| In the previous experiment,our research team found that the appropriate dose ofβ-hydroxybutyratethat one of the main components of ketone body can significantly reduces Infarct size in the brain and improves the neurological dysfunction of the the middle cerebral artery occlusion(MCAO)model rat through the lateral ventricle,and play a role in brain protection of the MCAO model.In order to clarify the cerebral protective effect and the mechanism of the acetoacetic acid and beta-hydroxybutyrate that main components of ketone bodies,in ischemic and hypoxic environment,this study was designed to investigate neurons derived from the cerebral cortex of Sprague dawley feta rats Whether have a protective effect and its possible related mechanisms on the oxidative damage of the cellular cells.In vitro,An model of oxidative injury of neurons in rat cerebral cortical neurons was established by hypoxia culture in serum-free,sugar-free medium and a three-gas incubator(healforce),and to ininvestigate the dose-effect,time-effect relationship of different levels and different pretreatment time of acetoacetate And β-hydroxybutyric acid on neurons that cultured under the glucose and oxygen deprivation.And this topic is mainly divided into the following two parts:Part IThe Effect of Acetoacetate and p-Hydroxybutyrate on the Activity of Neurons derived from the cerebral cortex of SD feta rat Objective:the primary neurons was isolated,purified and cultured from the cerebral cortex of SD fetal rats(15-20d during pregnancy),and the purity of neurons were identified and observed the effect of cell survival rate and its dose-effect,time-effect relationship by acetoacetate and β-hydroxybutyrate.Methods:1.the primary neurons was isolated,purified and cultured from the cerebral cortex of SD fetal rats(15-20d during pregnancy).2.The purity of neurons were identified at 7 days after culture.3.The primary cultured neurons were divided into the normal group and the experimental group.The experimental group were divided into group I(acetoacetate group)and group II(beta-hydroxybutyrate group),each group contains 10 concentrations of the subgroup(1 mM,2 mM,3 mM,5 mM,10 mM,20 mM,30 mM,40 mM,50 mM,100 mM),each subgroup was set at 12 h,24 h and 36 h points,and 5 parallel samples were set in each group.4.CCK8 test was utilized to test the proliferation and cytotoxicity of the neuron,and to indirectly reflect the survival rate.Results:1.On the 7th day,the primary neuron derived from the cerebral cortex of Sprague dawley feta rats grew well,the cell bodies were larger,and the trunk and branches of the cytoplasm were significantly prolonged and thickened,and the protrusions formed a dense neural network.2.The immunocytochemistry of NSE is a classic method to identify neurons.The results showed neurons are the main body,and the Purity accounts for more than 95%.3.CCK8 test was utilized to detect the survival rate of neurons,The results showed that with the increase dose of acetoacetate concentration in the range of 1-10 mM,the survival rate of neuronal cells was not statistically different from that of the normal group(P>0.05).In the range of 20-100 mM,the survival rate of neurons was significantly lower than that of the normal group(P<0.01),and the survival rate of neuronal cells showed a decreasing trend in the range of 30-100 mM(P<0.05),there was no significant difference in the survival rate of neurons in the same concentration(P>0.05).With the increase dose of(3-hydroxybutyrate concentration in the range of 1-5 mM and the increase time of action time from 12 to 36 hours,the survival rate of neuronal cells was not statistically different from that of the normal group(P>0.05);At 10 mM concentration,from 12h to 24 hours,the survival rate of neurons was not statistically different from normal(P>0.05),but the survival rate of neurons was significantly lower than that of the control group when the time was prolonged to 36 hours(P<0.05).When the concentration was in the range of 20-100 mM,the survival rate of neurons was significantly lower than that of the normal control group(P<0.01),and the survival rate of neuronal cells decreased as the concentration increased(P<0.05).In the range of 20-50 mM,the survival rate of neurons showed a progressive decrease