姜黄素调节NMDAR/Ca2+/CaMKⅡ信号通路对异氟醚诱导的幼龄小鼠术后认知功能障碍的影响

doi:10.11958/20221719

天津医药 ›› 2023, Vol. 51 ›› Issue (9): 948-954.doi: 10.11958/20221719

姜黄素调节NMDAR/Ca²⁺/CaMKⅡ信号通路对异氟醚诱导的幼龄小鼠术后认知功能障碍的影响

马慧敏(), 柳璐^△(), 熊英, 赵慧, 孔繁丽

华中科技大学同济医学院附属武汉儿童医院（武汉市妇幼保健院）麻醉科手术室（邮编430014）

收稿日期:2022-11-01 修回日期:2023-02-16 出版日期:2023-09-15 发布日期:2023-09-13
通讯作者: △E-mail：365038103@qq.com
作者简介:马慧敏（1983），女，主治医师，主要从事麻醉学方面研究。E-mail：mahuimin972@163.com
基金资助:
湖北省卫生健康委科研立项项目(WJ2019M012)

Effect of curcumin regulating NMDAR/Ca²⁺/CaMKⅡ signaling pathway on postoperative cognitive dysfunction induced by isoflurane in young mice

MA Huimin(), LIU Lu^△(), XIONG Ying, ZHAO Hui, KONG Fanli

Anesthesia Department Operating Room, Wuhan Children's Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology (Wuhan Maternal and Child Health Hospital), Wuhan 430014, China

Received:2022-11-01 Revised:2023-02-16 Published:2023-09-15 Online:2023-09-13
Contact: △E-mail：365038103@qq.com

马慧敏, 柳璐, 熊英, 赵慧, 孔繁丽. 姜黄素调节NMDAR/Ca²⁺/CaMKⅡ信号通路对异氟醚诱导的幼龄小鼠术后认知功能障碍的影响[J]. 天津医药, 2023, 51(9): 948-954.

MA Huimin, LIU Lu, XIONG Ying, ZHAO Hui, KONG Fanli. Effect of curcumin regulating NMDAR/Ca²⁺/CaMKⅡ signaling pathway on postoperative cognitive dysfunction induced by isoflurane in young mice[J]. Tianjin Medical Journal, 2023, 51(9): 948-954.

摘要/Abstract

摘要：

目的探讨姜黄素（Cur）调节N-甲基-D-天冬氨酸受体（NMDAR）/钙离子（Ca²⁺）/钙调素依赖性蛋白激酶Ⅱ（CaMKⅡ）信号通路对异氟醚（ISO）诱导的幼龄小鼠术后认知功能障碍（POCD）的影响。方法将72只C57BL/6J小鼠分为对照组、ISO组、低剂量Cur组（Cur-L组，50 mg/kg）、中剂量Cur组（Cur-M组，100 mg/kg）、高剂量Cur组（Cur-H组，200 mg/kg）、Cur-H+NMDA（NMDAR激活剂）组（200 mg/kg+8 mg/kg），每组12只。经对应给药处理30 min后，对照组小鼠吸入含30%氧气和空气的混合气体2 h，其余各组小鼠吸入2% ISO 2 h，每天1次，持续14 d。末次给药24 h后，Morris水迷宫实验检测小鼠学习与空间记忆能力；HE染色检测海马CA1区病理学变化；免疫荧光染色检测小鼠海马CA1区神经元特异核蛋白（NeuN）阳性表达；TUNEL染色检测神经细胞凋亡；酶联免疫吸附试验检测海马CA1区组织中白细胞介素-1β（IL-1β）和肿瘤坏死因子-α（TNF-α）水平；蛋白印迹法检测海马CA1区组织中NMDAR1和CaMKⅡ蛋白表达；荧光探针检测海马CA1区Ca²⁺浓度。结果与对照组比较，ISO组小鼠海马CA1区病理损伤严重，逃避潜伏期延长，神经细胞凋亡率升高，海马CA1区组织中IL-1β和TNF-α水平升高，NMDAR1和CaMKⅡ蛋白表达及Ca²⁺浓度升高（P<0.05），穿越平台次数和NeuN阳性细胞数减少（P<0.05）；与ISO组比较，Cur-L组、Cur-M组、Cur-H组小鼠海马CA1区病理损伤减轻，逃避潜伏期缩短，神经细胞凋亡率降低，海马CA1区组织中IL-1β和TNF-α水平降低，NMDAR1和CaMKⅡ蛋白表达及Ca²⁺浓度降低（P<0.05），穿越平台次数和NeuN阳性细胞数增加（P<0.05），且呈剂量依赖性；NMDA减弱了高剂量Cur对ISO诱导的小鼠POCD的改善作用（P<0.05）。结论 Cur可能通过抑制NMDAR/Ca²⁺/CaMKⅡ信号通路改善ISO诱导的小鼠POCD。

