地塞米松对脓毒症幼年大鼠大脑皮质神经元LC3表达的影响

Effect of Dexamethasone on LC3 expression of neurons in cerebral cortex of juvenile rats with sepsis

目的探讨地塞米松对脓毒症幼年Wistar大鼠大脑皮质细胞和神经元LC3表达的影响。方法阑尾结扎穿孔术（CLP）建立幼年Wistar脓毒症大鼠模型。60只30日龄健康雄性Wistar大鼠按随机数字表法随机分为假手术组（10只）、脓毒症未治疗组（25只）和地塞米松组（25只）。地塞米松组在CLP后12h时行尾静脉注射地塞米松（1mg／kg），隔天1次，共3次，脓毒症未治疗组注射同剂量的9g／L盐水。定期对大鼠进行神经行为学评分。大鼠40日龄时被统一处死，取大鼠大脑皮质组织，免疫荧光检测大脑皮质细胞LC3和神经元核抗原（NeuN）的表达，图像分析系统软件统计表达阳性的细胞数量，Westernblot检测LC3-I和LC3-II蛋白的表达。结果CLP后3h时Wistar大鼠开始出现蜷缩少动、竖毛、寒战等表现，脓毒症未治疗组大鼠神经行为学评分低于假手术组（t=9．895，P=0．000）。CLP后10d（即大鼠40日龄），地塞米松组25只大鼠死亡12只，死亡率48％，脓毒症未治疗组25只大鼠死亡8只，死亡率32％，2组死亡率差异无统计学意义（χ2=1．333，P=0．248）。免疫荧光染色和图像分析提示：与假手术组和地塞米松组比较，脓毒症未治疗组大鼠大脑皮质LC3阳性细胞的比例明显增多（0．6067±0．0301比0．3533±0．0258，t=15．644，P=0．000；0．6067±0．0301比0．2703±0．0194，t=22．450，P=0．000）。Westernblot检测表明：脓毒症未治疗组大鼠大脑皮质细胞LC3．II表达上调，LC3-II／LC3-I比值明显高于假手术组和地塞米松组（0．4133±0．0225比0．2050±0．0152，t=18．802，P=0．000；0．4133±0．0225比0．1850±0．0235，t=17．206，P=0．000）。假手术组和地塞米松组大鼠大脑皮质LC3阳性神经元较少，与假手术组和地塞米松组比较，脓毒症未治疗组大鼠大脑皮质LC3阳性神经元明显增多（0．5800±0．0200比0．2983±0．0147，t=27．783，P=0．000；0．5800±0．0200比0．2617±0．0172，t=28．614，P=0．000）。结论幼年Wistar大鼠在脓毒症时大脑皮质表达LC3的细胞数量增多，LC3-II表达上调，LC3-II／LC3-I比值增加，LC3阳性神经元数量也增多，而地塞米松对其具有抑制作用。

Objective To investigate the effect of Dexamethasone on microtubule - associated protein 1 light chain 3 （ LC3 ） expression of cells and neurons in cerebral cortex of juvenile Wistar rats with sepsis. Methods Models of juvenile Wistar rats with sepsis were established through cecal ligation and puncture （CLP）. Totally 60 cases of 30 - dayold juvenile male Wistar rats were randomly divided into sham- operation group （10 cases）, non- treated group （25 cases）and Dexamethasone group （25 cases）. Twelve hours after CLP, rats in Dexamethasone group were injected with Dexamethasone （ 1 mg / kg） via tail vein every other day,with a total of 3 times. The same dose of saline was used in the non - treated group. All rats were killed at the age of 40 days. Expressions of LC3 and neuronal nuclei （NeuN） of cells in cerebral cortex of rats were detected by using immunofluorescence assay, and the number of positive cells was calculated by using image analysis system software. Expressions of LC3 - I and LC3 - II protein were measured by a- dopting Western blot. Results Three hours after CLP,rats appeared to be curled up and showed piloerection and shi- vering and the neurobehavioral score in non - treated group was significantly lower than that in sham - operation group （t =9. 895,P =0.000）. Twelve of 25 rats in Dexamethasone group died in 10 days after CLP （48%）,while 8 of 25 rats in non -treated group died （32%） ,and the difference was not statistically significant between the 2 groups （χ2 = 1. 333 ,P = 0. 248 ）. The immunofluorescence staining and image analysis showed the percentage of LC3 positive cells in non -treated group was significantly increased （0. 606 7 ±0. 030 1 vs 0. 353 3 ±0. 025 8,t = 15. 644,P =0. 000; 0.606 7± 0.030 1 vs O. 270 3± 0.019 4,t = 22. 450, P = 0. 000 ）. In non - treated group, the LC3 expression of cells in the cerebral cortex of rats was up - regulated, and the LC3 - Ⅱ/LC3 - I ratio was significantly higher than that in sham operation group and Dexamethasone group （0.413 3 ±0. 022 5 vs O. 205 0 ± 0. 015 2, t = 18. 802, P = 0. 000 ; 0.413 3± 0.022 5 vs 0.185 0 ±0. 023 5, t = 17. 206, P = 0. 000 ）. The LC3 positive neurons in the cerebral cortex of rats were less in sham operation group and Dexamethasone group. The LC3 positive neurons were more in non - treated group than that in sham operation group and Dexamethasone group （0. 580 0 ±0. 020 0 vs 0. 298 3 ±0. 014 7,t = 27. 783 ;P = 0.000 ;0.580 0 ± 0.020 0 vs 0. 261 7 ±0.017 2 ,t = 28. 614 ;P = 0.000）. Conclusions The LC3 expres-sion of cells in the cerebral cortex of juvenile Wistar rats with sepsis was up - regulated, LC3 - Ⅱ/LC3 - I ratio in- creased, and the number of LC3 positive neurons also increased, while Dexamethasone could have inhibitory effect on them.