姜黄素干预通过促进自噬改善氯化锰所致的大鼠神经行为损伤

doi:10.19723/j.issn.1671-167X.2022.03.003

摘要/Abstract

摘要：

目的: 探讨姜黄素(curcumin，CUR)对锰中毒大鼠神经损伤的干预效应及其机制。方法: 成年雄性SD大鼠60只，随机分为5组，每组12只。(1)空白对照组: 通过腹腔注射(intraperitoneal injection，ip) 给予0.9%(质量分数)生理盐水，并通过灌胃(intragastric, ig)给予双蒸水(double distilled water，ddH₂O)；(2)锰中毒模型组: ip给予MnCl₂ 15 mg/kg(Mn²⁺ 6.48 mg/kg)，ig给予ddH₂O；(3)单独姜黄素组: ip给予0.9%生理盐水，ig给予CUR 300 mg/kg；(4)MnCl₂+CUR1组：ip给予MnCl₂ 15 mg/kg，ig给予CUR 100 mg/kg；(5)MnCl₂+CUR2组: ip给予MnCl₂15 mg/kg，ig给予CUR 300 mg/kg)。5组大鼠每周给药5 d, 连续给予4周。旷场实验、转棒实验检测动物的探索行为、焦虑抑郁状态、运动及平衡能力，Morris水迷宫(Morris water maze, MWM)实验检测动物的学习、记忆能力。每组6只大鼠进行脑纹状体HE染色和免疫组织化学(immunohistochemistry, IHC)检查，并且其中2只大鼠一侧纹状体进行透射电镜(transmission electron microscope，TEM)观察。每组另外6只大鼠的一侧纹状体利用电感耦合等离子体质谱(ICP-MS)测定锰含量，另一侧纹状体用免疫印迹(Western blotting, WB)检测α-突触核蛋白(α-Synuclein，α-Syn)、细胞质和细胞核转录因子EB(transcription factor EB，TFEB)、雷帕霉素靶蛋白(mammalian target of rapamycin，mTOR)、p-mTOR、Beclin1、P62和微管相关蛋白轻链蛋白3(microtubule-associated protein light chain-3，LC3)表达水平，脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(terminal deoxynucleotidyl transterase-mediated dUTP nick end labeling; TUNEL)检测大鼠纹状体多巴胺能神经元凋亡。结果: 旷场和转棒实验中，锰中毒模型组水平运动距离、直立次数、中央区运动时间和运动距离、在棒时间、在棒圈数较空白对照组显著降低(P < 0.05)，而MnCl₂+CUR2组的上述指标均较锰中毒模型组显著增加(P < 0.05)。MWM定位航行实验第3、4天时，锰中毒模型组逃避潜伏期和游泳距离显著高于空白对照组，而MnCl₂+CUR2组则显著高于锰中毒模型组(P < 0.05)。MWM探索实验中，锰中毒模型组大鼠穿越平台次数、目标象限游泳时间、目标象限游泳时间百分比、目标象限距离及目标象限距离百分比显著低于对照组(P < 0.05)，而MnCl₂+CUR2组大鼠这些指标均较锰中毒模型组显著增加(P < 0.05)。脑纹状体IHC和HE染色发现，锰中毒模型大鼠脑纹状体中酪氨酸羟化酶(tyrosine hydroxylase，TH)阳性多巴胺能神经细胞显著减少，嗜酸性细胞数量显著增加，MnCl₂+CUR1和MnCl₂+CUR2组TH阳性细胞显著增加，嗜酸性细胞减少，TEM观察发现，锰中毒模型组纹状体神经细胞出现染色质凝结，核固缩；线粒体水肿或空泡，可见溶酶体以及自噬泡。MnCl₂+CUR2组染色质凝结，核固缩，线粒体水肿、空泡等均较锰中毒大鼠明显改善，并可见更多的溶酶体以及自噬泡。免疫印迹检测发现，锰中毒模型组α-Syn表达水平较空白对照组显著增加，而MnCl₂+CUR2组聚集性α-Syn表达则较锰中毒模型组显著减少(P < 0.05)。TFEB及自噬相关蛋白免疫印迹检测发现，与空白对照组比较，单独CUR组、锰中毒模型组、MnCl₂+CUR1和MnCl₂+CUR2组纹状体胞核TFEB表达均显著增加(P < 0.05)，与锰中毒模型组比较，MnCl₂+CUR1和MnCl₂+CUR2组其表达均显著增加(P < 0.05)；与空白对照组比较，各组mTOR、p-mTOR、P62蛋白表达水平显著降低，Beclin1、LC3Ⅱ/LC3Ⅰ表达水平则显著增加(P < 0.05)；与锰中毒模型组相比，MnCl₂+CUR1和MnCl₂+CUR2组大鼠脑纹状体中mTOR、p-mTOR及P62蛋白表达水平显著降低，Beclin1、LC3Ⅱ/LC3Ⅰ表达水平显著增加(P < 0.05)。纹状体TUNEL检测结果发现，与空白对照组比较，锰中毒模型组和MnCl₂+CUR1组TUNEL阳性细胞显著增加(P < 0.05)；而与锰中毒模型组相比，MnCl₂+CUR2组TUNEL阳性细胞则显著降低(P < 0.05)。结论: 一定剂量的姜黄素干预可以减轻锰中毒大鼠纹状体多巴胺能神经元损伤，改善锰中毒大鼠的神经行为症状，减少α-Syn聚集，减少细胞凋亡，其可能的机制是通过促进TFEB核转位增强细胞自噬。

