Pharmacological Effects Of Genistein On Vascular Endothelial Function

Phloretin is a major compound of dihydrochlcone, according to a large amount of studies, it has various biological activities, including anti-oxidant, anti-aging, anti-tumor, improving immune function, hyperglycemic and so on, and phloretin is widely used in the food and medicine industries, particularly, in the cosmetics field. Phloretin has been used as the main whitening antioxidant additive, because it has strong ability of scavenging free radicals. At the same time, it has reported that the choline could be metabolized into trimethylamine (TMA) by the gut flora, while the TMA is oxidized to oxidation trimethylamine (TMAO) in the liver, TMAO can slow or reduce removal rates of cholesterol in the blood vessels, and causes endothelial damage, finally accelerating the production of atherosclerosis.There for, we selected phloretin as the experiment material to study it’s vascular endothelial function and hepatoprotective activity. It comprehensively and intensively showed phloretin’s effect in physiological efficacy and mechanism of blood vessels, through its in vitro and in vivo experiments.The main conclusions and results of these studies were as follows:(1) The biochemical analysis of serum and liver tissues homogenate have shown that the mice were provided with 3% high choline water for consecutive 10 weeks, could cause the upward trend of blood lipid level, endothelial cell and liver inflammation, finally leading to vascular endothelial dysfunction and liver injury. However, after given different dosages of phloretin orally once a day, the mice serum of total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL), alanine aminotransferase (ALT), aspertate aminotransferase (AST), endothelin-1 (ET-1) and thromboxane B2 (TXB2) levels have markedly decreased. Moreover, the high density lipoprotein (HDL), nitric oxide (NO) and 6-ketone prostaglandin Fla (6-keto-PGFla) in the serum of the mice have decreased.(2) At the same time, the mice after 10 weeks treated with high choine, through analyzing the activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and levels of non-esterified fatty acid (NEFA), malondialdehyde (MDA) of liver tissues homogenate of high choline-treated mice, we could further demonstrated that high choline could cause liver injury and vascular endothelial dysfunction in mice. Then, after administration of phloretin orally daily obviously improved the above figures, and with the increased doses of phloretin the effect was stronger. Moreover, histopathological test with the H&E and Oil Red O staining of liver sections confirmed the liver pathologic changes associated with intake of high choline water, as the liver cells with fat accumulation, shrink and even necrosis. However, phloretin showed a strong hepatoprotective activity. These findings illustrated that phloretin could relieve or reduce the high choline-induced liver injury and vascular endothelial dysfunction.(3) To further explore the molecular mechanism of phloretin on vascular endothelial dysfunction and injury, human umbilical vein endothelial cell (HUVEC) injured model by being exposed to high dosage of 0.4 mmol/L of TMAO was developed. HUVEC viability and morphyology were measured to observe the influence of phloretin on HUVEC cultured by high TMAO. NO and ET-1 levels in cell supernatant were tested by using kits or ELISA, and HUVEC apoptosis was tested by flow cytometry detection and DAPI staining. The results showed that the high 0.4 mmol/L of TMAO could reduce the NO level and increase ET-1 level in cell supermatant, respectively from16.09±1.10 μmol/gprot and 2.81±0.20 pg/mL of normal group to 7.14±1.02 μmol/gprot and 6.12±0.38 pg/mL of model group (p<0.01). The Annexin V-FITC/PI assay tested that phloretin protected HUVEC survival rates from injury, HUVEC treated with high TMAO in 24 h, the apoptosis rate reached 23.6% and the cell survival rate was 76.23%, however, along treated with different dosages of phloretin, the apoptosis rate of HUVEC has improved, cell survival rates were 79.52%,82.97% and 86.80%. DAPI staining illustrated that the apoptosis situation of HUVEC apoptosis body, the damage and shrinkage of cell nucleus, finally leading to HUVEC apoptosis. However, various dosages of 12.5,25,50μmol/L of phloretin slightly elevated the HUVEC viability, restored the cell morphology, and increased the NO level and decreased ET-1 level, inhabited the HUVEC impairment by being exposed to high TMAO. These results suggest phloretin could adjust the expression of NO and ET-1, balance NO system function, and protect HUVEC from damage by high TMAO.