Protection Of Ultra-filtration Extract From The Mixture Of Anglica Sinensis And Hedysarum Polybotrys On Adriamycin-induced Myocardial Cell Damage

Objective:To discuss the possible protection mechanism of Ultra-filtration Extract from the mixture of Anglica sinensis and Hedysarum polybotrys(UFE-AH) on adriamycin (ADR)-induced myocardial damage by observing its impact on DNA damage, mitochondrial membrane potential changes and the protein expression of Bcl-2, Bax and Caspase-3induced by ADR in the myocardial cell of neonatal rats.Methods:Myocardial cells of neonatal rats (1-2d, Wistar) were primarily extracted by differential adhesion method. Cells with concentration of1×106/mL were cultured in closed incubator with37℃and5%CO2for72h. Cardiomyocyte apoptosis model was established by2.5mg·L-1ADR treatment. UFE-AH (1:5) was made by ceramic membrane and PAN (Ultra-filtration membrane) of the hollow fiber (cut to stay molecular weight to measure for100000). Subjects were divided into5groups:normal control group, model group (myocardial cell with adriamycin, final concentration2.5mg·L-1),UFE-AH low-dose intervention group (pretreatment myocardial cells with UFE-AH of0.38g·L-1for2h); UFE-AH middle-dose intervention group(pretreatment myocardial cells with UFE-AH of0.75g·L-1for2h); UFE-AH high-dose intervention group(pretreatment myocardial cells with UFE-AH of1.5g·L-1for2h); the growth Inhibition Rate of Cardiomyocytes of Neonatal Rat was detected by MTT assay; the DNA damage was detected by single cell gel electrophoresis; the mitochondrial membrane potential was detected with a confocal microscopy; the protein expression of Bcl-2,Bax and Caspase-3were detected by Western blotting assay.Results:1. MTT assay for myocardial cells survival rate detection:Compared with the normal control group, survival number of myocardial cells was decreased, and the growth inhibition of myocardial cells was significantly increased in model group (P<0.05). Compared with the normal group, the growth inhibition rate was significantly decreased in all UFE-AH groups (P<0.05).2. Single cell gel electrophoresis test results:Cells in normal group showed circular fluorescent core; comet tail was seen in cells of model group. The values of HDN A%, TDN A%, TL, TM, OTM in model group were significantly higher than in the normal group (P<0.05) Compared with model group, the the values of HDNA%, TDNATL, TM, OTM in UFE-AH intervention groups were significantly lower in the UFE-AH intervention group (P<0.05)3.Confocal laser scanning results:The Rh123fluorescence intensity of myocardial cells in normal group was very high; compared with normal group the Rh123fluorescence intensity of myocardial cells in the model group was significantly reduced, but in UFE-AH intervention group enhanced, mainly the ΔΨm increased(P<0.0.5).4. Western blot results:There was a certain amount of expression of Bcl-2, Bax, Caspase-3protein in normal group. Bcl-2expression in model group significantly was decreased(P<0.05), but Bax and Caspase-3expression was increased(P<0.05). In UFE-AH intervention group, Bcl-2protein expression was significantly increased(P<0.05), Bax, Caspase-3protein expression was significantly lower(P<0.05), and the radio of Bcl-2/Bax was significantly higher(P<0.05).Conclusions:1. DNA damage and the Bax, Caspase-3activation are involved in the adriamycin-induced injury of myocardial cell.2. The UFE-AH can reverse the adriamycin-induced myocardial cell injury, possibly by reducing the DNA damage induced by adriamycin, repairing the damaged mitochondrial membrane potential and suppressing the release of apoptosis factors.3. The UFE-AH may play a part in the treatment of cardiovascular disease through inhibition of cardiomyocyte apoptosis.