NF-κB“圈套”对癫痫大鼠脑内COX-2调控作用的研究
摘要
癫痫(Epilepsy)给患者带来的巨大伤害是由癫痫反复发作(Seizure)所引起的脑损伤,而癫痫的脑损伤的主要原因是由癫痫发作所引起的脑内炎症反应。关于癫痫的脑内炎症反应机制,近年来的研究报道显示,癫痫的炎症反应与脑内环氧化酶-2(Cyclooxygenase-2,COX-2)的表达增加及前列腺素(PGs)水平增高有着密切关系,COX-2为PGs合成的限速酶,被激活后不仅可产生氧化应激反应,还可将花生四烯酸转化为PGs等有害物质,虽然COX-2可被促炎性因子和某些与炎症反应有关的生长因子所诱导,但在中枢神经系统及癫痫动物脑内COX-2的激活调节机制还并不十分清楚。
核转录因子(NF-κB)的激活可高效诱导靶基因的转录,调节着诸多参与免疫、炎症反应因子如细胞粘附分子、细胞因子等的表达,在炎症和免疫反应中起着调控作用,已有研究证明在癫痫的脑损伤中NF-κB起着重要作用。但是在癫痫发作过程中是否由于NF-κB的激活,从而调控了其下游靶基因之一COX-2的高表达,才导致了脑内的炎症反应和一系列病理变化尚无明确报道。
目的:本研究设计并合成与核转录因子NF-κB顺式元件相一致的双链寡聚脱氧核苷酸(κB-decoy)和相应的无关序列(错配-decoy),用这种核转录因子“圈套”策略抑制NF-κB的活性,通过研究癫痫大鼠脑内NF-κB的活性被抑制时COX-2的表达情况,明确癫痫的脑损伤病理变化与NF-κB活性和COX-2表达的相关性以及NF-κB在癫痫中对COX-2表达的调控作用,以进一步了解癫痫的脑内病理改变机制。
方法:选用体重为250-300g的健康成年SD雄性大鼠,将其随机分为3组即(1)实验组(注射κB-decoy组)、(2)对照组(注射错配-decoy组)、(3)正常组(注射生理盐水组)。κB-decoy-ExGen500复合物和错配-decoy-ExGen500复合物由大鼠脑立体定位仪于脑室内微量注射,采用氯化锂-匹罗卡品(40mg/kg)制备大鼠癫痫模型,癫痫发作后选取不同的时间点的大鼠灌流固定,免疫组化方法测定NF-κB和COX-2在脑内癫痫敏感脑区的活性,RT-PCR测定COX-2的表达。
结果: NF-κBp65免疫组化结果显示:与对照组相比,实验组大鼠海马结构CA1区、CA3区和齿状回门区神经元胞浆中免疫阳性染色明显增强; NF-κB未发生核转移,其活性被抑制。COX-2免疫组化结果显示:与对照组相比,实验组大鼠海马神经元的免疫阳性染色明显减弱; COX-2的表达水平低于对照组。RT-PCR结果显示:与对照组相比,在癫痫发作后4h时实验组的大鼠海马结构COX-2mRNA表达水平明显降低。
结论:
1. NF-κB“圈套”具有抑制癫痫大鼠脑内NF-κB活性的作用。
2. NF-κB“圈套”具有抑制癫痫大鼠脑内COX-2表达的作用。
3.在癫痫大鼠脑内,NF-κB对其下游靶基因COX-2具有调控作用,即NF-κB被激活后使COX-2的转录和表达增加,NF-κB被抑制后使COX-2的转录和表达减少。
The epileptic harm to victims is brain injury due to recurrent seizure, and epileptic brain injury is mainly resulted from the inflammatory reaction in brain. Concerning the mechanism of the inflammatory reaction in brain, previous studies indicated that it was closely related to the increased expression of cyclooxygenase-2 (COX-2) and the elevated level of prostaglandins(PGs). COX-2 is the rate-limiting enzymes of PGs’synthesis, being activated ,it could not only lead to oxidative stress reaction ,but convert arachidonic acid into harmful substance such as PGs. Althoug COX-2 can be induced by some inflammatory factors and inflammatory relevant cytokines, its mechnism of activation and regulation in central nervous system and epileptic rat’s brain is not clear.
