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Objective:To investigate the inhibitory effect of phloretin on inflammation and oxidative stress in interleukin (IL)-1β induced orbital fibroblasts (OFs) from Graves orbitopathy (GO) patients and its mechanism.Methods:The orbital fat and connective tissue from 6 eyes of 6 patients diagnosed as inactive GO who underwent orbital decompression in Henan Eye Hospital from January 2019 to December 2020 were collected.Primary OFs were isolated and passaged by explant culture and were identified by cell immunofluorescence assay.OFs were divided into control group, IL-1β induced group, and groups of various phloretin concentrations (25, 50, 75, 100 and 200 μmol/L). The viability of OFs after 24- and 48-hour treatment of the various phloretin concentrations was determined by cell counting kit-8 (CCK-8). OFs were induced by IL-1β to simulate an inflammatory environment of GO in vitro.Intracellular reactive oxygen species (ROS) levels of the normal control group, IL-1β induced group, 50 μmol/L phloretin group and 100 μmol/L phloretin group were detected by fluorescent probe (H 2DCF-DA). The concentrations of pro-inflammatory cytokines IL-6, IL-8 and monocyte chemoattractant protein-1 (MCP-1) in cell culture supernatant of the normal control group, IL-1β induced group and phloretin treated groups (25, 50, 75, and 100 μmol/L) were examined by enzyme-linked immunosorbent assay (ELISA). The expressions of heme oxygenase-1 (HO-1), nuclear factor erythroid 2-related factor (Nrf2) proteins, as well as P38, extracelluar regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) proteins as well as their phosphorylated proteins in the MAPK signal pathway of the normal control group, IL-1β induced group and 100 μmol/L phloretin group, were detected by Western blot.The purpose and methods of the study were explained to the patients and their family members.Written informed consent was obtained.The study protocol was approved by the Ethics Committee of Henan Provincial People's Hospital (No.HNEECKY-2020[07]). Results:For cultured OFs, the mesenchymal origin was confirmed by positive expression of vimentin and fibroblasts were identified by negative expression of desmin, S-100 and cytokeratin-18.CCK-8 showed that there was no significant difference in absorbance value after 24- and 48-hour treatment between 25 μmol/L, 50 μmol/L, 75 μmol/L and 100 μmol/L phloretin groups and control group (all at P>0.05). The ROS levels of 50 μmol/L and 100 μmol/L phloretin groups were 21.95±1.71 and 10.01±1.03, respectively, which were significantly lower than 39.27±4.01 of IL-1β induced group (both at P<0.01). ELISA showed that IL-6 concentrations in 25 μmol/L, 50 μmol/L, 75 μmol/L and 100 μmol/L phloretin groups were (4 544.25±572.98), (1 000.25±133.96), (724.25±98.63), (519.50±118.02)pg/ml, respectively, which were all significantly lower than (7 581.75±565.93)pg/ml in IL-1β induced group (all at P<0.01). IL-8 concentrations in 50 μmol/L, 75 μmol/L and 100 μmol/L phloretin groups were (3 679.50±676.76), (2 143.75±616.20), (1 174.75±284.18)pg/ml, respectively, which were all significantly lower than (8 411.00±939.67)pg/ml in IL-1β induced group (all at P<0.01). The concentrations of MCP-1 in 50 μmol/L, 75 μmol/L and 100 μmol/L phloretin groups were (3 783.25±610.24), (1 565.75±457.89), (745.75±227.01)pg/ml, respectively, which were all significantly lower than (5 533.00±602.87)pg/ml in IL-1β induced group (all at P<0.01). The relative expression levels of HO-1 and Nrf2 were significantly higher and the relative expression levels of p-P38, p-ERK, and p-JNK were significantly lower in 100 μmol/L phloretin group than IL-1β induced group (all at P<0.01). Conclusions:Phloretin reduces the oxidative stress level of IL-1β induced OFs from GO patients and inhibits the production of pro-inflammatory cytokines.The mechanism is related to the activation of Nrf2/HO-1 and the inhibition of the MAPK signal pathway.
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Objective: To compare the cardioprotective efficacy of equimolar doses (50 mM/kg, p.o.) of phloretin and genistein against doxorubicin-induced cardiotoxicity in rats. Methods: Cardiotoxicity was induced in rats by intraperitoneal injection of 6 mg/kg doxorubicin on alternative days till the cumulative dose reached 30 mg/kg. This study included four treatment groups of rats (n=6): the control group (0.5% carboxymethyl cellulose solution-treated), the doxorubicin- treated group (0.5% carboxymethyl cellulose solution along with doxorubicin), the genistein-treated group (50 mM/kg/day; p.o. along with doxorubicin) and phloretin-treated group (50 mM/kg/day; p.o. along with doxorubicin). On the 10th day of dosing, rats were anesthetized for recording ECG, mean arterial pressure, and left ventricular function. Oxidative stress, nitric oxide levels, and inflammatory cytokines were estimated in the cardiac tissue. Cardiac function parameters (creatine kinase MB, lactate dehydrogenase, aspartate aminotransferase, and alanine transaminase) were estimated in the serum samples. Results: Phloretin treatment inhibited doxorubicin-induced oxidative stress and also reduced nitric oxide levels in cardiac tissues of rats. Phloretin administration attenuated doxorubicin- induced alterations in hemodynamic parameters (heart rate, mean arterial blood pressure, and left ventricular function) and suppressed the expression of pro-inflammatory cytokines. The cardiac injury markers like creatine kinase MB, lactate dehydrogenase, aspartate aminotransferase, and alanine transaminase were reduced by both genistein and phloretin. All these effects of phloretin were more prominent than genistein. Conclusions: Phloretin offers cardioprotection that is comparable to genistein, a clinically validated cardioprotectant against doxorubicin-induced cardiotoxicity. Further studies are needed to confirm and establish the therapeutic utility of phloretin as a chemopreventive adjuvant to doxorubicin chemotherapy.
