【作者】 赵清；

【导师】 何朝晖；

【作者基本信息】 重庆医科大学 ， 外科学（神经外科）， 2016， 硕士

【摘要】 目的作为体内重要的促氧化蛋白,硫氧还蛋白相互作用蛋白(Thioredoxin-interacting protein,TXNIP)可以在多种应激状态下导致细胞凋亡和炎症发生。课题组前期研究表明内质网应激(Endoplasmic eticulum stress,ER stress)介导的脑细胞死亡是加重蛛网膜下腔出血(subarachnoid hemorrhage,SAH)后早期脑损伤(early brain injury,EBI)的重要机制。近来的研究发现,ER stress可以在转录和转录后水平调节TXNIP的表达。因此本研究拟对ER stress和TXNIP进行干预,以期阐明ER stress-TXNIP信号通路参与SAH后EBI的发生过程。方法采用SD雄性大鼠血管内穿刺法建立SAH模型,检测TXNIP及下游因子SAH后72h内动态变化;采用TXNIP siRNA、TXNIP抑制剂Resveratrol、PERK抑制剂GSK2656157、IRE1α抑制剂STF-083010对TXNIP及ER stress感受体进行干预。Western blot检测干预前后TXNIP及通路相关因子表达变化,三维荧光检测脑细胞TXNIP定位,荧光TUNEL检测TXNIP在凋亡细胞中的定位、干预前后凋亡细胞数量变化情况,同时评估死亡率、SAH评分、神经功能评分、脑水肿、血脑屏障通透性变化情况。结果SAH后TXNIP及下游相关因子在出血早期即出现增高(0.729±0.548,P<0.05)。荧光共聚焦发现TXNIP在神经元、小胶质细胞和星形胶质细胞表达,荧光TUNEL发现TXNIP与凋亡细胞出现共定位。对TXNIP及ER stress干预后,TXNIP的表达被显著抑制(P<0.05),并伴随下游凋亡因子和炎症因子释放减少(P<0.05),SAH大鼠预后得到改善。结论作为体内重要的命运调控细胞器,内质网参与多种死亡调节机制。本研究证实ER stress介导的TXNIP表达能明显加重细胞凋亡发生,促进炎症介质释放,加重SAH后EBI。因此抑制TXNIP可能是今后SAH治疗新的措施。更多还原

【Abstract】 Objective: As an important pro-oxidant protein in vivo, Thioredoxin-interacting protein(TXNIP) had been identified could induce apoptosis and inflammatation under various stress. Our previous study had confirmed Endoplasmic eticulum stress(ER stress) participated in pathological procession of early brain injury(EBI) after subarachnoid hemorrhage(SAH). Intriguingly, recently some elegant researches found ER stress significantly induced TXNIP expression at transcription and post-transcription levels. So this experiment applied appropriate interventions on TXNIP and ER stress, tried to authenticate the relationship between ER stress-TXNIP signaling pathway and EBI.Methods: SAH models were performed by endovascular perforation, TXNIP expression in early 72 hours and its downstream factors were measured. TXNIP siRNA and its inhibitor-Resveratrol were applied to downregulate TXNIP generation. The special inhibitors-GSK2656157, STF-083010 of ER stress sensors PERK, IRE1α were utilized. Immunofluorescence and TUNEL fluorescent double-labeling detected the location of TXNIP in cerebral and apoptotic cells. TXNIP and its upstream and downstream factors were measured by Western blot, and TUNEL positive cells were counted. Meanwhile, mortality, SAH grade, neurological deficits, brain water content and blood-brain barrier(BBB) permeability had been evaluated.Results: The expression of TXNIP was significantly elevated in early period after SAH, along with its downstream factors. Fluorescence showed that TXNIP expressed in neurons, microglia and astrocytes, colocalized with TUNEL positive cells. Downregulation of TXNIP could significantly reduce downstream apoptosis factors generation and inflammatory cytokines releasing. Suppressing TXNIP expression could attenuate the prognosis of SAH rats.Conclusion: As the destiny executor, Endoplasmic eticulum participates in variety death regulation. Here, we confirmed that TXNIP induced by ER stress could aggravate EBI after SAH through promoting apoptosis and inflammatory amplification. Inhibition of TXNIP may be a new effective strategy for treatment of SAH.更多还原