Spautin-1通过抑制杏仁核区星形胶质细胞焦亡改善小鼠TBI后焦虑样行为

Spautin-1 in improving anxiety-like behaviors in mice after traumatic brain injury through inhibiting astrocytic pyroptosis in the amygdala

目的探讨自噬抑制剂Spautin-1对小鼠创伤性颅脑损伤(TBI)后焦虑样行为的影响及其机制。方法36只C57BL/6小鼠采用随机数字表法分为假手术组、TBI组、TBI+自噬抑制剂Spautin-1组,每组12只。后2组小鼠采用改良的Feeney自由落体硬膜外撞击法建立TBI模型。造模后10 min,TBI+自噬抑制剂Spautin-1组小鼠侧脑室注射2μL Spautin-1溶液(10 mmol/L),另外2组注射等量溶剂。造模后1、7、14 d采用神经系统症状评分(NSS)评估小鼠的运动、感觉和反射功能。造模后15、16 d分别采用旷场实验、高架十字迷宫实验评估小鼠的焦虑样行为。造模后16 d采用尼氏染色检测小鼠杏仁核区尼氏小体的数量,免疫荧光染色检测小鼠杏仁核区神经元特异性核蛋白(NeuN)阳性细胞数、白细胞介素(IL)-18及IL-1β阳性星形胶质细胞数,Western blotting实验检测小鼠杏仁核区自噬、焦亡相关蛋白的表达。结果(1)造模后1、7 d,与假手术组比较,TBI组、TBI+自噬抑制剂Spautin-1组小鼠的NSS评分增加,差异均有统计学意义(P<0.05)。(2)造模后15、16 d,与假手术组比较,TBI组、TBI+自噬抑制剂Spautin-1组小鼠旷场实验的跨格次数、中央区停留时间百分比下降,高架十字迷宫实验的进入开放臂次数(OE)百分比、开放臂停留时间(OT)百分比下降;与TBI组比较,TBI+自噬抑制剂Spautin-1组小鼠的跨格次数、中央区停留时间百分比、OE百分比、OT百分比升高,差异均有统计学意义(P<0.05)。(3)与假手术组比较,TBI组、TBI+自噬抑制剂Spautin-1组小鼠杏仁核区尼氏小体、NeuN阳性细胞数减少;与TBI组比较,TBI+自噬抑制剂Spautin-1组小鼠杏仁核区尼氏小体、NeuN阳性细胞数增加,差异均有统计学意义(P<0.05)。(4)与假手术组比较,TBI组、TBI+自噬抑制剂Spautin-1组小鼠杏仁核区IL-1β、IL-18阳性星形胶质细胞百分比增加;与TBI组比较,TBI+自噬抑制剂Spautin-1组小鼠杏仁核区IL-1β、IL-18阳性星形胶质细胞百分比减少,差异均有统计学意义(P<0.05)。(5)与假手术组比较,TBI组、TBI+自噬抑制剂Spautin-1组小鼠杏仁核区NOD样受体家族蛋白3(NLRP3)、活化半胱氨酸天冬氨酸蛋白酶-1(Caspase-1)、成孔蛋白D-N末端片段(GSDMD-N)、泛素特异性肽酶(USP)13及B淋巴细胞瘤-2相互作用蛋白(Beclin1)的表达上调,微管相关蛋白1轻链3(LC3)Ⅱ/LC3Ⅰ值升高;与TBI组比较,TBI+自噬抑制剂Spautin-1组小鼠杏仁核区NLRP3、活化Caspase-1、GSDMD-N、USP13、Beclin1蛋白的表达下调,LC3Ⅱ/LC3Ⅰ值降低,差异均有统计学意义(P<0.05)。结论自噬抑制剂Spautin-1能够改善小鼠TBI后的焦虑样行为,其机制可能与抑制小鼠杏仁核区星形胶质细胞的自噬依赖性焦亡相关。

Objective To investigate the effect of Spautin-1(an inhibitor of autophagy)on improving anxiety-like behaviors and its mechanism in mice after traumatic brain injury(TBI).Methods Thirty-six C57BL/6 mice were randomly divided into sham-operated group,TBI group,and TBI+Spautin-1 group(n=12);TBI models in the latter two groups were established by modified Feeney free fall epidural impingement method.Mice in TBI+Spautin-1 group were administered with Spautin-1(2μL,10 mmol/L)into the lateral ventricle 10 min after modeling,but mice in the other two groups were only injected with same volume of solvent.Neuropathy Symptom Score(NSS)was used to evaluate the functions of motor,sensory and reflexes of mice on 1st,7th and 14th d of modeling.On 15th and 16th d of modeling,open field test and elevated plus maze test were used to evaluate the anxiety-like behaviors in mice.The number of Nissl bodies in the amygdala of mice was calculated by Nissl staining 16 d after modeling.The numbers of neuron specific nucleoprotein(NeuN)positive cells,interleukin(IL)-18 and IL-1βpositive astrocytes in the amygdala were detected by immunofluorescent staining.Western blotting was used to detect the autophagy-and pyrotopic-associated protein expressions in the amygdala region of mice.Results(1)As compared with the sham-operated group,the TBI group and TBI+Spautin-1 group had significantly increased NSS scores on 1st and 7th d of modeling(P<0.05).(2)Open field test showed that as compared with the sham-operated group,the TBI group and TBI+Spautin-1 group had significantly smaller number of crossing grids,significantly decreased percentage of time spending in the central zone([central area residence time/total time]×100%),significantly decreased percentage of frequencies entering into opening arm(OE)(OE/[OE+frequencies of entering closing arm]×100%)and opening arm time(OT)percentage(OT/[OT+time of closing arm]×100%);as compared with the TBI group,TBI+Spautin-1 group had significantly larger number of crossing grids,and significantly increased time percentage spending in the central zone,OE percentage,and OT percentage(P<0.05).(3)As compared with sham-operated group,the TBI group and TBI+Spautin-1 group had significantly smaller numbers of Nissl bodies and NeuN positive cells in the amygdala of mice(P<0.05);as compared with TBI group,TBI+Spautin-1 group had significantly larger numbers of Nissl bodies and NeuN positive cells in the amygdala of mice(P<0.05).(4)As compared with the sham-operated group,the TBI group and TBI+Spautin-1 group had significantly increased percentages of IL-1βand IL-18 positive astrocytes in amygdala of mice(P<0.05);as compared with the TBI group,the TBI+Spautin-1 group had significantly decreased percentages of IL-1βand IL-18 positive astrocytes in amygdala of mice(P<0.05).(5)As compared with sham-operated group,the TBI group and TBI+Spautin-1 group had significantly higher protein expressions of NOD-like receptor family protein 3(NLRP3),activated cysteine aspartate protease-1(Caspase-1),pore-forming protein D-N terminal fragment(GSDMD-N),ubiquitin specific peptidase(USP)13 and B-lymphocytoma-2 interacting protein(Beclin1),and statistically higher ratio of microtubule-associated protein 1 light chain(LC)3 II/LC3 I(P<0.05);as compared with TBI group,the TBI+Spautin-1 group had significantly decreased protein expressions of NLRP3,activated Caspase-1,GSDMD-N,USP13 and Beclin1 in the amygdala,and statistically lower ratio of LC3 II/LC3 I(P<0.05).Conclusion Spautin-1 improves the anxiety-like behaviors in mice after TBI,whose mechanism may be associated with the inhibition of astrocytic pyroptosis in the amygdala.