with the prolongation of the action time in the same concentration(P<0.05).At the concentration of 100 mM,the survival rate of neuronal cells was not counted.Difference at each time point(P>0.05).Conclusions:The experimental results showed that the concentration of acetoacetate less than 10 mM had no cytotoxicity on the survival rate of neurons,and indicating that this concentration range can be used as a safe dose of acetoacetate for subsequent experiments.the concentration of β-Hydroxybutyric acid less than 5 mM had no cytotoxicity on the survival rate of neurons,and when the concentration ofβ-hydroxybutyrate was 10 mM and the action time was less than 24 hours,it also had no cytotoxicity,but when the duration of action was prolonged to 36 hours,it had cytotoxic,which indicating that the concentration of p-hydroxybutyric acid less than 10 mM could be used as a safe dose for follow-up experiments,and the duration of action should be less than 24 hours.Part IIThe protective effects and molecular mechanism of Acetoacetate and Beta hydroxybutyrate in cytotoxicity of oxygen glucose deprivation on neurons derived from the cerebral cortex of SD fetal ratsObjective:To observe the effect and the mechanism of acetoacetate and β-hydroxybutyrate on the apoptosis of neurons after glucose and oxygen deprivation in the cerebral cortex of SD fatal rats.Methods:1.The primary cultured neurons divided into two groups:the normal cultured group and the glucose-oxygen-deprivation cultured group(sugar and oxygen-deprived serum-free and sugar-free medium was inoculated with cells.Incubator(healforce)was subjected to hypoxic culture).Each group was divided into the control group,high-dose acetoacetate group,middle-dose acetoacetate group,low-dose acetoacetate group,high-dose β-hydroxybutyrate group,middle-dose β-hydroxybutyrate group and the low-dose(3-hydroxybutyrate group,there are 14 groups in total.Based on these 14groups,set three time points for 1h,5h and 24h.In the acetoacetate groups,the low,middle and high doses of the acetoacetate groups were 2 mM,5 mM and 8 mM respectively.The low,medium and high doses of the β-hydroxybutyrate groups were 2 mM,5 mM and 10 mM respectively,each subgroup was set at 1 h,5 h and 24 h points.2.CCK8 test was utilized to test the proliferation and cytotoxicity of the neuron,which indirectly reflected the survival rate of neurons.3.The analysis of Mitochondrial respiratory chain complex Ⅲ(Coenzyme Q-cytochrome C reductase)was performed.4.The immunofluorescence histochemical marker of NSE was used to observe the morphology and calculate the purity of neurons.5.Tunel staining was used to observe the apoptosis of neurons.6.The extraction of neurons in each groups was used for Western Blot process to test the expression of Apoptosis proteins caspase3,bcl2,bax and Cytochrome C(CytC).Results:1.CCK8 test was utilized to test the survival rate of neurons in different groupsThe survival rate of neurons only in the high-dose acetoacetate group was significantly higher than that in the normal culture group at the 24 h time point(P<0.05),but there was no statistical difference in the other groups(P>0.05).The survival rate of neurons at each time point in the glycogen-deprived group was significantly lower than that in the normal culture group(P<0.05);at the 24h time point,the survival rate of neurons in the middle dose group and high dose group of the acetoacetate group andβ-hydroxybutyrate group were significantly decreased.The survival rate of the neurons was significantly higher than that in the glycogen deprivation group(P<0.01).The increase trend was more significant in the high-dose group(P<0.01),but which still lower than that in the normal culture group(P<0.05).The survival rate of neurons in the low-dose group was not significantly different from that in the glycogen deprivation group(P>0.05).The survival rate of neurons was no significant difference between the groups at the 1 h and 5h time points(P>0.05).2.Mitochondrial respiratory chain complex III activity(Coenzyme Q-cytochrome C reductase)detectionMitochondrial analysis showed that:In normal culture large groups,there were no significant difference in the activity of mitochondrial respiratory chain complex III in the low,middle and high dose of