Toll样受体4在蛛网膜下腔出血大鼠早期脑损伤中的作用及对海马区神经元自噬的影响

The role of toll-like receptor 4 in early brain injury in rats with subarachnoid hemorrhage and its effect on the autophagy of neurons in the hippocampus

目的探讨Toll样受体4(TLR4)在蛛网膜下腔出血(subarachnoid hemorrhage,SAH)大鼠早期脑损伤中的作用及其对海马CA1区神经元自噬的影响。方法 192只SD大鼠按随机数字表法分为假手术组、SAH模型组(模型组)、SAH+DMSO溶剂组(溶剂组)、SAH+TLR4抑制剂组(CLI-095组),每组再依据取材时间不同分为24 h和48 h两个时间点。采用颈内动脉穿刺法建立SAH模型,溶剂组与CLI-095组在制备SAH模型前侧脑室分别注射DMSO溶液10 μL或100 μg/mL的CLI-095溶液10 μL。Garcia评分检测大鼠行为学改变;干湿重法检测脑水肿情况;HE染色观察海马CA1区神经元形态;免疫组化和免疫印迹法检测大鼠海马CA1区TLR4、LC3和Beclin1蛋白表达。结果与假手术组比较,模型组各时间点Garcia评分均降低(P<0.05),脑水肿程度加重,存活神经元数量减少(P<0.05),海马CA1区TLR4、LC3及 Beclin1 表达增多(P<0.05)。与模型组比较,CLI-095组各时间点Garcia 评分增多(P<0.05),脑水肿程度减轻,存活神经元数量增多(P<0.05),海马CA1区TLR4、LC3及Beclin1表达降低(P<0.05)。结论 TLR4可能通过调控SAH诱导的自噬,参与并加重SAH继发性脑损伤的过程。

Objective To investigate the role of Toll-like receptor 4 (TLR4) in early brain injury in rats with subarachnoid hemorrhage and its effect on autophagy in hippocampus CA1 area. Methods Totally 192 SD rats were randomly divided into sham operation group,SAH model group (model group),SAH+DMSO solvent group (solvent group),and SAH+TLR4 inhibitor group (CLI-095 group). Each group was divided into 24 h and 48 h 2 time points. The SAH model was established by internal carotid artery puncture. The drug-administered group was injected with 10 μL of DMSO solution or 100 μg/mL of CLI-095 solution of 10 μL before the preparation of SAH model. The behavioral changes of the rats were detected by Garcia score;the cerebral edema was detected by wet and dryweight method. The morphology of hippocampal CA1 neurons was observed by HE staining. Immunohistochemistry and immunoblotting were used to detect the expressions of TLR4,LC3 and Beclin1 in the hippocampus CA1 area. Results Compared with the sham group,Garcia score was reduced at each time point ( P <0.05). The degree of brain edema was increased and the number of viable neurons was reduced ( P <0.05). The expressions of TLR4, LC3 and Beclin1 were increased in the hippocampus CA1 area in the model group ( P <0.05).Compared with the model group,the Garcia score was increased at each time point ( P <0.05). The degree of brain edema was reduced and the number of viable neurons was increased ( P <0.05). The expressions of TLR4,LC3 and Beclin1 were reduced in the hippocampus CA1 area in the CLI-095 group ( P <0.05). Conclusion TLR4 may participate in the regulation of SAH-induced autophagy and aggravate the process of secondary brain injury in SAH.