, is a dihydrochalcone, which is present in the apple. It exhibits a wide variety of activities such as antioxidative, anti-inflammatory, anti-microbial, anti-allergic, anticarcinogenic, anti-thrombotic, and hepatoprotective, besides being involved in the activation of apoptotic associated gene expression and signal transduction in molecular pathways. Despite a multitude of clinical studies, new efforts are needed in clinical research to determine the complete therapeutic potential of phloretin.

The modulatory role of phloretin in Aß25-35 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 Aß25-35 peptide sequence intracerebroventricularly (icv) in Wistar rats. To achieve this, male Wistar rats were injected with aggregated Aß25-35 peptide icv, followed by 21 days phloretin (2.5 mg/kg, 5 mg/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 Aß25-35-injected rat brains. The above experimental findings evinced the promising role of phloretin in Aß25-35-injected rats and which further envisage its potential to be explored in the treatment of AD.

Phloretin attenuates mucus hypersecretion and airway inflammation induced by cigarette smoke.

BACKGROUNDS: Cigarette 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. METHODS: Phloretin 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-1ß) were quantified. Moreover, the effects of phloretin on cigarette smoke extract (CSE)-induced expression of MUC5AC and IL-1ß 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. RESULTS: Phloretin 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-1ß 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. CONCLUSIONS: This 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.

Glucose Transporter 1-Dependent Glycolysis Is Increased during Aging-Related Lung Fibrosis, and Phloretin Inhibits Lung Fibrosis.

Aging is associated with metabolic diseases such as type 2 diabetes mellitus, cardiovascular disease, cancer, and neurodegeneration. Aging contributes to common processes including metabolic dysfunction, DNA damage, and reactive oxygen species generation. Although glycolysis has been linked to cell growth and proliferation, the mechanisms by which the activation of glycolysis by aging regulates fibrogenesis in the lung remain unclear. The objective of this study was to determine if glucose transporter 1 (GLUT1)-induced glycolysis regulates age-dependent fibrogenesis of the lung. Mouse and human lung tissues were analyzed for GLUT1 and glycolytic markers using immunoblotting. Glycolytic function was measured using a Seahorse apparatus. To study the effect of GLUT1, genetic inhibition of GLUT1 was performed by short hairpin RNA transduction, and phloretin was used for pharmacologic inhibition of GLUT1. GLUT1-dependent glycolysis is activated in aged lung. Genetic and pharmacologic inhibition of GLUT1 suppressed the protein expression of α-smooth muscle actin, a key cytoskeletal component of activated fibroblasts, in mouse primary lung fibroblast cells. Moreover, we demonstrated that the activation of AMP-activated protein kinase, which is regulated by GLUT1-dependent glycolysis, represents a critical metabolic pathway for fibroblast activation. Furthermore, we demonstrated that phloretin, a potent inhibitor of GLUT1, significantly inhibited bleomycin-induced lung fibrosis in vivo. These results suggest that GLUT1-dependent glycolysis regulates fibrogenesis in aged lung and that inhibition of GLUT1 provides a potential target of therapy of age-related lung fibrosis.

Phloretin attenuates LPS-induced acute lung injury in mice via modulation of the NF-κB and MAPK pathways.

Phloretin, which can be isolated from apple trees, has demonstrable anti-inflammatory and anti-oxidant effects in macrophages. We previously reported that phloretin could inhibit the inflammatory response and reduce intercellular adhesion molecule 1 (ICAM-1) expression in interleukin (IL)-1ß-activated human lung epithelial cells. In the present study we now evaluate whether phloretin exposure could ameliorate lipopolysaccharide (LPS)-induced acute lung injury in mice. Intra-peritoneal injections of phloretin were administered to mice for 7 consecutive days, prior to the induction of lung injury by intra-tracheal administration of LPS. Our subsequent analyses demonstrated that phloretin could significantly suppress LPS-induced neutrophil infiltration of lung tissue, and reduce the levels of IL-6 and tumor necrosis factor (TNF)-α in serum and bronchoalveolar lavage fluid. We also found that phloretin modulated myeloperoxidase activity and superoxide dismutase activity, with decreased gene expression levels for chemokines, proinflammatory cytokines, and ICAM-1 in inflamed lung tissue. Phloretin also significantly reduced the phosphorylation of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK), thus limiting the inflammatory response, while promoting expression of heme oxygenase (HO)-1 and nuclear factor erythroid 2-related factor 2, both of which are cytoprotective. Our findings suggest that, mechanistically, phloretin attenuates the inflammatory and oxidative stress pathways that accompany lung injury in mice via blockade of the NF-κB and MAPK pathways.