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Objective:To explore the absorption and transport properties of flavanomarein in the Madin-Darby canine kidney(MDCK) monolayer cell model. Method:Methyl thiazolyl tetrazolium(MTT) assay was used to investigate the toxicity of flavanomarein in MDCK cells. The resistance value of MDCK monolayer cell model was detected by Millicell-ERS-2 cell resistometer. The effects of mass concentration of flavanomarein,administration time,sodium-glucose cotransporter(SGLTs) inhibitor and glucose transporter 2(GLUT2) inhibitor on the transmembrane transport of flavanomarein were investigated. The concentration of flavanomarein was determined by UPLC-MS/MS, and the apparent permeability coefficient(Papp) and the efflux ratio(ER) were calculated. Result:When the concentration of flavanomarein was 5.625-120 mg·L-1, there was no significant toxic effect on MDCK cells. The transport of flavanomarein in MDCK monolayer cell model was time-dependent and concentration-dependent. The Papp values of flavanomarein were basically between 1×10-6 cm·s-1 to 10×10-6 cm·s-1. Compared with the blank group, the phlorizin group significantly reduced the transport of flavanomarein on the MDCK monolayer cell model at 60 min and 90 min. Conclusion:Flavanomarein is a moderately absorbed drug in the intestine, its transmembrane transport mechanism is dominated by passive transport along with active transport. SGLTs may be involved in mediating the transport of flavanomarein on the MDCK monolayer cell model.
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Objective: To determine the transdermal absorption of phloretin and ligustilide in Fufang phloretin cream.Methods: In vitro percutaneous absorption test using the Franz intelligent transdermal absorption apparatus was adopted, an HPLC method was used to determine the contents of phloretin and ligustilide in the accepting solution at different time points, and then the cumulative release amount and the total penetration amount were calculated to evaluate the percutaneous absorption behavior.Results: Phloretin and ligustilide showed a good linearity within the range of 0.044 2-4.42 μg·ml-1(r=0.999 9) and 0.048 7-4.87 μg·ml-1(r=0.999 9), respectively, the average recovery was 99.85% and 99.92%, and RSD was 0.67% and 0.66% (n=9), respectively.The in vitro permeation behavior of phloretin and ligustilide through the skin of rats fit Higuchi equations(r>0.9), suggesting a good linear correlation between the cumulatine penetration amount (Qn) and (time).Conclusion: Fufang phloretin cream has a good percutaneous permeation property in vitro.
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Phloretin is a kind of natural dihydrochalcone from the root bark, fruits, and leaves of the apple tree. It’s also found in a variety of plants. Phloretin has remarkable curative effect in anti-inflammatory, antibacterial, antitumor, and other biological activities. So it has a wide range of application prospects. The derivatization of phloretin contributes to the improvement of its biological activity and water solubility. This paper reviews the progress of the derivatization of phloretin and provides a reference for its research and utilization.
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Objective To investigate proliferative and apoptotic effects of phloretin on hepatoma carcinoma cells, hepatoma carcinoma cells HepG-2 was used as research materials.Methods This research observed morphological alterations using phase contrast microscopy and electron microscopy, cell proliferation were detected by MTT assay, and using flow cytometry detected apoptotic rates, cell cycle progression, mitochondrial trans-membrane potential and intracellular calcium homeostasis.ResuIts Apoptotic cells appeared morphological alterations.Phloretin exerted a inhibitory the proliferation of HepG-2 cell line, and induced its apoptosis in a dosage and duration dependent manner.Cell cycle was arrested at G1 phase, mitochondrial trans-membrane potential dropped, intracellular free Ca2 + increased.ConcIusion Phloretin can induce apoptosis of HepG-2 via arresting cell cycle progression, reducing mitochondrial trans-membrane potential and disturbing intracellular calcium homeostasis.
