| Objective:To study if the Naoshenkang capsule could influence expression of 8-OHdG, HIF-1α, ROS, VEGF of rat hippocampal neurons in anoxia and high glucose and investigate its protection for rat hippocampal neurons.Methods:1. Preparation of groups and medicated serum24 adult male Wistar rats, in accordance with the random number table, were assigned into four groups, each group of six. Respectively given low(5mL/kg), middle(10mL/kg), high(20mL/kg)concentration of Naoshenkang Capsule, and the same dosage Sodium Chloride by intragastric administration as the previous. Blood was obtained to prepare serum and save in the refrigerator at -20℃.2. Hippocampal neurons in primary cultureDecollated the newborn Wistar rats (male and female unlimited) within 24h after they were sterilized with 0.75 volume fraction of alcohol. Taked out full brain tissue,isolated bilateral hippocampa under aseptic conditionsl, cut them into pieces, digested, filtered, centrifuged, resuspended, inoculated, put in the incubator under 37℃,5%CO2. After4-6h, the planting medium was changed into DMEM/F12 medium including 2%B27. Replace half of the cell medium every other day and observe the cell metamorphosis by the invert microscope.3. Authenticate the primary hippocampal neurons by MAP-2 immunofluore scence staining.When the cells climbing to the carry sheet glass for 9 days, the slides of cells were lavated, fixed, incubated and seal with the buffered glycerol. Observ ed the positive expression cells of MAP-2 and take a microphotograph.4. Preparation of oxidative damaged and high glucose modelAfter 8 days, the experimental groups and different means was carried out as follows:(1)High glucose and xanthine/xanthine oxidase (x/xo) group:The toco-oxygen system which including X (1mmol/L)and XO (20U/L)were added into this group. After 45 minutes, neurons were raised in 25mmol/L DMEM without serum. Indexes were detected after 12 hours.(2)The low, middle and high dosage of Naoshenkang groups:the low, middle, and high dosage of Naoshenkang medicated serum were respectively added into the DMEM for 30 minutes. Then change the X/XO system for 25mmol/L DMEM without serum after 45 minutes. Indexes were detected after 12 hours.(3)The normal control group:Neurons were raised in 5.5mmol/L DMEM containing 10% saline serum. Indexes were detected after 12 hours.5. The proteins of 8-OHdG, ROS, HIF-1α were detected by the Elisa Kit. The mRNA expression of HIF-1α, ROS, VEGF were mesured by the Real-time PCR. Results:1. The results of MAP-2 immunofluorescence staining shows that the cultured cells were the rat hippocampal neurons, the positive cell percentage was 85%-90%. Through the observation found that the cells cultured in high glucose and hypoxia decreased and swelled. The high dosage of Naoshenkang group exhibited good growth.2. The Elisa Kit method shows that compared with those of the normal control group, the production of 8-OHdG, ROS, HIF-1α were higher (P<0.01); compared with those of the x/xo group,the production of 8-OHdG, ROS, HIF-1α in all of the Naoshenkang groups were lower(P<0.05), the large dosage of Naoshenkang group showing a superior outcome compared with those of the other two Naoshenkang groups.3. The Real-time PCR for fluorescence quantification method shows that c ompared with those of the normal control group, the expression of HIF-1α, R OS, VEGF were higher (P<0.01); compared with those of the x/xo group, the expression of HIF-1α, ROS, VEGF in all of the Naoshenkang groups were low er (P<0.05), the large dosage of Naoshenkang group showing a superior outco me compared with those of the other two Naoshenkang groups.Conclusions:Naoshenkang capsule may play a resultful role on hippocampal neurons in high glucose with oxidative damage. The Naoshenkang capsule can efficiently reduce the expression of 8-OHdG, ROS, HIF-1α, VEGF mRNA, then finally decrease the oxidative stress injury. |
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