Experimental Study On The Effects Of Quercetin On Hippocampal Neurons Of Sprague Dawley Rats Cultured With High Glucose

[Objective]To explore the effect of Quercetin on expressions of GRP78, Caspase-12and apoptosis of hippocampal neurons of Sprague Dawley rats cultured with high glucose.[Methods]Hippocampus were obtained from newborn24h Sprague Dawley rats, then separated 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, the neurons were cultured respectively in following conditions including DMEM, high glucose(50mmol/L) media,50mmol/L glucose media containing0.5、1.25、2.5、5、7.5、10、20、100umol/L Quercetin.DMEM served as negative control. After72h, MTT was used to decide the optimum concentration of Quercetin.In the above condition, the neurons were cultured respectively in following conditions including DMEM, high glucose(50mmol/L) media,50mmol/L glucose media containing2.5、5.7.5umol/L Quercetin and10μmol/LZ-VAD-FMK (ZVAD).After being cultured for72h under different conditions, MTT colorimetric assay was adopted to measure vitality of cultivated neurons, flow cytometry was used to detect apoptosis of neurons, and western blotting was explored to determine the expression of GRP78and Caspase-12.[Results]1. Hippocampal neurons were culturated successfully, and the purity of neurons wasabove95%and it can be used in the experiment study.2. MTT colorimetric assay:After72h, compared with control group, the OD values ofhigh glucose group and different Quercetin groups all diminished significantly(P<0.01);The OD values of Q5(5umol/L)and Q7.5(7.5umol/L)groups increased greatlycompared with high glucose group (P<0.05) and there were no significant differences between these two treated groups and ZVAD group(P>0.05).3. Flow Cytometry:The apoptosis rate of hippocampal neurons of high glucose group, different Quercetin groups and ZVAD group all significantly increased compared with that of control group(P<0.01);the apoptosis rate of different Quercetin groups and ZVAD group significantly decreased compared with high glucose group (P<0.01,P<0.05), and there was no significant difference between Q5group、 Q7.5group and ZVAD group (P>0.05).4. Western Blotting:Compared with control group, the expressions of GRP78and Caspase-12in high glucose group, different Quercetin groups and ZVAD group all significantly increased(P<0.01); Compared with high glucose group, the expressions of GRP78and Caspase-12in all Quercetin groups decreased noticeably (P<0.01), which was more significant in Q5group and Q7.5group(P<0.01); there were no significant differences between Q5group and Q7.5group; the expressions of Caspase-12of Q5group、Q7.5group and ZVAD group showed no significant differences(P>0.05).[Conclusion]1、High glucose (50mmol/L) promoted the apoptosis of hippocampal neurons and increased the expressions of GRP78and Caspase-12at72h, while the vitality of hippocampal neurons was inhibited.2、Quercetin could increase the vitality of hippocampal neurons, inhibit the apoptosis of hippocampal neurons and the role of Quercetin was similar to that of Z-VAD-FMK group.In the case of Quercetin inhibiting the apoptosis of hippocampal neurons,the expressions of GRP78and Caspase-12decreased,which showed that the mechanism of action of Quercetin may be associated with the endoplasmic reticulum stress.[Innovation]To study the effect of of high glucose on expressions of endoplasmic reticulum stress protein of GRP78,Caspase-12and apoptosis in hippocampal neurons incubated in vitro, as well as to study the effect of Quercetin on expressions of GRP78,Caspase-12and apoptosis in hippocampal neurons cultured in high glucose from the aspects of cellular level and molecular level, which hasn’t been reported home and abroad.