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[Abstract ] Objective To investigate effect of phloretin on apoptotic of hepatoma carcinoma cells SMMC-7721, and explore its mechanisms.Methods Logarithmic phase of hepatoma carcinoma cells SMMC-7721 were cultured separately with 30, 60, 120 mg/L phloretin, morphological alterations of apoptotic were observed by phase contrast microscopy and AO/EB double fluorescence staining method was used to observe were low, medium and high concentration trentment group, respectively.the cells treated by phloretin.Apoptotic rates, cell cycle progression, mitochondrial trans-membrane potential and intracellular calcium homeostasis were detected by flow cytometry.Results Cells appeared typical apoptosis morphological alterations.Phloretin induced SMMC-7721 cell line apoptosis in a dosage and duration dependent manner.Cell cycle was arrested at G1 phase, mitochondrial trans-membrane potential decreased, intracellular free Ca2 +increased.Conclusion Phloretin induce apoptosis of SMMC-7721 by affecting cell cycle progression, reducing mitochondrial trans-membrane potential and changing intracellular calcium homeostasis.
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OBJECTIVE: To evaluate the effect of phloretin on the uptake of ginsenoside Rb1 by the cell model, primary rat brain microvascular endothelial cells, (rBMEC) in vitro. METHODS: Experiments were divided into two groups, ginsenoside Rb1 with and without the addition of phloretin intervention. The cellular uptake of ginsenoside Rb1 was determined by LC-MS/MS. Protein content in cultured cells was measured by the method of BCA. RESULTS: The uptake amount of ginsenoside Rb1 in rBMEC with phloretin was significantly lower(P < 0.05). CONCLUSION: The uptake of ginsenoside Rb1 by rBMEC is significantly inhibited by phloretin.
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Objective To investigate the proliferative and apoptotic effects of phloretin on gastric cancer cell andthe possible mechanisms. Methods SGC-7901 were treated with different concentrations of phloretin(40,80,160 mg/L), and the cell morphological alterations were detected by using Hoechst33258 staining, cell activity were detected by MTT assay, cell apoptosis, cell cycle progression, mitochondrial trans-membrane potential and intracellular calcium homeostasis were detected by flow cytometry.ResuIts After treated with different concentrations of phloretin at different times, SGC-7901 cell showed morphological alterations.Phloretin could inhibite the proliferation of SGC-7901 cell line, and induced its apoptosis in a dosage and duration dependent manner.Cell cycle was arrested at G1 phase, mitochondrial trans-membrane potential dropped, intracellular free Ca2 +increased.ConcIusion phloretin can induce apoptosis of SGC-7901 via arresting cell cycle progression, reducing mitochondrial trans-membrane potential and disturbing intracellular calcium homeostasis.
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AIM To investigate whether the relaxation characteristics of phytoestrogens resveratrol and phloretin on contractile response of aortic strips are similar to that of estrogen and the mechanisms underground. METHODS Aortic strips from rabbits were suspended in organ baths containing Krebs solution, and then isometric tension was measured. RESULTS Resveratrol and phloretin inhibited the contractile responses to norepinephrine (NE), KCl and CaCl2, shifted their concentration-response curves rightward with pD2′ values of 2.89, 3.34, 3.37 for resveratrol and 3.23, 3.52, 3.77 for phloretin respectively. Also both of them concentration-dependently relaxed KCl-precontracted aortic strip. The relaxing response of resveratrol but not of phloretin in aortic strip was significantly reduced by removal of endothelium or incubation with Nω-L-nitro-arginine and methylthioninium chloride, however both their relaxant effects were not affected by indometacin and propranolol. In Ca2+-free Krebs solution containing 0.01 mmol·L-1 EGTA, resveratrol and phloretin inhibited NE-induced contraction which was caused by Ca2+ release from intracellular store, but did not affect the contraction which was induced by Ca2+ influx. CONCLUSION Resveratrol and phloretin can induce vasorelaxations which may relate to inhibition of Ca2+ influx through potential-dependent calcium channels and Ca2+ release from intracellular stores, and the relaxing response of resveratrol is endothelium-dependent in part, but of phloretin is not endothelium-dependent.
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It has been reported that ascorbic acid[AA]appears to be actively taken up by the corneal endothelium and protect the endothelium against harmful effects of the oxidative reactions. To investigate the effect of ascorbic acidon the corneal endothelial function, rabbit`s corneas were mounted in the in vitro specular microscope. Corneal endothelium was perfused with ascorbic acid, then switched to AA plus ouabain solution, and vice versa. Also, phloretin was perfused onto the endothelium with AA and ouabain. Andcorneal endothelium was perfused with GBR or AA solution followed by perfusion with ouabain. Corneal thickness was measured during the perfusion and the corneal swelling rate calculated. Corneal endothelial permeability was also measured after perfusion of ascorbic acid. Perfusion with AA showed no corneal swelling, but swelling rate was even lower than GBR control. Corneal endothelial permeability did not change upon AA perfusion. In corneas preperfused with ouabain, AA added to ouabain solution decreased corneal swelling rates induced by ouabain solution[19.9 vs. 40.5 micrometer/hr]. The corneas preperfused with AA also showed decreased swelling rates with subsequent perfusion of ouabain added to AA solution[21.7 vs.28.6 micrometer/ hr]. Phloretin inhibited the effect of AA.However, when ouabain was removed, the corneal swelling plateaued but did not return to baseline thickness in both AA and GBR perfusion.The results of this study showed that AA can increase corneal endothelial pump function and reduce corneal swelling caused by ouabain.