The Effect And Mechanism Of Rhodioside And Puerarin On The Apoptosis Of Hippocampal Neurons Of Sprague Dawley Rats Cultured With High Glucose Medium

[Objective]1. To observe the effect of high glucose on apoptosis of rat hippocampal neurons and the p38MAPK and JNK signaling pathway.2. To explore the effect of rhodioside and puerarin on apoptosis of hippocampal neurons cultured with high glucose medium and the p38MAPK and JNK signaling pathway.[Methods]Hippocampus were obtained from newborn24h Sprague Dawley rats, then cultivated and purified by methods of trypsin digestion, gradient centrifugation and differential attachment. Immunofluorescence of neuron-specific enolase was adopted for the identification of the cultivated hippocampal neurons. Set25mmol/L glucose as the control group and the conditioned medium50,75,100,125mmol/L of different concentrations of glucose. CCK-8was used to decide the optimum concentration and reactive time of high glucose. In50mmol/L Glu high glucose medium,50μmol/L rhodioside,25μmol/L puerarin, lOμmol/L p38MAPK pathway inhibitor SB239063, lOμmol/L JNK pathway inhibitor SP600125was separately added into the medium.72hours later, CCK-8was used to detect vitality of cultivated neurons, TUNEL was used to detect apoptosis of neurons, and western blotting was adopted to detect the expressions of p-p38, p38, p-JNK and JNK.[Results]1. Hippocampal neurons were culturated successfully, and the purity of neurons was95.4±2.3%.2. CCK-8used to detect the vitality of neurons:Hippocampal neurons in vitro cultivation of24h and48h, the cells relative vitality of each high glucose groups compared with the normal group was no significant difference (P>0.05).The cells relative vitality of all high glucose groups were suppressed significantly compared with the normal group at72h and48h (P<0.05). The cells relative vitality of salidroside group and puerarin group significantly increased compared with50mmol/L Glu as the high glucose group at72h(P <0.05).3. TUNEL Apoptosis detection:The apoptosis rate of high glucose group increased compared with the normal group (P<0.01). The apoptosis rate of rhodioside group, p38MAPK inhibitor group and JNK inhibitor group decreased compared with high glucose group (P<0.01). The apoptosis rate of puerarin group decreased compared with high glucose group (P<0.05). The apoptosis rate of rhodioside group and puerarin group increased compared with the normal group (P<0.01). The apoptosis rate of p38MAPK inhibitor group and JNK inhibitor group increased compared with the normal group (P<0.05). There is no significant difference between rhodioside group, puerarin group, p38MAPK inhibitor group and JNK inhibitor group (P>0.05).4. Western Blotting:The p-p38/p38of high glucose group increased compared with the normal group (P<0.01). The p-p38/p38of p38MAPK inhibitor decreased compared with the normal group (P<0.01). The p-p38/p38of rhodioside group, puerarin group, p38MAPK inhibitor group and JNK inhibitor group decreased compared with high glucose group (P<0.01). The p-p38/p38of rhodioside group, puerarin group and JNK inhibitor group increased compared with the normal group (P<0.01). There is no significant difference between rhodioside group and puerarin group(P>0.05). The p-p38/p38of rhodioside group and puerarin group increased compared with p38MAPK inhibitor group (P<0.01) and decreased compared with JNK inhibitor group (P<0.01).The p-JNK/JNK of high glucose group increased compared with the normal group (P <0.01). The p-JNK/JNK of JNK inhibitor decreased compared with the normal group(P <0.01). The p-JNK/JNK of rhodioside group, puerarin group, JNK inhibitor group and p38MAPK inhibitor group significantly decreased compared with high glucose group (P <0.01). The p-JNK/JNK of rhodioside group, puerarin group and p38MAPK inhibitor group increased compared with the normal group (P<0.01). The p-JNK/JNK of puerarin group significantly decreased compared with rhodioside group(P<0.01). The p-JNK/JNK of rhodioside group and puerarin group increased compared with JNK inhibitor group (P<0.01) and decreased compared with p38MAPK inhibitor group (P<0.01).[Conclusion]1. When hippocampal neurons cultured in high glucose (50mmol/L) for72h, high glucose inhibited its vitality. Rhodioside (50μmol/L) and puerarin (25μmol/L) can increase the vitality of hippocampal neurons cultured in high glucose.2. High glucose promoted the apoptosis of hippocampal neurons, and the p-p38/p38and p-JNK/JNK of high glucose group significantly increased compared with normal group. It prompts that p38MAPK and JNK signaling pathways were activated, and specific inhibitor SB239063, SP600125could block the pathway and improve the condition of cell apoptosis.3. Under the condition of high glucose, rhodioside and puerarin can alleviate the apoptosis of hippocampus neurons, reduce the phosphorylation of p38and JNK in p38MAPK and JNK signaling pathway.[Innovation]To study the effect of rhodioside and puerarin on apoptosis of SD rats hippocampal neurons cultured with high glucose medium and the p38MAPK and JNK signaling pathway. Although proapoptotic effect of high glucose on cultured hippocampal neurons has been reported before, whether high glucose via p38MAPK and JNK pathway induced the apoptosis of hippocampal neurons cultured in vitro, the effect and mechanism of rhodioside and puerarin on apoptosis of hippocampal neurons cultured with high glucose medium in vitro haven’t been reported home and abroad.