Activated nuclear factor kappa B ( NF-κB) could efficiently induce the transcription of extensive target genes, adjust the expression of immune and inflammatory response factor such as cell adhesion molecules and cytokines , play a regulating role in the course of inflammation and immunoreaction. Ample results of studies could prove that NF-κB is significant in epileptic brain injury , however whether it is the activation of NF-κB that makes one of its downstream target genes ---- COX-2’s high expression in seizure or not is also not clear .
Objective: Our study uses a“decoy”stratetgy to inhibit the activity of NF-κB. We designed and synthesized a double-stranded oligodeoxynucleo- tides(ODNs) ----κB-decoy ---- which matches with the cis-element of NF-κB and a corresponding ODNs with irrelevant scrambled sequence ---- scrambled-decoy, UsingκB-decoy to inhibit the activity of NF-κB in seizure, we detected the expression of COX-2. To do that we hope to explore the relationship of pathological changes in brain injury with the activity of NF-κB and the expression level of COX-2 ,clearify the regulating effect of NF-κB on COX-2 in seizure, and further understand the mechanism of brain pathological changes.
Methods:Choose adult male SD rats whose body weight is in the range from 250g to 300g, and divide them randomly into three groups: experimental group (κB-decoy group) ,control group (Scrambled- decoy group) and normal group (saline-control group) .Use the rat stereotaxic instrument to take intraventricular injection ofκB-decoy- ExGen500 or Scrambled-decoy-ExGen-500 compound . Use Lithium- Pilocarpine to make rat’s seizure model (40mg/kg). After seizure choose different time point to irrigate and fix the rats , use immuno-histochemistry to detect the activity of NF-κB and the expression of COX-2 in epileptic sensitive brain regions, use RT-PCR to detect the expression of COX- 2mRNA.
Results: Immunohistochemistry of NF-κBp65 showed that: compared with control group, the positive staining in cytoplasm in experimental group was obviously deeper which indicated that NF-κB in experimental group was not translocated from cytoplasm into nucleus after stimulation, its activity was inhibited byκB-decoy. Immunohistochemistry of COX-2 showed that: compared with control group, the positive staining in experimental group was obviously shallower which indicated that the COX-2 expression level in experimental group was lower than in control group. RT-PCR of COX-2mRNA showed that: the expression level of COX-2mRNA in experimental group was lower than in control group (for the time point 4h after seizure).
Conclusions:
1.κB-decoy could inhibit the activity of NF-κB in epileptic rat’s brain.
2.κB-decoy could inhibit COX-2’s expression in epileptic rat’s brain.
3. In epileptic rat’s brain , NF-κB could positively regulate one of its downstream target genes ---- COX-2 gene.
核转录因子(NF-κB)的激活可高效诱导靶基因的转录,调节着诸多参与免疫、炎症反应因子如细胞粘附分子、细胞因子等的表达,在炎症和免疫反应中起着调控作用,已有研究证明在癫痫的脑损伤中NF-κB起着重要作用。但是在癫痫发作过程中是否由于NF-κB的激活,从而调控了其下游靶基因之一COX-2的高表达,才导致了脑内的炎症反应和一系列病理变化尚无明确报道。
目的:本研究设计并合成与核转录因子NF-κB顺式元件相一致的双链寡聚脱氧核苷酸(κB-decoy)和相应的无关序列(错配-decoy),用这种核转录因子“圈套”策略抑制NF-κB的活性,通过研究癫痫大鼠脑内NF-κB的活性被抑制时COX-2的表达情况,明确癫痫的脑损伤病理变化与NF-κB活性和COX-2表达的相关性以及NF-κB在癫痫中对COX-2表达的调控作用,以进一步了解癫痫的脑内病理改变机制。
方法:选用体重为250-300g的健康成年SD雄性大鼠,将其随机分为3组即(1)实验组(注射κB-decoy组)、(2)对照组(注射错配-decoy组)、(3)正常组(注射生理盐水组)。κB-decoy-ExGen500复合物和错配-decoy-ExGen500复合物由大鼠脑立体定位仪于脑室内微量注射,采用氯化锂-匹罗卡品(40mg/kg)制备大鼠癫痫模型,癫痫发作后选取不同的时间点的大鼠灌流固定,免疫组化方法测定NF-κB和COX-2在脑内癫痫敏感脑区的活性,RT-PCR测定COX-2的表达。
结果: NF-κBp65免疫组化结果显示:与对照组相比,实验组大鼠海马结构CA1区、CA3区和齿状回门区神经元胞浆中免疫阳性染色明显增强; NF-κB未发生核转移,其活性被抑制。COX-2免疫组化结果显示:与对照组相比,实验组大鼠海马神经元的免疫阳性染色明显减弱; COX-2的表达水平低于对照组。RT-PCR结果显示:与对照组相比,在癫痫发作后4h时实验组的大鼠海马结构COX-2mRNA表达水平明显降低。
结论:
1. NF-κB“圈套”具有抑制癫痫大鼠脑内NF-κB活性的作用。
2. NF-κB“圈套”具有抑制癫痫大鼠脑内COX-2表达的作用。
