| This paper used blue fox (Alopex Lagopus) gall bladder bile as experiment material and analysed composition of fox bile by reversed-phase high-performance liquid chromatography, explored effect of fox bile on oxidative damage induced by hydrogen peroxide in neonatal rat cardiac myocytes. The research content and method:Firstly, established the method for composition bile acid in fox bile by reversed-phase high-performance liquid chromatography through orthogonal test. Secondly, established oxidative damage model in cardiac myocytes with different concentrations of hydrogen peroxide, different concentrations of fox bile preprocessed myocardial cells, cell survival rate was determined by MTT assay. Thirdly, we detected biochemical indicators including lactate dehydrogenase, superoxide disambiguation enzyme, malondialdehyde and reduced glutathione for analysising fox bile on myocardial cells against oxidative damage.Results:Firstly, a column was Hypersil BDS Ci8(4.6mm×250mm,5μm). Methanol-30mmol/L potassium dihydrogen phosphate(V:V,70:30, pH=3.8) was used as mobile phase in isocratic elution. Flow rate was1.0mL/min. The detector wavelength was set at205nm and the column temperature was25℃. The fox bile contained taurocholic acid and taurochenodeoxy--cholic acid and their peak area ratios respectively were35.05%and9.23%. But we didn’t detect the free bile acid. Each component had good liner relationship at the range of3.33-10g/L. SecondLy, cell survival rate determined by MTT assay showed that cell survival rate gradually decreased with the increase of hydrogen peroxide concentration and it was concentration of dependencies with hydrogen peroxide. When the concentration of hydrogen peroxide was400μmol/L and its treatment time was six hours, cell survival rate was57.78%, it was used to establish oxidative damage. Compared with oxidative injury group, the cell survival rate of different concentrations (100、200、400μg/mL) fox bile groups was significantly increased (P<0.05). Biochemical analysis showed that:The permeability of cell membrance and lipid peroxidation were increased by H2O2treatment. So the levels of LDH (136.31±18.94U/L) in culture medium and the contents of MDA in cells (28.53±0.46nmol/mgprot) were significantly higher than the control group respectively (42.24±2.143U/L、11.33±0.494nmol/mgprot), difference had statistics significance (P<0.05). The activity of SOD (24.24±2.776U/mL) and the content of GSH (48.59±0.8392μmol/gprot) in cells were significantly lower than the control group respectively (39.43±0.7404U/mI、59.85±1.751μmol/gprot)(P<0.05). Fox bile could significantly reduce the leakage of LDH by H2O2-induced and the content of MDA.400μg/mL-dose group (LDH52.68±5.056U/L, MDA12.58±0.7049nmol/mgprot),200μg/mL-dose group (LDH69.11±0.5021U/L, MDA13.59 ±0.4672μmol/gprot) and100μg/mL-dose group (LDH101.94±0.198U/L, MDA16.12±0.2305μmol/gprot) were significantly higher than the H2O2damage group (P<0.05). Fox bile could significantly increase the activity of SOD and the content of GSH in cells.400u.g/mL-dose group (SOD36.81±0.5304U/mL,GSH56.77±1.619μmol/gprot),200μg/mL-dose group (SOD35.10±0.6451U/mL,GSH54.46±0.9518μmol/gprot) and1OOμg/mL-dose group (SOD31.84±0.2864U/mL, GSH51.92±1.023μmol/gprot) were significantly lower than the H2O2damage group (P<0.05).To sum up, high performance liquid chromatography method can be simple, rapid and effective separation combined type bile acid of the fox bile. And fox bile had protective effect on oxidative damage induced by H2O2in myocardial cell. |
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