Study Of The Influence Of Ginsenosiderb1,Huangqi On The Apoptosis Of Cultured Neonate Rat Cerebral Cortical Neurons Caused By Hypoxia And The Mechanisms Of The Influence

Objective:To investigated the effect of ginsenosideRb1 and huangqi on the apoptosis of cultured cerebral cortical neurons caused by hypoxia and the mechanism of the protctive effect in order to supply some experimental bases for clinical application.Methods:① The primary culure of cerebral neurons of postnatal rats was established with neurobasal medium suppled 2%B27 supplement .② Neuronal viability was investigated by trypan blue exclusion to determine the effect of hypxia of different time(6,12,24 hours) and reperfusion on cultured cerebral neurons．③ Neuronal hypoxic culture and drugs interventive experiment:At 4 Div ,the cultured neurons of postnatal rat cerebral cortex were used to hypoxic investigation .there were divided to such research groups: (1)the normal control group (the negtve control group):the neurons of which was cultured in 5%CO2/95%air atmosphere at 37℃without hypoxia; (2)the induced apoptotic group(the postive control group):the neurons were deprivated oxygen and then returned to normal culture condition;(3)GinsenosideRb1 experimental group1(10μg/ml)；(4)GinsenosideRb1experimental group2(50μg/ml)；(5)GinsenosideRb1 experimental group3(100μg/ml)；(6)huangqi injection experimental group 1(10mg/ml)；(7)huangqi injection experimental group 2(50mg/ml)；(8)huangqi experimental group 3(100mg/ml)；(3)-(8)groups were drugs interventive groups,the drugs of specific concentration were added to the medium of cultured neurons at 4 Div,then the neurons together with that of the postive control group were placed in a gas-tight tank flushed with 5%CO2/95%N2 at 37℃ for 12 hours.After 12h of hypoxia the neurons were further incubated up to 48h in 5%CO2/95%air atmosphere at 37℃.Then apoptotic incident of cultured neurons was evaluated by Hoechst 33342 staining ,flow cytometer , DNA agarose gel electrophoresis. ④The change of expression of apoptotic controlling genes bcl-2/bax were detected by immunocytochemistry among drugs intereventive groups,the postive control group and the negetive control group.RESULTS:①After cultured neurons were hypoxiaed for different times(6,12,24 hours) and reperfused for 48 hours, compared with the normal control group, there were less live cells. The cell viability of the normal control group , 6 hours hypoxia group and 12 hours hypoxia group was more than 80%,but that of 24 hours hypoxia group was less than 50%. ② Little apoptosis was detected by FACS,DNA agarose gel electrophoresis and Hoechst 33342 staining in negative control group. In the apoptosis induced group, many of the neurons had condensed chromatin(some of them broken into debris) which emitted brighter green fluorescence by using the the fluorescence dye Hoechst 33342; Sub-G1 peak was detected by flow cytometry and typical apoptotic DNA ladders were also detected by electrophoresis. The apoptotic rates of neurons in different concentration the ginsenoside Rb1 group and huangqi injection group were decreaser compared with that in the postive group by using the fluorescence dye Hoechst 33342 and flow cytometry,furthermore apoptotic rates of neurons were lower and lower with the increase of contreation of added drugs . typical apoptotic DNA ladders could hardly be detected by DNA agarose gel electrophoresis in the ginsenoside Rb1 group 2and huangqi injection group2.The expression of bcl-2 in cultured cerebral neurons was found to lower level in contrasts with that of bax measured by immunocytochemistry after hypoxia and reperfusion . Ginsenoside Rb1 and huangqi could up-regulate bcl-2 protein and down–regulate bax protein expression.CONCLUSION: Ginsenoside Rb1 and huangqi could prevent the apoptotic incidence trigger by hypoxia.Ginsenoside Rb1 and huangqi could up-regulate the expression of bcl-2 protein of hypoxic neurons and down–regulate that of bax.The primary culure of crebral neurons of postnatal rats with neurobasal medium suppled 2%B27 supple was a good model for investigation of the drugs that prevent from hypoxic apoptosis of neuron and the mechnism o...