关键词: 姜黄素, 受体, N-甲基-D-天冬氨酸, 钙通道, 钙-钙调素依赖性蛋白激酶2型, 认知功能障碍, 术后认知并发症, 异氟醚

Abstract:

Objective To investigate the effect of curcumin (Cur) regulating N-methyl-D-aspartate receptor (NMDAR)/calcium ion (Ca²⁺)/calmodulin dependent protein kinase Ⅱ (CaMKⅡ) signaling pathway on isoflurane (ISO) -induced postoperative cognitive dysfunction (POCD) in young mice. Methods Seventy-two C57BL/6J mice were separated into the control group, the ISO group, the low-dose Cur group (Cur-L group, 50 mg/kg), the medium-dose Cur group (Cur-M group, 100 mg/kg), the high-dose Cur group (Cur-H group, 200 mg/kg) and the Cur-H+NMDA (NMDAR activator) group (200 mg/kg+8 mg/kg), with 12 animals in each group. After 30 min of corresponding drug treatment, the mice in the control group inhaled a mixed gas containing 30% oxygen and air for 2 hours, and mice in the other groups inhaled 2% ISO for 2 hours, once a day for 14 days. Twenty-four hours after the last treatment, Morris water maze test was used to detect the learning and spatial memory abilities of mice. HE staining was used to detect the pathological changes in hippocampal CA1 area. Immunofluorescence staining was used to detect the positive expression of neuron specific nucleoprotein (NeuN) in hippocampus CA1 region of mice. TUNEL staining was used to detect neuronal apoptosis. Enzyme linked immunosorbent assay was used to detect levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in hippocampal CA1 tissue. Western blot assay was used to detect NMDAR1 and CaMKⅡ protein expression in mouse hippocampal CA1 tissue. Intracellular Ca²⁺ concentration in hippocampal CA1 region was detected by fluorescence probe. Results Compared with the control group, the pathological damage of hippocampal CA1 region of mice was severe in the ISO group, and the escape latency was prolonged. The apoptosis rate of neural cells, levels of IL-1β and TNF-α, expression levels of NMDAR1 and CaMKⅡ protein, and the concentration of Ca²⁺ in hippocampal CA1 region were increased (P<0.05). Times of crossing platform and the number of NeuN positive cells were decreased in the ISO group (P<0.05). Compared with the ISO group, pathological damages of hippocampal CA1 region of mice were alleviated in the Cur-L group, the Cur-M group and the Cur-H group, the escape latency was shortened, and the apoptosis rate of neural cells, levels of IL-1β and TNF-α, expression levels of NMDAR1 and CaMKⅡ protein, and the concentration of Ca²⁺ in hippocampal CA1 region were decreased (P<0.05). Times of crossing platform and the number of NeuN positive cells were increased (P<0.05), which was dose-dependent. NMDA attenuated the improvement effect of high-dose Cur on ISO induced POCD in mice (P<0.05). Conclusion Curcumin may improve ISO-induced POCD in mice by inhibiting NMDAR/Ca²⁺/CaMKⅡ signaling pathway.

