Effects Of ERK Inhibitor On Neuronal Apoptosis And The Expression Of Apoptosis-related Protein In The Rats Of Early Intermittent Hypoxia

Objective Through building the model of intermittent hypoxia in rats to observe neuronal apoptosis and apoptosis-related proteins, expression in the area of hippocampus nerve cells of early intermittent hypoxia. Injecting ERK inhibitor U0126 to block ERK signaling pathway and to explore the effects of early intermittent hypoxia on neuronal apoptosis and apoptosis-related proteins, expression in the CA1 area of hippocampus nerve cells.Method Using the random number randomly divided 135 adult male Wistar rats into 3groups: control group(UC group), intermittent hypoxia group(IH group), ERK inhibitor groups(U0126 group). Every group were divided 1d, 3d, 7d, 10 d, 14 d five times subgroups, and each subgroup selected nine rats. Rats in ERK inhibitor groups were injected ERK inhibitor U0126(0.2mg/kg)at 7:00 am every day, 30 min after injection with IH hypoxic rats together put into the box, gave a different flow rate of nitrogen and compressed air cyclically. each cycle time was 120 s, the fluctuation of oxygen concentration was between 5-21%, every day exposured 7h. UC rats were placed in hypoxic box, sustained administration of an oxygen concentration of 21% air.After the end of Experiment, the rats were placed in the same rearing conditions and living conditions of ordinary terrarium, gave enouph fodder and water. Rats were killed at the time which designed. We used immunohistochemistry to detect the expression of ERK1/2、P53、Bax and Bcl-2 and Western blot to detect the expression of ERK1/2、P53 in hippocampal CA1 area. The apoptosis of neurons was detected by TUNEL method.The result of immunohistochemistry is represented with the IOD value. Image J software analyzed the Western Blot bands absorbance(A) value. GAPDH was selected as an internal consultation, and each sample was taken the result of repeated three times. A value of each sample is compared with its internal reference value A is obtained the final result.Tunel result in the same multiples(40 × 10) were observed, each slice randomly opted five different horizons,we calculated the total number of neural cells and the number of apoptotic cells in hippocampal CA1 area respectively.The number of apoptotic cells divided by the total number of cells × 100% is the apoptosis index(AI). Using SPSS17.0 statistical package for data analysis, the resulting data are expressed as mean ±standard deviation(xˉ ±s), among groups were compared using one-way ANOVA(Oneway ANOVA), with P<0.05 was considered statistically significant.Result 1 The results of neuronal apoptosis in rat hippocampal CA1 area : The morphology of apoptotic cells were round or oval observed by optical microscope, nuclei showed brown or tan, which in irregular shapes, and various sizes, and nuclei of nonapoptotic cells were blue after stained by hematoxylin, nuclei were larger than that in nonapoptotic cells, the shapes and sizes were more consistent. The comparation among different groups: Compared with UC group, the neuronal apoptosis index in IH group and ERK inhibitor group was no significant difference in the first day(P>0.05) and was significantly increased in 3d,7d,10 d,14d(P<0.05); Compared with IH group, the neuronal apoptosis index in ERK inhibitor group decreased significantly in 3d,7d,10 d,14d(P<0.05);The comparation within group: the neuronal apoptosis index in UC group at all time points was not significantly different(P>0.05); The neuronal apoptosis index in IH group and ERK inhibitor group at all time points had statistical significance(P<0.05),the neuronal apoptosis index tended to increase with the extension of hypoxia time. 2 The results of nerve cells ERK1/2 protein expression in rat hippocampal CA1 area :Immunohistochemistry and Western Blot results showed, The comparation among different groups: Compared with UC group, the expression of ERK1/2 protein in IH group were significantly higher in 1d,3d,7d,10 d,14d(P<0.05), but the ERK1/2 protein expression at all time points had no significant difference in ERK inhibitor group(P>0.05); Compared with IH group, ERK inhibitor group ERK1/2 protein expression decreased significantly at each time point(P<0.05); The comparation within group: the expression of ERK1/2 protein in UC group and ERK inhibitor group at all time points was not significantly different(P>0.05); The expression of ERK1/2 protein in IH group at all time points had statistical significance(P<0.05),with the extension of time,it first rises,peaked at10 d, then decreases at14 d. 3 The results of nerve cells P53 protein expression in rat hippocampal CA1 area:Immunohistochemistry and Western Blot results showed, The comparation among different groups: Compared with UC group, the expression of P53 protein in IH group and ERK inhibitor group were significantly increased in1 d,3d,7d,10 d,14d(P<0.05); Compared with IH group, ERK inhibitor group P53 protein expression decreased significantly at each time point(P<0.05); The comparation within group: the expression of P53 protein in UC group at all time points was not significantlydifferent(P>0.05); The expression of ERK1/2 protein in IH group and ERK inhibitor group at each time point had statistical significance(P<0.05),with the extension of time,it first rises, peaked at10 d, then decreases at14 d. 4 The results of nerve cells Bax protein expression in rat hippocampal CA1 area : Immunohistochemistry results showed, The comparation among different groups: Compared with UC group, the expression of Bax protein in IH group and ERK inhibitor group were significantly increased in1 d,3d,7d,10 d,14d(P<0.05); Compared with IH group, ERK inhibitor group Bax protein expression decreased significantly at each time point(P<0.05); The comparation within group: the expression of Bax protein in UC group at each time point was not significantly different(P>0.05); The expression of Bax protein in IH group and ERK inhibitor group at each time point had statistical significance(P<0.05), with the extension of time, it first rises, peaked at10 d, then decreases at14 d. 5 The results of nerve cells Bcl-2 protein expression in rat hippocampal CA1 area : Immunohistochemistry results showed, The comparation among different groups: Compared with UC group, the expression of Bcl-2protein in IH group and ERK inhibitor group were significantly increased in1 d,3d,7d,10 d,14d(P<0.05); Compared with IH group, ERK inhibitor group Bcl-2 protein expression significantly increased at each time point(P<0.05); The comparation within group: the expression of Bcl-2 protein in UC group at all time points was not significantly different(P>0.05); The expression of Bcl-2 protein in IH group and ERK inhibitor group at all time points had statistical significance(P<0.05),with the extension of time,it first rises, peaked at10 d, then decreases at14 d.Conclusion 1 The early intermittent hypoxia can cause neuronal apoptosis and the expression of apoptosis-related protein in the rats of hippocampal CA1 area, while activation of ERK signaling pathway. 2 ERK inhibitor U0126 blocked ERK signaling pathway and reduced the expression of pro-apoptotic gene P53, while Bcl-2 and Bax ratio increased, thereby reducing the apoptosis of neurons in the early intermittent hypoxia.