高浓度七氟醚通过抑制VEGF/PI3K/AKT信号通路诱导神经干细胞凋亡的研究

doi:10.3877/cma.j.issn.2095-1221.2018.05.005

目的

探讨妊娠中期的七氟醚暴露对神经干细胞凋亡过程的影响和作用机制。

方法

将孕中期大鼠随机分为3组，每组48只孕鼠：对照组，低浓度七氟醚组，高浓度七氟醚组。在妊娠第14天，以2﹪或3.5﹪七氟醚麻醉怀孕大鼠2?h。通过免疫荧光检查神经干细胞凋亡，收集麻醉后6、24和48?h以及出生后第0天（P?0），第14天（P?14）和第28天（P?28）的脑组织进行Nestin-TUNEL免疫荧光双标染色以及Nestin、血管内皮生长因子（VEGF）和磷酸肌醇3-激酶（PI3K）AKT通路相关蛋白的免疫印迹检测。采用单因素方差分析和Bonferroni事后检验进行统计学分析。

结果

麻醉后6、24和48?h以及P?0，P?14和P?28，脑组织中Nestin和TUNEL阳性细胞的百分比增加[6?h：对照组0.91±0.07，低浓度组1.01±0.08，高浓度组2.62±0.21（F?=?399，P?<?0.01）；24?h：对照组0.96±0.04，低浓度组1.09±0.13，高浓度组2.49±0.17（F?=?364.37，P?<?0.01）；48?h：对照组0.95±0.05，低浓度组1.24±0.11，高浓度组2.51±0.13（F?=?524.52，P?<?0.01）；P?0：对照组0.97±0.03，低浓度组1.01±0.09，高浓度组2.21±0.15（F?=?378.31，P?<?0.01）；P?14：对照组0.96±0.03，低浓度组1.22±0.12，高浓度组1.89±0.14（F?=?158.33，P?<?0.01）；P?28：对照组0.95±0.05，低浓度组1.09±0.13，高浓度组1.69±0.19（F?=?66.83，P?<?0.01）。但高浓度组Nestin蛋白水平降低[6?h：对照组0.95±0.08，低浓度组0.81±0.11，高浓度组0.62±0.13（F?=?18.60，P?<?0.01）；24?h：对照组0.92±0.06，低浓度组0.85±0.13，高浓度组0.74±0.12（F?=?5.66，P?=?0.0108）；48?h：对照组0.95±0.04，低浓度组0.80±0.08，高浓度组0.72±0.14（F?=?11.86，P?<?0.01）；P?0：对照组0.97±0.06，低浓度组0.72±0.09，高浓度组0.67±0.09（F?=?31.31，P?<?0.01）；P?14：对照组0.94±0.03，低浓度组0.69±0.07，高浓度组0.65±0.13（F?=?26.11，P?<?0.01）；P?28：对照组0.95±0.08，低浓度组0.91±0.12，高浓度组0.58±0.13（F?=?26.25，P?<?0.01）]。并且在麻醉后6?h，胎鼠大脑中VEGF，PI3K和磷酸化AKT（p-AKT）水平降低。但在低浓度七氟醚暴露组中并未出现变化。

结论

妊娠中期暴露于高浓度的七氟醚会降低VEGF，PI3K和p-AKT蛋白水平并诱导神经干细胞发生凋亡，从而导致后代的学习和记忆功能障碍。

关键词: 七氟醚, 血管内皮生长因子, 磷酸肌醇3-激酶, AKT, 神经干细胞, 凋亡, 妊娠

Objective

To investigate the effects of sevoflurane exposure on apoptosis of neuronal cells and the mechanism in mid-pregnant rats.

