Effects Of Jinmaitong On Apoptosis Of Hippocampal Neurons Of Sprague Dawley Rats Cultured With High Glucose Medium

[Objective]To explore the effect of Jinmaitong (JMT) on expressions of Bax, Bcl-2and Caspase-3and apoptosis in hippocampal neurons cultured with high glucose medium form.[Methods]Hippocampus were obtained from newborn24h Sprague Dawley rats, then cultivated and purified by methods of trypsin digestion, differential attachment and gradient centrifugation. Immunofluorescence of neuron-specific enolase was adopted for the identification of the cultivated hippocampal neurons. In the third day of cultivation,25,50,75,100,125,150mmol/L glucose were added into the medium separately. Then MTT was used to decide the optimum concentration and reactive time of high glucose. In the third day of cultivation,50mmol/L glucose was added into the medium, and ZVAD group with other addition of10μmol/L Z-VAD-FMK, inhibitor of caspase, and JMTH, JMTM and JMTL groups with other addition of10%high-, middle-, low-dose Jinmaitong serum separately.72hours later, MTT was used to detect vitality of cultivated neurons, flow cytometry was used to detect apoptosis of neurons, and western blotting was adopted to detect the expressions of Bax, Bcl-2and Caspase-3.[Results]1. Hippocampal neurons were culturated successfully, and the purity of neurons was above95%.2. MTT Colorimetric Analysis:The OD values of all high glucose groups increased with the extension of culture time, and started to decrease at72h. And the OD values of50,75,100groups decreased significantly at72h compared with that of48h (P<0.05); The OD values of all hige glucose groups decreased significantly compared with that of normal group at72h (P<0.01). Compared with50mmol/L Glu group, the OD values of JMTH, JMTM and ZVAD increased significantly at72h (P<0.05), and there was no significant difference between JMTH and ZVAD groups. 3. Flow Cytometry:The apoptosis rate of high glucose group, ZVAD, JMTH, JMTM and JMTL groups are35.97%,28.85%,29.01%,31.22%,32.68%seperately, significantly increased compared with that of normal group(19.45%)(P<0.01); compared with high glucose group, apoptosis rate of ZVAD and JMT groups significantly decreased (P<0.01), and there is no significant difference between JMTH and ZVAD groups (P>0.05).4. Western Blotting:Compared with normal group, the expressions of Bax and Caspase-3in high glucose group, ZVAD and JMT groups significantly increased(P<0.05), and the expression of Bcl-2significantly decreased in high glucose group and ZVAD groups (P<0.05); compared with high glucose group, the expressions of Caspase-3and Bax significantly decreased in ZVAD and JMT groups (P<0.01), and the expression of Bcl-2significantly increased (P<0.05); compared with ZVAD group, the expressions of Caspase-3and Bax significantly decreased in JMT groups (P<0.05), and the expression of Bcl-2significantly increased in JMTH and JMTM groups(P<0.05).[Conclusion]1. Hippocampal neurons were cultivated and purified by methods of trypsin digestion, differential attachment and gradient centrifugation. Immunofluorescence of neuron-specific enolase was adopted for the identification of the neurons, and the results showed the purity was above95%.2. High glucose inhibited the vitality of hippocampal neurons. JMTH and JMTM groups can increase the vitality of hippocampal neurons cultivated in high glucose, and the role of JMTH group is similar to that of ZVAD group.3. High glucose promoted the apoptosis of hippocampal neurons. JMT inhibited the apoptosis of hippocampal neurons cultivated in high glucose, and the role of JMTH group is similar to that of ZVAD group.4. High glucose promoted expressions of Bax and Caspase-3of hippocampal neurons, and depressed the expression of Bcl-2. JMT corrected the abnormal expressions of Bcl-2, Bax and Caspase-3, and the roles of JMTH and JMTM groups were better than that of ZVAD group.[Innovation]To study the effect of Chinese compound medicine, Jinmaitong, on expressions of Bax, Bcl-2and Caspase-3and apoptosis in hippocampal neurons cultured with high glucose medium form. Although proapoptic effect of high glucose on cultured hippocampal neurons has been reported before, expressions of apoptosis-related proteins of hippocampal neurons, and the intervention of compound Chinese medicine on apoptosis and abnormal expressions of Bax, Bcl-2and Caspase-3of hippocampal neurons cultured with high glucose medium haven’t been reported home and abroad.