Effect Of Naoshenkang Capsule On Oxidative Stress Of Rat Hippocampal Neurons With Anoxic Injury In High Glucose

Objective:1. To observe the effects of Naoshenkang Capsule-Traditional Chinese Medicine with function of Invigorating Qi and Tonifying Kidney on the appearance, survival and apoptosis rates of cultured rat hippocampal neurons with anoxic injury in high glucose.2. To research the effects of Naoshenkang Capsule on the mRNA expressions of HIF-1α, Nox4and p47phox of cultured rat hippocampal neurons with anoxic injury in high glucose.Methods:1. Preparation of medicated serumAdult male Wistar rats were randomly divided into five groups, each group of6, respectively given low (5ml/kg), middle (10ml/kg), high (20ml/kg) concentration of Naoshenkang Capsule, astragalus particles solution (10ml/kg) and the same dosage Sodium Chloride by administration. Blood was obtained to prepare serum.2. The cultivation of the primary hippocampal neurons and MAP-2antibody test.Newborn Wistar rats (male and female unlimited) within24h were sterilized with0.75volume fraction of alcohol, take out full brain tissue, stripping meningeal and connective tissue. Then, the bilateral hippocampus was separated and hippocampal neurons were cultured. Arabinosyl cytosine was added into culture medium to inhibite overgrowth of non-neurocytes and to purify neurons. After cultivating8days, we confirmed that the cultured cells into neurons by the immunohistochemistry staining(MAP-2antibody test) and determine its purity. 3. On the eighth day, The primarily cultured rat hippocampal neurons were randomly divided into the following six groups:(1) The normal control group:Neurons were raised in5.5mmol/L DMEM containing10%saline serum. Indexes were measured after twelve hours.(2)High glucose and xanthine/xanthine oxidase (x/xo) group:The toco-oxygen system that the final concentration of X and XO respectively were1mmol/L and20U/L were added into this group. After a quarter to one hour, neurons were raised in25mmol/L DMEM without serum. Indexes were measured after twelve hours.(3) Astragalus group, the low, middle and high dosage of Naoshenkang groups: astragalus, the low, middle, and high dosage of Naoshenkang medicated serum were respectively added into the DMEM before half an hour. After the X/XO system affected for a quarter to one hour, neurons were raised in25mmol/L DMEM without serum. Indexes were measured after twelve hours.4. The survival rate of hippocampal neurons was measured by MTT. The apoptosis rate of hippocampal neurons was tested by flow cytometry. The method of real-time RT-PCR was used to detect the mRNA expression of HIF-la, Nox4and p47phox.Results:1. Neurons microtubule associated protein (MAP-2) immunofluorescence staining test results showed that the cells cultured in vitro was the rat hippocampal neurons, the positive cell percentage was80%-90%. Observed the cells under inverted phase contrast microscope, high glucose and xanthine/xanthine oxidase group was in poor growth and decrease in the number. In contrast, high dosage of Naoshenkang group was in small damage and in good condition.2. MTT shows that the survival rate of neurons of High glucose and xanthine/xanthine oxidase (x/xo) group were significantly lower than the normal control group (P<0.01). The survival rate of neurons of astragalus group and all dosage of Naoshenkang groups were significantly higher than the X/XO group, and the high dosage group was the highest with statistical significance (both P<0.05).The flow cytometry test shows that the apoptosis rate of neurons of High glucose and xanthine/xanthine oxidase (x/xo) group were significantly higher than the normal control group (P<0.01). The apoptosis rate of neurons of the astragalus group and all dosage of Naoshenkang groups were significantly lower than the X/XO group, and the high dosage group was the lowest with statistical significance (both P<0.05).3. The RT-PCR method shows that the expression of HIF-1a, Nox4and p47phox mRNA of the X/XO group were significantly higher than the normal control group (P<0.01). The expression of HIF-la, Nox4and p47phox mRNA of the astragalus group and all dosage of Naoshenkang groups were significantly lower than the X/XO group, and the high dosage group was the lowest with statistical significance (both P<0.05).Conclusion:Naoshenkang capsule can make a protective effect on hippocampal neurons in high glucose with oxidative damage. Its mechanism may be that Naoshenkang capsule can down regulate HIF-1a, Nox4and p47phoxmRNA expression, reduce the oxidative stress injury.