高渗盐水对脑缺血大鼠Notch信号通路的影响

The effect of hypertonic saline on notch signaling pathway in experimentally induced cerebral ischemic rats

目的：探讨高渗盐水（ hypertonic saline， HS）对大鼠脑缺血后小胶质细胞Notch信号通路的影响。方法 SPF级-雄性SD大鼠随机（随机数字法）分为假手术组，脑缺血组，生理盐水（ NS）组，10％高渗盐水（10％HS）组。除假手术组外，其他各组采用线栓法复制右侧大脑中动脉栓塞脑缺血模型，缺血2 h后实施再灌注24 h， NS组和10％HS组按0.3 mL／h经尾静脉分别匀速泵入NS和10％HS治疗24 h。然后采用免疫荧光法、 RT-PCR、 Western blot检测各组大鼠脑缺血灶周围Notch1及Notch受体的胞内片段NICD的表达。数据采用单因素方差分析，用LSD法进行组间两两比较，以P＜0.05为差异具有统计学意义。结果免疫荧光表明与假手术组比较，脑缺组和NS组缺血灶周围小胶质细胞Notch1与NICD的表达明显增加；10％HS组与脑缺血组及NS组比较，缺血灶周围小胶质细胞Notch1与NICD的表达明显减少。 RT-PCR表明脑缺血组及NS组与对照组比较， Notch1 mRNA的表达明显增加（对照组：1.000±0.076；脑缺血组：2.203±0.283； NS组：1.616±0.185； P＜0.01）；10％HS组与脑缺血组及NS组比较， Notch1 mRNA的表达均明显减少（脑缺血组：2.203±0.283； NS 组：1.616±0.185； HS 组：1.202±0.177； P ＜0.05）。 Western blot表明脑缺血组和 NS 组与对照组相比，脑缺血灶周围 Notch1蛋白表达明显增加（对照组：0.290±0.079；脑缺血组：0.750±0.029； NS 组：0.765±0.182； P ＜0.01）；10％HS 治疗后， Notch1蛋白表达较脑缺血组和NS组明显减少（脑缺血组：0.750±0.029； NS组：0.765±0.182；HS组：0.390±0.195； P＜0.05）。脑缺血组和NS组与对照组比较，大鼠脑缺血灶周围NICD蛋白表达明显增加（对照组：0.401±0.196；脑缺血组：0.906±0.359； NS组：0.847±0.153； P＜0.01）；10％HS治疗后， NICD蛋白表达较脑缺血组和NS组明显减少（脑缺血组：0.906±0.359；NS组：0.847±0.153； HS组：0.561±0.165； P＜0.05）。结论 HS可抑制大鼠脑缺血后小胶质细胞上Notch信号通路的激活。

Objective To explore whether hypertonic saline would partake in regulating Notch signaling in microglia in experimentally induced cerebral ischemic rats.Methods Male SD rats were randomly divided into sham group, cerebral ischemia group, normal saline group （ NS group ） , 10%hypertonic saline group （10%HS group） , the model of cerebral ischemia were established in all rats except the sham group by using middle cerebral artery occlusion （ MCAO） .After 2 hours of MCAO, the rats were through reperfusion for 24 h.In addition, rats in the normal saline group and 10% HS group were respectively treated with a continuous intravenous injection of normal saline （0.3 mL/h） and 10%HS （0.3 mL/h） by tail vein for 24 h.Immunofluorescence methods, RT-PCR and Western blot were used to detect the expression of Notch1 and intracellular Notch receptor domain （ NICD） .All data was analyzed by one-way analysis of variance （ ANOVA） , The intergroup comparisons were analyzed by the least-significant-difference （LSD） tests.Differences were considered statistically significant if P〈0.05.Results Immunofluorescence showed that the expression of Notch1 and NICD were significantly increased in the microglia around peri-ischemia area in cerebral ischemia group and normal saline group compared to sham group;the expression of Notch1 and NICD in the microglia around peri-ischemia area were significantly reduced in 10% HS group compared to ischemia group and NS group.RT-PCR showed that the mRNA expression of Notch1 was significantly increased in ischemia group and NS group compared to sham group （ sham group： 1.000 ± 0.076; ischemia group： 2.203 ±0.283; NS group： 1.616 ±0.185; P 〈0.01 ）; however, it was significantly reduced in 10% HS group compared to ischemia group and NS group （ ischemia group：2.203 ±0.283; NS group： 1.616 ±0.185; 10%HS group： 1.202 ±0.177; P 〈0.05 ） .Western blot showed that the protein expression of Notch1 was significantly increased in ischemia group and NS group compared to sham group （ sham group： 0.290 ±0.079; ischemia group： 0.750 ±0.029; NS group：0.765 ±0.182;P〈0.01）;but was significantly reduced in 10%HS group compared to ischemia group and NS group （ ischemia group：0.750 ±0.029; NS group：0.765 ±0.182;10%HS group：0.390 ±0.195;P〈0.05 ） .The protein expression of NICD was significantly increased in ischemia group and NS group compared to sham group （ sham group： 0.401 ±0.196; ischemia group： 0.906 ±0.359; NS group：0.847 ±0.153;P〈0.01）;but was significantly reduced in 10%HS group compared to ischemia group and NS group （ ischemia group：0.906 ±0.359; NS group：0.847 ±0.153;10%HS group：0.561 ±0.165;P〈0.05 ） .Conclusion Our results suggest that HS markedly suppresses Notch signaling in microglia around the ischemia tissue area in experimental induced cerebral ischemic rats.