关键词: 锰中毒, 姜黄素, 转录因子EB, 自噬

Abstract:

Objective: To investigate the protective effects of curcumin(CUR) and its mechanism on a rat model of neurotoxicity induced by manganese chloride (MnCl₂), which mimics mangnism. Methods: Sixty male SD rats were randomly divided into 5 groups, with 12 rats in each group. Control group received 0.9% saline solution intraperitoneally (ip) plus double distilled water (dd) H₂O intragastrically (ig), MnCl₂ group received 15 mg/kg MnCl₂(Mn²⁺ 6.48 mg/kg) intraperitoneally plus dd H₂O intragastrically, CUR group received 0.9% saline solution intraperitoneally plus 300 mg/kg CUR intragastrically, MnCl₂+ CUR1 group received 15 mg/kg MnCl₂ intraperitoneally plus 100 mg/kg curcumin intragastrically, MnCl₂+ CUR2 group received 15 mg/kg MnCl₂ intraperitoneally plus 300 mg/kg CUR intragastrically, 5 days/week, 4 weeks. Open-field and rotarod tests were used to detect animals' exploratory behavior, anxiety, depression, movement and balance ability. Morris water maze (MWM) experiment was used to detect animals' learning and memory ability. ICP-MS was used to investigate the Mn contents in striata. The rats per group were perfused in situ, their brains striata were removed by brains model and fixed for transmission electron microscope (TEM), histopathological and immunohistochemistry (ICH) analyses. The other 6 rats per group were sacrificed. Their brains striata were removed and protein expression levels of transcription factor EB (TFEB), mammalian target of rapamycin (mTOR), p-mTOR, Beclin, P62, microtubule-associated protein light chain-3 (LC3) were detected by Western blotting. Terminal deoxynucleotidyl transterase-mediated dUTP nick end labeling (TUNEL) staining was used to determine neurocyte apoptosis of rat striatum. Results: After exposure to MnCl₂ for four weeks, MnCl₂-treated rats showed depressive-like behavior in open-field test, the impairments of movement coordination and balance in rotarod test and the diminishment of spatial learning and memory in MWM (P < 0.05). The striatal TH⁺ neurocyte significantly decreased, eosinophilic cells, aggregative α-Syn level and TUNEL-positive neurocyte significantly increased in the striatum of MnCl₂ group compared with control group (P < 0.05). Chromatin condensation, mitochondria tumefaction and autophagosomes were observed in rat striatal neurocytes of MnCl₂ group by TEM. TFEB nuclear translocation and autophagy occurred in the striatum of MnCl₂ group. Further, the depressive behavior, movement and balance ability, spatial learning and memory ability of MnCl₂+ CUR2 group were significantly improved compared with MnCl₂ group (P < 0.05). TH+ neurocyte significantly increased, the eosinophilic cells, aggregative α-Syn level significantly decreased in the striatum of MnCl₂+ CUR2 group compared with MnCl₂ group. Further, compared with MnCl₂ group, chromatin condensation, mitochondria tumefaction was alleviated and autophagosomes increased, TFEB-nuclear translocation, autophagy was enhanced and TUNEL-positive neurocyte reduced significantly in the striatum of MnCl₂+ CUR2 group (P < 0.05). Conclusion: Curcumin alleviated the MnCl₂-induced neurotoxicity and α-Syn aggregation probably by promoting TFEB nuclear translocation and enhancing autophagy.