Method

Rats in the second trimester of pregnancy were randomly divided into three groups, 48 pregnant rats in each group: control group, low concentration sevoflurane group and high concentration sevoflurane group. On the 14th day of pregnancy, pregnant rats were anesthetized with 2﹪ or 3.5﹪ sevoflurane for 2 hours. Neural stem cell apoptosis was examined by immunofluorescence. Brain tissues were collected at 6, 24 and 48 hours after anesthesia and on days 0, 14 and 28 after birth for Nestin-TUNEL double labeling immunofluorescence staining and immunoblotting for Nestin, vascular endothelial growth factor (VEGF) and phosphoinositol 3-kinase (PI3K)-AKT pathway-related proteins. One-way ANOVA and Bonferroni post-test were used for statistical analysis.

Results

The percentage of Nestin and TUNEL positive cells in the brain tissue increased at 6, 24 and 48 hours after anesthesia, as well as P?0, P?14 and P?28 (At 6 h, the control group was 0.91±0.07; the low concentration group was 1.01±0.08; and the high concentration group was 2.62±0.21, F?=?399, P?<?0.01; at 24?h, the control group was 0.96±0.04; the low concentration group was 1.09±0.13; and the high concentration group was 1.89±0.14, F?=?364.37, P?<?0.01; at 48?h the control group was 0.95±0.05; the low concentration group was 1.09±0.13; and the high concentration group was 1.69±0.19, F?=?524.52, P?<?0.01; at P?0 the control group was 0.97±0.03; the low concentration group was 1.01±0.09; the high concentration group was 2.21±0.15, F?=?378.31, P?<?0.01; at P?14, the control group was 0.96±0.03; the low concentration group was 1.22±0.12; and the high concentration group was 1.89±0.14, F?=?158.33, P?<?0.01; at P?28, the control group was 0.95±0.05; the low concentration group was 1.09±0.13; and the high concentration group was 1.69±0.19, F?=?66.83, P?<?0.01, but the level of Nestin protein in the high concentration group decreased. At 6?h the control group was 0.95±0.08, the low concentration group was 0.81±0.11, the high concentration group was 0.62±0.13, F?=?18.60, P?<?0.01; at 24?h the control group was 0.92±0.06, the low concentration group was 0.85±0.13, the high concentration group was 0.74±0.12, F?=?5.66, P?=?0.0108; the high concentration group was 0.74±0.12; at 48?h the control group was 0.95±0.04, the low concentration group was 0.80±0.08, the high concentration group was 0.72±0.14, F?=?11.86, P?<?0.01; at P?0 the control group was 0.97±0.06, the low concentration group was 0.72±0.09, the high concentration group was 0.67±0.09, F?=?31.31, P?<?0.01; at P?14 the control group was 0.94±0.03, the low concentration group was 0.69±0.07, the high concentration group was 0.65±0.13, F?=?26.11, P?<?0.01; at P?28 the control group was 0.95±0.08, the low concentration group was 0.91±0.12, the high concentration group was 0.58±0.13, F?=?26.25, P?<?0.01. The levels of vascular endothelial growth factor, PI3K and phosphorylated AKT (p-AKT) in the fetal rat brain decreased 6 hours after anesthesia (P < 0.05). However, there was no significant change in low concentration sevoflurane exposure group.

Conclusion

Exposure to sevoflurane in the second trimester of pregnancy can decrease the levels of vascular endothelial growth factor, PI3K and p-AKT proteins and induce apoptosis of neural stem cells, leading to learning and memory dysfunction in the offsprings.

