凝血酶诱发的脑积水中TNF-α与IL-1β对水孔蛋白-1与水孔蛋白-4表达水平的影响

Thrombin-induced hydrocephalus:the effect of TNF-α and IL-1β on the expression of aquaporin1 and 4

目的探讨蛛网膜下腔出血(Subarachnoid hemorrhage, SAH)中凝血酶(Thrombin, TH)诱导脑积水形成时肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)、白细胞介素-1β(Interleukin-1β,IL-1β)在脑积水病理发展过程中对水孔蛋白1(Aquaporin 1,AQP1)和水孔蛋白4(Aquaporin 4,AQP4)表达水平的调控作用。方法选用健康雄性远交群(Sprague Dawley, SD)大鼠40只,随机分成4组(n=10):假手术组、凝血酶(Thrombin, TH)组、TH/TNF-α抑制剂组、TH/IL-1β抑制剂组;假手术组大鼠枕大池注入0.3 mL生理盐水;TH组大鼠枕大池注入0.3 mL(10 U/mL)凝血酶;TH/TNF-α抑制剂组大鼠枕大池注入0.3 mL(10 U/mL)凝血酶+(0.25μM)TNF-α抑制剂Simponi, TH/IL-1β抑制剂组大鼠枕大池注入0.3 mL(10 U/mL)凝血酶+(0.25μM)TH/IL-1β抑制剂GIBH-130;每组大鼠至少5只以上,按照缺失补足以保证充足的样本量;建模后的第1、3 d对大鼠行神经功能评分;第3 d将大鼠处死,计算相对侧脑室面积,免疫组织化学染色评价大鼠TNF-α,IL-1β,AQP1及AQP4的阳性表达水平。结果 (1)第3 d假手术组、TH组、TH/TNF-α抑制剂组、TH/IL-1β抑制剂组大鼠侧脑室面积分别为(3.58±0.40)、(6.09±0.82)、(5.06±0.53)、(5.25±0.68)%;与假手术组比较,TH组、TH/TNF-α抑制剂组、TH/IL-1β抑制剂组相对侧脑室面积增大(P<0.01);与TH组比较,TNF-α抑制剂组与IL-1β抑制剂组相对侧脑室面积减少(P<0.01);(2)免疫组织化学染色检测:1)大鼠TNF-α与IL-1β的免疫阳性表达主要分布于胞浆内,TNF-α主要位于侧脑室周围组织的星形胶质细胞及神经元,IL-1β主要位于侧脑室周围组织的小胶质细胞及星形细胞;与假手术组比较,TH组、TH/TNF-α抑制剂组、TH/IL-1β抑制剂组TNF-α与IL-1β的阳性表达增加(P<0.01);与TH组比较,TH/TNF-α抑制剂及TH/IL-1β抑制剂组TNF-α与IL-1β的阳性表达减少(P<0.01);2)AQP1与AQP4的免疫阳性表达主要分布于胞膜,其中AQP1主要位于脉络丛上皮细胞,AQP4主要位于侧脑室周围组织的室管膜细胞;与假手术组比较,TH组、TH/TNF-α抑制剂组及TH/IL-1β抑制剂组AQP1,AQP4的阳性表达增加(P<0.01);与TH组比较,TH/TNF-α抑制剂及TH/IL-1β抑制剂组AQP1,AQP4的阳性表达减少(P<0.01)。结论 (1)在TH诱发的SAH脑积水病理发展中TH可诱导TNF-α、IL-1β表达水平上调,从而增加AQP1的表达,促进脉络丛分泌脑脊液;AQP4表达水平升高可能为脑积水后的代偿反应;(2)通过抑制TNF-α,IL-1β表达可以下调AQP1,并引起继发性的AQP4的表达减少,改善大鼠神经功能缺损症状;TNF-α,IL-1β可作为临床治疗脑积水的重要靶点。

