The Effect Of High Glucose On Apoptosis Of Cultured Rat Hippocampal Neurons With Anoxic Injury And The Regulation Of Naoshenkang Capsule

Objective:1. To investigate the effects of high glucose on apoptosis of cultured rat hippocampal neurons with anoxic injury.2. To observe the effects of Naoshenkang Capsule on the apoptosis rate of rat hippocampal neurons with anoxic injury.3. To observe the effects of Naoshenkang Capsule on the mRNA expressions of HIF-1α, Bcl-2 and Bax.Methods:1. Adult male Wistar rats were randomly divided into five groups, and respectively given Ligustrazine Hydrochloride Injection with 40mg/kg by intraperitoneal injection, and low (5mL/kg),middle (10mL/kg),high (20mL/kg) concentration of Naoshenkang Capsule and the same dosage Sodium Chloride by administration. Blood was obtained 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 inhibite overgrowth of non-neurocytes and to purify neurons.2. On the 8th day, the neurocyte cultured in the normal and high glucose environment were used and randomly divided into eight groups: ①Control group:Neurons were raised in 5.5mmol/L DMEM.②High glucose group:Neurons were raised in 25mmol/L DMEM without serum. Indexes were measured after twelve hours.③X/XO group:The toco-oxygen system that the final concentration of X and XO respectively were lmmol/L and 20U/L were added into this group. After a quarter to one hour, neurons were raised in 25mmol/L DMEM without serum. Indexes were measured after twelve hours.④Igustrazine group, normal sodium group and the small, middle, and large dosage of Naoshenkang groups:Igustrazine, normal sodium and the small, middle, and large dosage of Naoshenkang groups were respectively added before half an hour. After the X/XO system affected for a quarter to one hour, neurons were raised in 25mmol/L DMEM without serum. Indexes were measured after twelve hours.3. The metabolic rate of hippocampal neurons was measured by MTT. The apoptosis rate of hippocampal neurons was tested by flow cytometry. The method of Real-time PCR was used to evaluate the mRNA expression of HIF-la, Bcl-2 and Bax, and the ratio of Bcl-2 and Bax.Results:1. The metabolic rate of neurons of the high glucose and X/XO group were significantly lower than the control group (P<0.01). The metabolic rate of neurons of the X/XO group was significantly lower than the high glucose group (P<0.05). The metabolic rate of neurons of igustrazine group and all dosage of Naoshenkang groups were significantly higher than the X/XO group, and the middle dosage group was the highest with statistical significance (both P<0.05). The igustrazine group was get close to the low group with no statistical significance (P>0.05).2. The apoptosis rate of neurons of the high glucose and X/XO group were significantly higher than the control group (P<0.01). The apoptosis rate of neurons of the X/XO group was significantly higher than the high glucose group (P＜0.05). The apoptosis rate of neurons of igustrazine group and all dosage of Naoshenkang groups were significantly lower than the X/XO group, and the middle dosage group was the lowest with statistical significance (both P＜0.05). The igustrazine group was get close to the low group with no statistical significance (P>0.05).3. The expression of Bcl-2 mRNA and the ratio of Bcl-2/Bax of the high glucose group and X/XO group were significantly lower, but the expression of HIF-la mRNA and Bax mRNA significantly higher than the control group (both P<0.01). The expression of Bcl-2 mRNA and the ratio of Bcl-2/Bax of the X/XO group were significantly lower, but the expression of HIF-la mRNA and Bax mRNA significantly higher than the high glucose group (both P<0.05). The expression of Bcl-2 mRNA and the ratio of Bcl-2/Bax of the ligustrazine group and all Naoshenkang groups were significantly higher, but the expression of HIF-la mRNA and Bax mRNA significantly lower than the X/XO group, and the middle dosage group was the most significant with statistical significance (both P<0.05). The igustrazine group was get close to the low group with no statistical significance (P>0.05).Conclusions:High glucose can cause the apoptosis of hippocamp neuron, and Naoshenkang Capsule can down-regulate the expression of HIF-1αmRNA and Bax mRNA, up-regulate the expression of Bcl-2 mRNA, inhibit the apoptosis of hippocamp neuron and has a protective effect on the anoxic injury in high glucose environment.