Neuroprotective Effects Of The Traditional Chinese Medicine With Function Of Invigorating Qi And Tonifying Kidney Against Anoxia-Reoxygenation Injury On Cultured Rat Hippocampal Neurons

Objective:1.To investigate the effects of Naoshenkang Capsule-Traditional Chinese Medicine with function of Invigorating Qi and Tonifying Kidney on the appearance, survival rate and lactate dehydrogenase(LDH) activity of cultured rat hippocampal neurons with anoxia-reoxygenation injury.2.To investigate the effects of Naoshenkang Capsule on the activity of superoxide dismutase(SOD) and content of malondialdehyde(MDA).3.To explore the effects of Naoshenkang Capsule on the apoptosis rate and mRNA expressions ofBcl-2,Bax and BDNF genes.Methods:1.Adult male Wistar rats were randomly divided into two groups and orally given 5%water extract of Naoshenkang Capsule and the same dosage Sodium Chloride respectively to prepare serum.Newborn rats were sterilized with 0.75 volume fraction of alcohol,and their brains were obtained.Then,the bilateral hippocampus was separated and hippocampal neurons were cultured.Arabinosyl cytosine was added into culture medium to suppress overpopulation of non-neurocytes,and increase the purification rate of neurons.After cultivating 7 days, we identified the cultured cells by the immunohistochemistry staining(MAP-2 antibody is used).On the 10th day,they could be used in experiments.2.The primarily cultured neonate rat hippocampal neurons were randomly divided into five groups:â‘ normal group:neurons were cultured with DMEM/F12 and incubated with 5%CO₂ and 95%O₂;â‘¡anoxia-reoxygenation group:anoxia-reoxygenation injury model was made: neurons were treated with non-serum DMEM/F12 solution and then cultured in an incubator(37℃,95%N₂,5%CO₂,oxygen content under 1%) for 6 hours,10%normal rat serum was added before 24h reoxygenation(5%CO₂,95%O₂);â‘¢small,middle and large dosage of Chinese Medicine-protective groups: anoxia-reoxygenation injury model was made,5%,10%,20%of Naoshenkang containing serum was added to small,middle and large dosage of Chinese Medicine-protective groups before 24h reoxygenation.3.The metabolic rate of MTT,content of MDA and the activity of SOD,LDH were measured by assay kits.The apoptosis rate of hippocampal neurons was tested by flow cytometry.RT-PCR was used to evaluate the mRNA expressions of Bcl-2, Bax and BDNF genes.Results:The activity of SOD,survival rate of cells,the Bcl-2/Bax and mRNA expression of Bcl-2 genes in anoxia-reoxygenation group were significantly lower(all P＜0.01), but the level of MDA and LDH,apoptosis rate and the mRNA expression of Bax, BDNF genes higher(all P＜0.01) than those in normal group.The activity of SOD, survival rate of cells,the mRNA expression of Bcl-2,BDNF genes and the ratio of Bcl-2/bax in all Chinese Medicine-protective groups were significantly higher(all P＜0.01),but the level of MDA and LDH,apoptosis rate and the mRNA expression of Bax genes lower(all P＜0.01) than those in anoxia-reoxygenation group and demonstrated obvious dosage-effect relationship.Conclusions:Collectively,these results indicated that Naoshenkang Capsule strongly protect primary cultured neurons against anoxia-reoxygenation-induced oxidative stress.It can also inhibit the apoptosis of injured hippocampal neurons by increasing the mRNA level of Bcl-2 and decreasing the mRNA level of Bax.The mechanism may be carried out by the effects of multi-levels,multi-steps and multi-targets.