3.在癫痫大鼠脑内,NF-κB对其下游靶基因COX-2具有调控作用,即NF-κB被激活后使COX-2的转录和表达增加,NF-κB被抑制后使COX-2的转录和表达减少。
The epileptic harm to victims is brain injury due to recurrent seizure, and epileptic brain injury is mainly resulted from the inflammatory reaction in brain. Concerning the mechanism of the inflammatory reaction in brain, previous studies indicated that it was closely related to the increased expression of cyclooxygenase-2 (COX-2) and the elevated level of prostaglandins(PGs). COX-2 is the rate-limiting enzymes of PGs’synthesis, being activated ,it could not only lead to oxidative stress reaction ,but convert arachidonic acid into harmful substance such as PGs. Althoug COX-2 can be induced by some inflammatory factors and inflammatory relevant cytokines, its mechnism of activation and regulation in central nervous system and epileptic rat’s brain is not clear.
Activated nuclear factor kappa B ( NF-κB) could efficiently induce the transcription of extensive target genes, adjust the expression of immune and inflammatory response factor such as cell adhesion molecules and cytokines , play a regulating role in the course of inflammation and immunoreaction. Ample results of studies could prove that NF-κB is significant in epileptic brain injury , however whether it is the activation of NF-κB that makes one of its downstream target genes ---- COX-2’s high expression in seizure or not is also not clear .
Objective: Our study uses a“decoy”stratetgy to inhibit the activity of NF-κB. We designed and synthesized a double-stranded oligodeoxynucleo- tides(ODNs) ----κB-decoy ---- which matches with the cis-element of NF-κB and a corresponding ODNs with irrelevant scrambled sequence ---- scrambled-decoy, UsingκB-decoy to inhibit the activity of NF-κB in seizure, we detected the expression of COX-2. To do that we hope to explore the relationship of pathological changes in brain injury with the activity of NF-κB and the expression level of COX-2 ,clearify the regulating effect of NF-κB on COX-2 in seizure, and further understand the mechanism of brain pathological changes.
Methods:Choose adult male SD rats whose body weight is in the range from 250g to 300g, and divide them randomly into three groups: experimental group (κB-decoy group) ,control group (Scrambled- decoy group) and normal group (saline-control group) .Use the rat stereotaxic instrument to take intraventricular injection ofκB-decoy- ExGen500 or Scrambled-decoy-ExGen-500 compound . Use Lithium- Pilocarpine to make rat’s seizure model (40mg/kg). After seizure choose different time point to irrigate and fix the rats , use immuno-histochemistry to detect the activity of NF-κB and the expression of COX-2 in epileptic sensitive brain regions, use RT-PCR to detect the expression of COX- 2mRNA.
Results: Immunohistochemistry of NF-κBp65 showed that: compared with control group, the positive staining in cytoplasm in experimental group was obviously deeper which indicated that NF-κB in experimental group was not translocated from cytoplasm into nucleus after stimulation, its activity was inhibited byκB-decoy. Immunohistochemistry of COX-2 showed that: compared with control group, the positive staining in experimental group was obviously shallower which indicated that the COX-2 expression level in experimental group was lower than in control group. RT-PCR of COX-2mRNA showed that: the expression level of COX-2mRNA in experimental group was lower than in control group (for the time point 4h after seizure).
Conclusions:
1.κB-decoy could inhibit the activity of NF-κB in epileptic rat’s brain.
2.κB-decoy could inhibit COX-2’s expression in epileptic rat’s brain.
3. In epileptic rat’s brain , NF-κB could positively regulate one of its downstream target genes ---- COX-2 gene.
引文
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