acetoacetate and beta-hydroxybutyrate groups(P>0.05).The activity of the mitochondrial respiratory chain complex III in the glucose and oxygen deprivation group was significantly lower than that in the normal culture group(P<0.01).The high and middle doses of acetoacetate and β-hydroxybutyrate groups were higher than those in the oxygen deprivation group(Control),and there were more significantly in the middle-dose group(P<0.05),while the low-dose group had no significant difference compared to the control group(P>0.05).3.The immunofluorescence histochemical marker of NSE to observe the morphology of neurons.In normal culture groups,the cytoplasm of neurons were round or triangular,the cytomembrane and nuclear membrane were integrity,the cytoplasm and nucleus are uniformly coloured,axons and processes stretching,the cell connection network are formed compactly between cells and cells.In glucose-oxygen-deprivation culture groups,the cytoplasm of neurons are became smaller,deformed,and the cytomembrane and nuclear membrane are integrity,but the cytoplasm of neurons appeared foaming phenomenon.With the phenomenon of foaming,the neurites appeared to be shortened or even disappeared,and the cells appeared to be round.In the acetoacetate group andβ-hydroxybutyrate groups,the cytoplasm deformation phenomenon and cytoplasm foaming phenomenon of neurons were reduced in the middle dose groups and high dose groups,and the shortening and regression phenomenon of neurites was improved,and there were more significantly in the high-dose groups,It is particularly evident that the cytoplasm of neurons foaming phenomenon is significantly reduced,the boundaries of the cell membrane and the nuclear membrane are clear,and the neurites of neurons are well extended,and the cell-cell connection network can be seen.There was no significant difference in cell morphology between the low-dose group and the glucose-oxygen-deprived culture group.4.Tunel staining to observe the apoptosis of neurons.The TUNEL index of neurons in each group was analyzed:the TUNEL index of neurons was significantly higher in each group of glucose-oxygen-deprived culture groups than those in normal culture groups.(P<0.05);the TUNEL index of neurons in the medium and high dose acetoacetic and β-hydroxyl groups was significantly lower than that in the glycogen deprivation group(P<0.01),and there were more significantly in the high-dose group(P<0.01),but still significantly higher than that in the normal culture group(P<0.05);but the TUNEL index of neurons was no significant difference in the low-dose acetoacetate and β-hydroxybutyrate groups compared with the glycogen deprivation group(P>0.05).5.Western Blot to test the expression of Apoptosis proteins caspase3,bcl2,bax and CytCprotein The results of Western Blot test were analyzed:compared with the normal culture group,the expression of caspase 3,bcl 2,bax and CytC wase significantly higher in glucose-oxygen deprivation groups(P<0.01).After glucose and oxygen deprivation,the expressions of caspase3,bax and CytC in middle and high-dose of acetoacetate andβ-hydroxybutyrate groups was significantly lower than those in glucose-oxygen deprivation group(P<0.05),and that was more significant in high-dose groups(P<0.05),but still higher than the normal culture group(P<0.05),then the expression of bcl2 in the middle and high-dose group of acetoacetate and β-hydroxybutyrate was significantly higher than that in the sugar-oxygen deprivation group(P<0.05),and that was More significant in the high-dose groups(P<0.05).The expression of caspase3,bax,bcl2 and CytC in the low-dose acetoacetate and β-hydroxybutyrate groups had no significant difference between the glucose-oxygen deprivation group(P>0.05).Conclusion:After glucose and oxygen deprivation,appropriate doses of acetoacetate andβ-hydroxybutyrate can reduce the toxic effects of glucose-oxygen deprivation on neurons derived from the cerebral cortex of fetal SD rats in vitro,that serves neuroprotective effects and anti-apoptosis induced by glucose-oxygen deprivation through regulating ratio of apoptosis related gene bcl-2/bax and enhancing the mitochondrial respiratory function and reducing the expression of caspase3,finally promote the survival of neurons. |
PDF Full Text Request