Key words: Sevoflurane, VEGF/PI3K/AKT, Neural stem cells, Apoptosis, Pregnancy

图1 荧光显微镜下观察各组胎鼠大脑神经干细胞（Nestin染色，×400）

图2 荧光显微镜下观察出生后各组小鼠大脑神经干细胞（Nestin染色，×400）

图3 免疫印迹检测不同时间点大脑组织中Nestin蛋白的表达水平

表1 在不同时间点Nestin染色阳性细胞所占比例的定量分析（±s）

分组	动物数	6?h	24?h	48?h	P?0	P?14	P?28
对照组	8	0.95±0.08	0.92±0.06	0.95±0.04	0.97±0.06	0.94±0.03	0.95±0.08
低浓度组	8	0.81±0.11^a	0.85±0.13	0.80±0.08^a	0.72±0.09^a	0.69±0.07^a	0.91±0.12
高浓度组	8	0.62±0.13^ab	0.74±0.12^a	0.72±0.14^a	0.67±0.09^a	0.65±0.13^a	0.58±0.13
F值	?	18.60	5.66	11.86	31.31	26.11	26.25
P值	?	< 0.01	0.0108	< 0.01	< 0.01	< 0.01	< 0.01

表1 在不同时间点Nestin染色阳性细胞所占比例的定量分析（±s）

分组	动物数	6?h	24?h	48?h	P?0	P?14	P?28
对照组	8	0.95±0.08	0.92±0.06	0.95±0.04	0.97±0.06	0.94±0.03	0.95±0.08
低浓度组	8	0.81±0.11^a	0.85±0.13	0.80±0.08^a	0.72±0.09^a	0.69±0.07^a	0.91±0.12
高浓度组	8	0.62±0.13^ab	0.74±0.12^a	0.72±0.14^a	0.67±0.09^a	0.65±0.13^a	0.58±0.13
F值	?	18.60	5.66	11.86	31.31	26.11	26.25
P值	?	< 0.01	0.0108	< 0.01	< 0.01	< 0.01	< 0.01

图4 荧光显微镜下观察各组妊娠中期七氟醚暴露后胎鼠大脑神经细胞（Nestin和TUNEL免疫荧光双标染色，×400）

图5 荧光显微镜下观察出生后各组七氟醚暴露后小鼠大脑神经细胞（Nestin和TUNEL免疫荧光双标染色，×400）

表2 不同时间点Nestin和TUNEL染色双阳性细胞定量分析（±s）

分组	动物数	6?h	24?h	48?h	P?0	P?14	P?28
对照组	8	0.91±0.07	0.96±0.04	0.95±0.05	0.97±0.03	0.96±0.03	0.95±0.05
低浓度组	8	1.01±0.08	1.09±0.13	1.24±0.11^a	1.01±0.09	1.22±0.12^a	1.09±0.13
高浓度组	8	2.62±0.21^ab	2.49±0.17^ab	2.51±0.13^ab	2.21±0.15^ab	1.89±0.14^ab	1.69±0.19^ab
F值	?	399.00	364.37	524.52	378.31	158.33	66.83
P值	?	< 0.01	< 0.01	< 0.01	< 0.01	< 0.01	< 0.01

表2 不同时间点Nestin和TUNEL染色双阳性细胞定量分析（±s）

分组	动物数	6?h	24?h	48?h	P?0	P?14	P?28
对照组	8	0.91±0.07	0.96±0.04	0.95±0.05	0.97±0.03	0.96±0.03	0.95±0.05
低浓度组	8	1.01±0.08	1.09±0.13	1.24±0.11^a	1.01±0.09	1.22±0.12^a	1.09±0.13
高浓度组	8	2.62±0.21^ab	2.49±0.17^ab	2.51±0.13^ab	2.21±0.15^ab	1.89±0.14^ab	1.69±0.19^ab
F值	?	399.00	364.37	524.52	378.31	158.33	66.83
P值	?	< 0.01	< 0.01	< 0.01	< 0.01	< 0.01	< 0.01

图6 妊娠中期七氟醚暴露6 h后，免疫印迹检测胎鼠脑组织中BAX，BAD，VEGF，PI3K，p-AKT和AKT的表达水平

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High concentration sevoflurane induces apoptosis of neural stem cells by inhibiting VEGF/PI3K/AKT signaling pathway

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High concentration sevoflurane induces apoptosis of neural stem cells by inhibiting VEGF/PI3K/AKT signaling pathway