赵清车旭东谭关平张红霞蒋登志孙晓川何朝晖

硫氧还蛋白相互作用蛋白表达下调减轻大鼠蛛网膜下腔出血后早期脑损伤☆

赵清*车旭东*谭关平*张红霞*蒋登志*孙晓川*何朝晖*

目的 观察大鼠蛛网膜下腔出血（subarachnoid hemorrhage，SAH）后早期硫氧还蛋白相互作用蛋白（thioredoxin-interacting protein，TXNIP）表达变化，检测干预前后TXNIP及下游凋亡因子表达，探讨TXNIP参与SAH后早期脑损伤（early brain injury，EBI）的可能机制。方法 血管内穿刺法建立SAH模型。97只成年雄性SD大鼠，随机分为假手术对照组（Sham组，17只）、SAH组（32只）、Control siRNA组（12只）、TXNIP siRNA组（12只）、白藜芦醇（resveratrol，RES）对照组（12只）、RES干预组（12只）。Western blot检测SAH后各时间点及干预前后TXNIP、p-ASK-1、Caspase-3表达变化，荧光共聚焦检测TXNIP在神经元定位，TUNEL法检测细胞凋亡与TXNIP共定位，同时进行脑水肿评估（6只/组）和行为学评分（12只/组）。结果 采用TXNIP siRNA和TXNIP抑制剂RES干预后死亡率、行为学评分及脑水肿（F=7.964，P＜0.05）得到改善。荧光共聚焦显示TXNIP在大鼠脑神经元广泛表达，且主要位于胞浆。荧光TUNEL提示TXNIP与皮层区及海马区凋亡细胞共定位。Western blot发现与Sham组（0.476±0.043，n=3）比较，TXNIP在SAH后12h（0.729±0.548）表达逐渐增高，72h（1.509±0.071）仍处于较高水平，同时伴随下游凋亡因子的增高，差异有统计学意义（F=7.806，P＜0.05）。采用TXNIP siRNA和TXNIP抑制剂RES干预后，TXNIP表达下调（F=900.849，P＜0.05，n=3），下游凋亡因子出现下降（p-ASK1，F= 32.897，P＜0.05；Caspase-3，F=89.120，P＜0.05）。结论 大鼠SAH后早期TXNIP表达增加，通过其促凋亡机制参与EBI发生，下调TXNIP能减轻大鼠SAH后EBI，这可能为临床SAH早期治疗提供新的治疗思路。

硫氧还蛋白相互作用蛋白蛛网膜下腔出血早期脑损伤细胞凋亡

【Abstract】Objective To explore the possible mechanism by which thioredoxin-interacting protein（TXNIP）participated in early brain injury（EBI）of subarachnoid hemorrhage（SAH）via examination of the expression of TXNIP and its downstream apoptotic factors before and after intervention.Methods Subarachnoid Hemorrhage（SAH）was performed by endovascular perforation.Total 97 adult male SD rats were randomly divided into 6 groups：sham-operation（17），SAH （32），control siRNA（12），TXNIP siRNA（12），resveratrol control（12）and resveratrol injection（12）.Western blot was used to examine the expression of TXNIP，p-ASK-1，Caspase-3 before and after intervention.Laser scanning confocal microscopy（LSCM）was used to detect the expression of TXNIP in neurons.The co-localization of TXNIP with apoptoticcells was examined by using fluorescent TUNEL.Mortality，behavior score and cerebral edema were also evaluated.Results Mortality，behavior scores and brain edema were improved after TXNIP siRNA and resveratrol injection（P＜0.05）. LSCM showed that TXNIP was widely expressed in brain and mainly located in cytoplasm of neurons in SAH rats.Fluorescent TUNEL revealed the co-localization of TXNIP with apoptotic cells.The expression level of TXNIP was significantly higher in SAH group than in sham operation（P＜0.05，n=3）.The expression level of TXNIP gradually increased at 12h and still remained at high level at 72h（P＜0.05）.This increase was simultaneously accompanied by the increase in downstream apoptosis factors，p-ASK-1 and Caspase-3.Inhibition of TXNIP by siRNA or resveratrol significantly reduced the expression of TXNIP，p-ASK-1 and Caspase-3（P＜0.05，n=3）.Conclusion TXNIP gradually increases in early period after SAH and aggravates brain damage through activation of ASK-1 apoptosis signaling pathway，whereas inhibition of TXNIP may attenuate EBI through reduction of p-ASK-1 and Caspase-3 after SAH.

【Key words】Thioredoxin-interacting protein Subarachnoid Hemorrhage Early brain injury Apoptosis

早期脑损伤（early brain injury，EBI）是影响蛛网膜下腔出血（subarachnoid hemorrhage，SAH）患者预后和生存的主要因素，EBI指的是SAH后早期（72h内）发生的一系列病理变化，但其具体机制尚未完全明确，而脑组织细胞凋亡占有重要地位［1］。……