Objective To investigate the effect of the inflammatory factors tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β) regulateing the expression of aquaporin 1(AQP1) and aquaporin 4(AQP4) on the pathological development of hydrocephalus induced by thrombin(TH).Methods Forty healthy male SD rats were randomly divided into four groups(n=10): sham operation group, TH group, TH/TNF-α inhibitor group, TH/IL-1β inhibitor group. Rats in the sham operation group were injected with 0.3 mL of normal saline into the cisterna magna, rats in the TH group were injected with 0.3 mL(10 U/mL) of TH into the cisterna magna, and the rats in the TH/TNF-α inhibitor group were injected with 0.3 mL(10 U/mL) of TH+0.25 μM TNF-α inhibitor(Simponi) into the cisterna magna, and the rats in the TH/IL-1β inhibitor group were injected with 0.3 mL(10 U/mL) of TH+0.25 μM TH/IL-1β inhibitor(GIBH-130) into the cisterna magna. There were at least 5 rats in each group, and sufficient sample size was guaranteed according to deficiency supplement. Neurological function scores were performed on the first and third days after odeling. On the third day, the rats were sacrificed and the relative lateral ventricle area was calculated. The positive expressions of TNF-α,IL-1β,AQP1 andAQP4 proteins were evaluated by immunohistochemical staining.Results(1) The area of lateral ventricle in the sham operation group, the TH group, the TH/TNF-α inhibitor group, the TH/IL-1β inhibitor group was(3.58±0.40) %,(6.09±0.82) %,(5.06±0.53) %,(5.25±0.68) %. Compared with the sham operation group, the relative lateral ventricle area of the TH group, the TH/TNF-α inhibitor group and the TH/IL-1β inhibitor group increased, and the difference was statistically significant(P<0.01).Compared with the TH group, the area of lateral ventricle in the TNF-α inhibitor group and the IL-1β inhibitor group decreased, and the difference was statistically significant(P<0.01).(2) Immunohistochemical test: 1) The immunopositive expression of TNF-α and IL-1β was mainly distributed in the cytoplasm of rats. TNF-α was mainly located in the astrocytes and neuronal cells of the peripheral tissues of the lateral ventricles. IL-1β was mainly located in the microglia and astrocytes of the peripheral tissues of the lateral ventricles. Compared with the sham operation group, the positive expression of TNF-α and IL-1β protein was increased in the TH group, the TH/TNF-α inhibitor group and the TH/IL-1β inhibitor group, and the difference was statistically significant(P<0.01).Compared with TH group, the positive expression of TNF-α and IL-1β protein in the TH/TNF-α inhibitor group and the TH/IL-1β inhibitor group decreased, with statistical significance(P<0.01). 2) The immunopositive expressions of AQP1 and AQP4 were mainly distributed in the cell membrane, AQP1 was mainly located in the choroid plexus epithelial cells, and AQP4 was mainly located in the ependymal cells of the periventricular tissues. Compared with the sham operation group, the positive expressions of AQP1 and AQP4 protein in the TH group, the TH/TNF-α inhibitor group and the TH/IL-1β inhibitor group were increased, with statistical significance(P<0.01).Compared with the TH group, the positive expressions of AQP1 and AQP4 protein in the TH/TNF-α inhibitor group and the TH/IL-1β inhibitor group were decreased, with statistical significance(P<0.01).Conclusion(1) In the pathological development of SAH hydrocephalus induced by TH, TH can induce the up-regulation of TNF-α and IL-1β, thereby increasing the expression of AQP1 and promoting the secretion of cerebrospinal fluid from the choroid plexus. The elevated expression of AQP4 may be a compensatory response after hydrocephalus.(2) By inhibiting TNF-α and IL-1β, AQP1 can be down-regulated, and secondary AQP4 expression can be reduced, and the symptoms of neurological impairment in rats can be improved. TNF-α and IL-1β may be important targets for clinical treatment of hydrocephalus.