Key words: curcumin, receptors, N-methyl-D-aspartate, calcium channels, calcium-calmodulin-dependent protein kinase type 2, cognitive dysfunction, postoperative cognitive complications, isoflurane

中图分类号:

R285.5，R614.2

图/表 8

表1 各组小鼠逃避潜伏期及穿越平台次数比较（n=12，$\bar{x}\pm s$）

Tab.1 Comparison of escape latency and crossing platform times of mice between the six groups

组别	逃避潜伏期/s	穿越平台次数/次
对照组	10.27±1.02	8.83±0.79
ISO组	58.45±7.34^a	1.73±0.12^a
Cur-L组	49.33±5.15^b	2.67±0.21^b
Cur-M组	38.82±2.77^bc	4.03±0.32^bc
Cur-H组	24.48±2.51^bcd	6.93±0.53^bcd
Cur-H+NMDA组	41.97±4.86^e	3.22±0.27^e
F	182.808^＊＊	474.270^＊＊

图1 HE染色检测小鼠海马CA1区病理损伤（×400）

Fig.1 HE staining of pathological lesions in hippocampus CA1 region of mice (×400)

图2 免疫荧光染色检测小鼠海马CA1区NeuN阳性表达（×400）

Fig.2 Detection of NeuN positive expression in hippocampal CA1 region of mice by immunofluorescence staining (×400)

图3 TUNEL染色检测小鼠神经细胞凋亡（×400）

Fig.3 TUNEL staining of apoptosis of mouse nerve cells (×400)

表2 各组小鼠海马CA1区NeuN阳性细胞数和神经细胞凋亡率比较（n=12，$\bar{x}\pm s$）

Tab.2 Comparison of the number of NeuN positive cells and apoptosis rate of nerve cells in CA1 region of hippocampus of mice between the six groups

组别	NeuN阳性细胞数/（个/视野）	细胞凋亡率/%
对照组	58.45±3.34	2.45±0.11
ISO组	10.27±1.02^a	18.62±1.21^a
Cur-L组	24.48±2.51^b	15.41±1.02^b
Cur-M组	35.82±2.77^bc	10.96±0.75^bc
Cur-H组	49.33±3.15^bcd	5.62±0.37^bcd
Cur-H+NMDA组	28.97±2.86^e	13.34±1.26^e
F	492.239^＊＊	554.614^＊＊

表3 各组小鼠海马CA1区组织中IL-1β、TNF-α水平比较（n=12，ng/g，$\bar{x}\pm s$）

Tab.3 Comparison of IL-1β and TNF-α levels in hippocampal CA1 region of mice between the six groups

组别	IL-1β	TNF-α
对照组	1.35±0.12	1.17±0.10
ISO组	12.28±1.02^a	8.45±0.62^a
Cur-L组	9.46±0.64^b	6.52±0.43^b
Cur-M组	6.82±0.47^bc	4.67±0.21^bc
Cur-H组	3.45±0.22^bcd	2.58±0.20^bcd
Cur-H+NMDA组	7.66±0.43^e	5.57±0.31^e
F	591.153^＊＊	663.438^＊＊

图4 各组小鼠海马CA1区组织中NMDAR1、CaMKⅡ蛋白印迹图 A：对照组；B：ISO组；C：Cur-L组；D：Cur-M组；E：Cur-H组；F：Cur-H+NMDA组。

Fig.4 Western blot results of NMDAR1 and CaMKⅡ in hippocampal CA1 region of mice in each group

表4 各组小鼠海马CA1区组织中NMDAR1、CaMKⅡ蛋白表达及Ca2+浓度比较（n=12，$\bar{x}\pm s$）

Tab.4 Comparison of NMDAR1 and CaMKⅡprotein expression and intracellular Ca2+ concentration in hippocampal CA1 region of mice between the six groups

组别	NMDAR1/ β-actin	CaMKⅡ/ β-actin	Ca²⁺/ （nmol/L）
对照组	0.31±0.02	0.23±0.02	68.52±3.16
ISO组	1.36±0.11^a	0.97±0.08^a	157.73±11.34^a
Cur-L组	1.05±0.08^b	0.72±0.08^b	129.24±9.62^b
Cur-M组	0.71±0.07^bc	0.49±0.04^bc	105.56±6.33^bc
Cur-H组	0.39±0.03^bcd	0.27±0.03^bcd	79.73±4.26^bcd
Cur-H+NMDA组	0.82±0.07^e	0.66±0.04^e	117.58±6.24^e
F	383.611^＊＊	334.224^＊＊	235.201^＊＊

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姜黄素调节NMDAR/Ca²⁺/CaMKⅡ信号通路对异氟醚诱导的幼龄小鼠术后认知功能障碍的影响

Effect of curcumin regulating NMDAR/Ca²⁺/CaMKⅡ signaling pathway on postoperative cognitive dysfunction induced by isoflurane in young mice

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