Objective: To investigate the neuroprotective effect of Huangpu Tongqiao Capsule (HPTQ) in a rat model of Wilson disease (WD) and explore the underlying mechanisms.
Methods: SD rat models of WD were established by feeding of coppersupplemented chow diet and drinking water for 12 weeks, and starting from the 9th week, the rats were treated with low-, moderate- and high-dose HPTQ, penicillamine, or normal saline by gavage on a daily basis for 3 weeks. Copper levels in the liver and 24-h urine of the rats were detected, and their learning and memory abilities were evaluated using Morris water maze test. HE staining was used to observe morphological changes of CA1 region neurons in the hippocampus, and neuronal apoptosis was detected with TUNEL staining. Hippocampal expressions of endoplasmic reticulum stress (ERS)-mediated apoptosis pathway-related proteins GRP78, CHOP, caspase-12, cleaved caspase-9, and cleaved caspase-3 at both the mRNA and protein levels were detected using RT-qPCR, immunofluorescence assay or Western blotting.
Results: Compared with normal control rats, the rat models with copper overload-induced WD exhibited significantly increased copper levels in both the liver and 24-h urine, impaired learning and memory abilities, obvious hippocampal neuronal damage in the CA1 region and increased TUNEL-positive neurons (P<0.01), with also lowered mRNA and protein expressions of GRP78, CHOP, caspase-12, cleaved caspase-9, and cleaved caspase-3 in the hippocampus (all P<0.01). Treatments with HPTQ and penicillamine significantly lowered copper level in the liver but increased urinary copper level, improved learning and memory ability, alleviated neuronal damage and apoptosis in the hippocampus, and decreased hippocampal expressions of GRP78, CHOP, caspase-12, cleaved caspase-9, and cleaved caspase-3 in the rat models (P<0.01 or 0.05).
Conclusion: HPTQ Capsule has neuroprotective effects in rat models of WD possibly by inhibiting ERS-mediated apoptosis pathway.
目的: 探究黄蒲通窍胶囊（HPTQ）对Wilson病（WD）铜负荷大鼠模型的神经保护作用及其可能的作用机制。
方法: SD大鼠随机分为6组：对照组、模型组、黄蒲通窍胶囊低剂量组（HPTQ-L，0.47 g/kg）、黄蒲通窍胶囊中剂量组（HPTQ-M，1.41 g/kg）、黄蒲通窍胶囊高剂量组（HPTQ-H，4.23 g/kg）、青霉胺组（PCA，0.09 g/kg），10只/组。通过含铜（1 g/kg）饲料、含铜（0.185%）水喂养12周构建WD铜负荷大鼠模型，铜离子（Cu）测定试剂盒检测肝铜、24 h尿铜水平；Morris水迷宫实验评估大鼠学习记忆能力；HE染色法观察海马组织CA1区神经元形态学改变；TUNEL染色观察海马组织CA1区神经元凋亡水平；免疫荧光法检测内质网应激（ERS）标志蛋白GRP78的表达情况；RT-qPCR和Western blot法检测GRP78、CHOP、caspase-12、cleaved caspase-9、cleaved caspase-3基因和蛋白表达水平。
结果: 与对照组比较，模型组肝铜及24 h尿铜水平明显升高（P<0.01）；学习记忆能力明显减弱（P<0.01）；CA1区海马神经元出现损伤；TUNEL阳性神经元数量增多（P<0.01）；GRP78、CHOP、caspase-12、cleaved caspase-9、cleaved caspase-3基因和蛋白表达水平升高（P<0.01）。与模型组比较，HPTQ各组及PCA组肝铜水平降低、24 h尿铜水平升高（P<0.01）；学习记忆能力改善（P<0.01，P<0.05）；海马神经元损伤减轻；TUNEL阳性神经元数量减少（P<0.01）；GRP78、CHOP、caspase-12、cleaved caspase-9、cleaved caspase-3基因和蛋白表达水平降低（P<0.01，P<0.05）。
结论: HPTQ对WD铜负荷大鼠模型具有神经保护作用，其机制可能与抑制ERS介导的凋亡途径有关。
Keywords: Huangpu Tongqiao Capsule; Wilson disease; apoptosis; cognitive impairment; endoplasmic reticulum stress.