| Objective: To explore the effect and mechanism of the ultra-filtration extracted fromangelica sinensis and hedysarum polybotrys on rat pheochromocytoma cells (PC12cells) damage induced by hydrogen peroxide.Methods: Different dosages of H2O2were used in the incubation of PC12cells fordifferent point of time to select the optimal dosage of H2O2and length of time toestablished the oxidative damage cell model, and it can be found that the optimaldosage of H2O2was200μmol·L-1and point of time was six hours. Then the oxidativedamaged cells were treated with three different dosages of ultra-filtration (1.5g·L-1,0.75g·L-1,0.375g·L-1) extracted from angelica sinensis and hedysarum polybotrysafter modeling for interference. Finally the PC12cells were divided into five groups:control group, model group, high-interference group, mid-interference group, andlow-interference group. Measuring the cell survival rate by MTT; testing the contentsof MDA, LDH and MDA by Ultraviolet spectrophotometry; testing the contents ofROS dyed by DCF-DA; detecting the rate of cell apoptosis by flow cytometry;measuring mitochondrial membrane potential RH123dyed by Laser scanningconfocal microscopy; detecting the expression of Caspase-3、Bax and Bcl-2byWestern blotting.Results:1.The changes of cell survival rate: The survival rate of PC12cells was decreasedsignificantly after damaged by H2O2, the survival rate of PC12cells of the modelinggroup was decreased obviously compared with the control group cells (p<0.05);survival rate of PC12cells interfered with ultra-filtration increased apparentlycompared with the modeling group cells (p<0.05), especially the high-interferencegroup clearly.2.The results of MDA, LDH and SOD of each groups of PC12cells: The contents ofMDA and LDH of the modeling group cells were increased, and but the activity ofSOD was decreased compared with the control group cells (p<0.05); In contrast, Thecontents of MDA and LDH of PC12cells interfered with ultra-filtration were increased and the activity of SOD was decreased compared with the modeling groupcells (p<0.05).3.The contents of ROS of each groups of PC12cells: The contents of ROS ofmodeling group cells was increased apparently compared with the control groupcells (p<0.05); The contents of ROS of PC12cells interfered with ultra-filtration wasdecreased compared with the modeling group cells (p<0.05).4.The rate of cell apoptosis of each groups of PC12cells: The rate of cell apoptosis ofmodeling group cells was increased obviously compared with the control group cells(p<0.05); The rate of cell apoptosis of PC12cells interfered with ultra-filtration wasdecreased compared with the modeling group cells (p<0.05).5.The results of mitochondrial membrane potential: The fluorescence intensity ofRH123of modeling group cells was apparently decreased, and the mitochondrialmembrane potential was also decreased compared with the control group cells(p<0.05); The fluorescence intensity of RH123of PC12cells interfered withultra-filtration was clearly increased, and the mitochondrial membrane potential wasalso increased compared with the modeling group cells (p<0.05).6.The expression of Caspase-3、Bax and Bcl-2: The expression of Caspase-3and Baxof the modeling group cells were increased, the expression of Bcl-2was decreased,and the ration between Bcl-2and Bax was also decreased compared with the controlgroup cells (p<0.05); The expression of Caspase-3and Bax of PC12cells interferedwith ultra-filtration were decreased, the expression of Bcl-2was increased, and theration between Bcl-2and Bax was increased compared with the modeling group cells(p<0.05).Conclusions:1.The ultra-filtration extracted from angelica sinensis and hedysarum polybotrys havethe protective effect on rat pheochromocytoma cells (PC12cells) damage induced byhydrogen peroxide.2.The ultra-filtration extracted from angelica sinensis and hedysarum polybotrys caneffectively protect cells by reducing the contents of MDA and ROS of the oxidativedamage cells, increasing the activity of SOD activity of the oxidative damage cells to raise antioxidant capacity, maintain the integrity of membrane structure and reducethe damage of oxygen free radicals to the cells.3.The ultra-filtration extracted from angelica sinensis and hedysarum polybotrys canmaintain mitochondrial membrane potential of PC12cells induced by H2O2, decreasethe expression of Caspase-3and Bax, increase the expressioin of Bcl-2to protect themitochondrial function and reduce apoptosis. |
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