外源性糖皮质激素致未成熟脑细胞过度凋亡的启动因素

目的 观测外源件糖皮质激素诱导婴幼鼠脑细胞过度凋亡中线粒体超氧化应激及胞内ca~(2+)]i超载的改变特征,确认激素启动未成熟脑过度凋亡的关键性因素.方法 分别设立与婴儿、1～2岁幼儿及成年人脑发育进程相匹配的生后7 d(PD7)、生后15 d(PD15)及生后60 d(PD60)不同日龄健康SD大鼠,共192只.模拟临床治疗婴儿痉挛泼尼松、促肾上腺皮质激素(ACTH)的剂量与疗程,两者剂量分别为6 mg·ks~(-1)·d~(-1)和150 U·m~(-2)d~(-1),连续4 d.各日龄组再被分为泼尼松、ACTH及正常对照组,以及给药后停药3周与相应对照组,每组均8只大鼠.分光光度计测定脑细胞线粒体内谷胱甘肽(GSH)、超氧化物歧化酶(SOD)、丙二醛(MDA)、Na~+ -K~+ -ATP酶活性和胞内Ca~(2+)]i.免疫组化法检测脑组织N-甲基-D-天门冬氨酸受体1(NMDA-R1).结果 泼尼松或ACTH均引起婴幼鼠额叶皮层神经细胞数减少、TUNEL阳性细胞数明显增多,以PD7、泼尼松组改变最突出:77.7±4.7(对照:102.3±5.9)(P=0.002),114.8±8.14(对照:56.3±5.6)(P=0.029).两激素均引起各组鼠脑细胞线粒体GSH含量、SOD活性及Na~+ -K~+ -ATP酶活性不同程度降低,而MDA含量不同程度增高,也均以PD7、泼尼松组改变最突出GSH(mg/g蛋白):158.3±6.1(对照:225.1±9.5),SOD(U/mg蛋白):155.8±4.3(对照:228.1±9.2),Na~+ -K~+ -ATP酶(U/mg蛋白):14.6±3.5(对照:20.8±5.5),MDA(nmol/mg蛋白):10.4±0.9(对照:4.8±1.9)(均P<0.01)].神经细胞ca~(2+)]i增高变化,仍以PD7、泼尼松组最显著:164.6±11.9 nmol/L(对照:125.8±6.0 nmol/L)(P=0.003).NMDA-R1密度分布与Ca~(2+)]i亦呈相似改变,其中PD7、泼尼松组平均吸光度值最高为0.36±0.03(对照:0.21±0.05),而PD60组仅为0.18±0.05(对照:0.15±0.02).停药3周后均恢复正常.结论 治疗剂量泼尼松和ACTH可导致婴鼠脑细胞线粒体超氧化-抗氧化失衡及能量代谢障碍.NMDA-R1、Bax表达增加以及线粒体氧化应激是激素诱发未成熟脑细胞过度凋亡的关键启动因素.婴儿脑细胞存在生理性胞内高Ca~(2+)及高密度NMDA-R1,可能是激素脑损伤主要发生在未成熟期的重要原因.

Activated factor of immature neurons for excessive apoptosis induced by exogenous glucocorticosteroid

Objective To confirm the key inducing factor of excessive apoptosis in immature brain under the exposure of exogenous glucocorticosteroid and observe the characteristics of mitochondrial oxidative stress and intracellular Ca~(2+)] i overload as compared to healthy adult rats. Methods A total of 192 healthy Sprague-Dawley (SD) rats selected for the study were divided into three groups including PD7, PD15 and PD60 corresponding human age stages including full-term newborn, one-year infant and adult respectively. To mimic the therapeutic regime for infantile spasms, 8 rats in each group was treaded with prednisone (6 mg· kg~(-1)·d~(-1)), ACTH (150 U·m~(-2)·d~(-1)), normal saline for4 days and drug withdrawal for 3 weeks.Rats were deeply anesthetized and sacrificed by decapitation. The mitochondrial concentrations of GSH, SOD, MDA, Na~+ -K~+-ATPase and intracellular Ca~(2+)i] were all determined by spectrophotometer. The cellular NMDA-Rl expression was measured by immunohistochemistry. Results Both prednisone and ACTH caused neuron reduced and TUNEL-positive cell increased in the frontal lobe, specially in PD7 group of prednisone, that were 77.7±4.7 neurons per VF (control group: 102.3±5.9) (P=0.002), 114.8± 8.14 (control group: 56.3±5.6) (P=0.029) respectively. The mitochondrial levels of GSH, SOD and Na~+ -K~+ -ATPase decreased after prednisone and ACTH administration at different level in different age groups whereas the concentration of MDA increased. These changes were significant in PD7 group after prednisone administration, that were 158.3±6.1 mg/gprot of GSH (control group: 225.1±9.5 mg/gprot) (P=0.006), 155.8±4.3 U/mgprot of SOD (control group: 228.1±9.2 U/mgprot) (P=0.006), 14.6± 3.5 U/mgprot of Na~+ -K~+ -ATPase (control group : 20.8±5.5 U/mgprot) and 10.4±0.9 nmol/mgprot of MDA (control group: 4.8±1.9 nmol/mgprot) (all P<0.01). Ca~(2+)] i level in neuron increased, specially in PD7 group after prednisone administration, that was 164.6±11.9 nmol/L (control group: 125.8±6.03 nmol/L) (P=0.003). Similar to Ca~(2+)] i changes, the density of NMDA-R1 also increased mostly in PD7 group after prednisone administration, that was 0.36±0.03 (control group: 0.21±0.05) while that was only 0.18±0.05 in PD60 group (control group: 0.15±0.02). At 3 weeks pest-dosing, all of them returned to normal level. Conclusion Dysfunctions of oxidation and antioxidation in mitochondria of immature neurocytes are induced by prednisone or ACTH within the therapeutic levels. Enhanced expressions of NMDA-R1, Bax and superoxide contribute to excessive apoptosis of immature neurocytes induced by exogenous glucocorticosteroid. The possible determining factors for immature brain is more vulnerable to exogenous glucocorticosteroid damage. It may be associated with the physiologically high intracellular level of calcium concentration and NMDA-R1 expression.