亚低温通过调控内质网应激激活自噬改善蛛网膜下腔出血大鼠早期脑损伤

Moderate Hypothermia Activates Autophagy and Reduces Early Brain Injury in Rats with Subarachnoid Hemorrhage by Regulating Endoplasmic Reticulum Stress

目的检测亚低温干预后蛛网膜下腔出血(SAH)大鼠神经细胞内质网应激及自噬的激活程度,探讨亚低温治疗的保护作用及机制。方法将48只成年雄性SD大鼠随机分为假手术组(S组)、SAH组、全身亚低温组(H组)和内质网应激抑制剂组(TUDCA组)。采用颈内动脉穿刺法建立大鼠SAH模型,H组于颈外动脉穿刺后即刻向大鼠全身喷洒乙醇进行体表降温,将直肠温度维持在30~32℃4 h;TUDCA组于颈动脉穿刺前1 h腹腔注射溶于生理盐水(100 mg/m L)的牛磺熊去氧胆酸(TUDCA,内质网应激抑制剂)溶液,剂量4.5 mg/kg。术后24 h取脑,HE染色法观察海马区神经细胞形态;免疫组织化学方法检测内质网应激相关因子GRP78、CHOP及自噬相关因子Beclin-1、LC3-Ⅱ的表达。结果与S组相比,SAH组海马区存活神经细胞数量减少(P<0.05),海马区GRP78、CHOP蛋白及Beclin-1、LC3-Ⅱ蛋白表达水平显著升高(P<0.05)。与SAH组相比,H组海马区存活神经细胞数量明显减少(P<0.05),GRP78、CHOP及Beclin-1、LC3-Ⅱ蛋白表达水平显著升高(P<0.05)。与SAH组相比,TUDCA组海马区存活神经细胞数量明显减少(P<0.05),GRP78、CHOP蛋白及Beclin-1、LC3-Ⅱ蛋白表达水平显著降低(P<0.05)。结论亚低温可通过调控内质网应激激活神经细胞自噬,减轻蛛网膜下腔出血的早期脑损伤。

Objective To detect endoplasmic reticulum stress and autophagy in neurons of rats with subarachnoid hemorrhage(SAH)and explore the protective effect of moderate hypothermia and its mechanism of action.Methods Forty-eight adult male Sprague-Dawley rats were randomly divided into sham group(S group),SAH group,systemic moderate hypothermia group(H group),and endoplasmic reticulum stress inhibitor group(TUDCA group).A rat model of SAH was established using an internal carotid artery puncture method.The body surface of rats in the H group was sprayed with alcohol immediately after puncture of the external carotid artery to reduce the temperature,and body temperature measured at the rectum was maintained at 30 to 32°C for 4 hours.In the TUDCA group,TUDCA solution in physiological saline(100 mg/mL)was intraperitoneally injected 1 h before carotid puncture at a dose of 4.5 mg/kg.The brains were removed,and the morphology of neurons in the hippocampus was observed using hematoxylin and eosin(HE)staining 24 hours after the operation.Immunohistochemistry was used to detect the expression of GRP78,CHOP,and the autophagy-related factors,Beclin-1 and LC3-Ⅱ.Results Compared with the SAH group,the survival rate of neurons in the hippocampus of the H group was significantly increased,and the expression of GRP78,CHOP,Beclin-1,and LC3-Ⅱproteins was significantly increased.However,the survival rate of neurons in the hippocampus and the expression of GRP78,CHOP,Beclin-1,and LC3-Ⅱproteins were significantly decreased in the TUDCA group than in the SAH group.Conclusion Moderate hypothermia can activate neuronal autophagy and reduce early brain injury following subarachnoid hemorrhage by regulating endoplasmic reticulum stress.