Key words: Manganism, Curcumin, Transcription factor EB (TFEB), Autophagy

中图分类号:

R135.1

来丽叶,窦长松,智翠娜,陈洁,马雪,赵鹏,姚碧云. 姜黄素干预通过促进自噬改善氯化锰所致的大鼠神经行为损伤[J]. 北京大学学报（医学版）, 2022, 54(3): 400-411.

Li-ye LAI,Chang-song DOU,Cui-na ZHI,Jie CHEN,Xue MA,Peng ZHAO,Bi-yun YAO. Curcumin alleviates the manganese-induced neurotoxicity by promoting autophagy in rat models of manganism[J]. Journal of Peking University (Health Sciences), 2022, 54(3): 400-411.

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Group	Week 1	Week 2	Week 3	Week 4
NC	216.92±12.6	281.38±12.30	347.31±17.74	406.46±21.64
CUR	219.17±9.10	286.61±11.88	354.83±25.39	397.87±25.37
MnCl₂	214.38±12.5	242.94±17.15^*	306.70±22.58^*	353.00±25.16^☆
MnCl₂+ CUR1	222.89±7.40	251.59±21.95	324.60±21.85	365.54±32.04^*
MnCl₂+ CUR2	221.74±9.40	259.04±16.30	323.93±16.30	369.27±22.27

Group	Mn²⁺ content/(ng/g)
NC	430.06±41.63
CUR	434.32±45.87
MnCl₂	842.39±68.05^☆
MnCl₂+ CUR1	781.99±82.86^☆
MnCl₂+ CUR2	817.24±49.14^☆

Test	Indicators	NC	CUR	MnCl₂	MnCl₂+CUR1	MnCl₂+CUR2
OFT	Numbers of rearing	18.25±5.37	17.63±5.85	10.63±2.45^☆	12.00±4.43^*	17.37±4.98^★
	Numbers of entries to the inner zone	5.38±3.42	6.63±3.25	4.00±2.83^*	4.83±1.83	5.13±2.64^#
	Speed in inner zone/ (m/s)	0.18±0.07	0.19±0.05	0.23±0.09	0.18±0.07	0.17±0.03
	Distance moved in the inner zone/m	2.32±0.59	2.22±0.49	1.17±0.25^*	1.44±0.30	2.25±0.38^#
	Time in the inner zone/s	13.16±2.40	13.38±3.04	5.69±1.22^*	8.02±1.49^*	13.26±2.10^#
Rotarod	Time on rotarod/s	98.86±19.5	99.41±17.98	61.98±23.04^☆	72.79±16.46^*	88.70±19.65^#
	Circles on rotarod	17.21±5.07	16.80±4.68	8.35±4.10^☆	10.63±3.87^*	12.73±5.02^#
	Fall speed/(m/s)	15.76±2.35	15.72±2.09	11.22±2.58	12.62±1.99	13.80±2.34

Items	NC	CUR	MnCl₂	MnCl₂+ CUR1	MnCl₂+ CUR2
Number of entries to the island	5.13±0.64	4.33±1.60	3.38±1.30^*	4.83±1.33	5.13±0.84^#
Number of entries to the target quadrant	10.13±0.84	9.75±1.03	9.13±1.81	10.67±1.21	10.50±2.51
Time in the target quadrant/s	41.24±5.56	42.50±5.59	34.75±6.17^*	33.98±2.92^*	42.84±6.65^#
Distance in the target quadrant/m	9.06±0.91	9.31±1.62	7.63±1.44^*	8.69±0.52	9.54±1.42^#
Target quadrant time/%	35.50±5.0	35.70±4.6	29.30±5.1^☆	28.20±2.5^☆	35.80±5.6^★
Target quadrant distance/%	29.10±2.1	30.40±3.5	26.30±4.4^*	27.40±2.4	29.40±2.7^#
Average speed/(m/s)	0.25±0.01	0.22±0.02	0.24±0.04	0.27±0.03	0.26±0.06
Total distance travelled/m	29.79±1.70	30.60±2.79	29.10±2.95	32.51±3.98	32.51±4.95

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