Abstract: BACKGROUND: Wilson’s disease not only affects liver function but can also cause oxidative damage to neurons in the brain through copper accumulation. Studies have found that the glucose-regulated protein 78/inositol-requiring enzyme 1α/tumor necrosis factor receptor-associated factor 2/c-Jun N-terminal kinase signaling pathway plays a key role in the neuropathological process of Wilson’s disease. Huangpu Tongqiao Capsule may reduce neuronal damage caused by copper overload and improve learning and memory function by scavenging oxygen free radicals, inhibiting neuroinflammation, and regulating endoplasmic reticulum stress-related pathways.
OBJECTIVE: To further validate the effect of Huangpu Tongqiao Capsule on cognitive function in a rat model of Wilson’s disease and to explore its mechanism of action.
METHODS: Forty SPF-grade male Sprague-Dawley rats were randomly divided into a normal group, a model group, a Huangpu Tongqiao Capsule group, and a positive control group, with 10 rats in each group. Rats in the latter three groups were fed a copper sulfate-loaded diet to establish a Wilson’s disease model. The normal and model groups received intragastric administration of normal saline. The Huangpu Tongqiao Capsule group received intragastric administration of 1.41 g/kg Huangpu Tongqiao Capsule decoction. The positive control group received intragastric administration of 0.09 g/kg penicillamine solution. All administrations were given continuously for 28 days. After administration, 24-hour urinary copper levels were measured. The Morris water maze test was used to evaluate learning and memory function. Hematoxylin-eosin staining was used to observe the structural integrity of neurons in the hippocampus. TUNEL staining was used to observe the number of positive neurons in the hippocampus. ELISA was used to detect the levels of superoxide dismutase and malondialdehyde in the rat hippocampus. Western blot was used to detect the expression of proteins related to the endoplasmic reticulum stress-associated glucose-regulated protein 78/inositol-requiring enzyme 1α/tumor necrosis factor receptor-associated factor 2/c-Jun N-terminal kinase signaling pathway in the hippocampus.
RESULTS AND CONCLUSION: (1) Compared with the normal group, 24-hour urinary copper levels were increased in the model group (P < 0.05). Compared with the model group, 24-hour urinary copper levels were further increased in the Huangpu Tongqiao Capsule group and the positive control group (P < 0.05). (2) Compared with the normal group, rats in the model group showed increased escape latency and decreased number of crossings over the original platform (P < 
0.05). Compared with the model group, rats in the Huangpu Tongqiao Capsule group and the positive control group showed shortened escape latency and increased number of crossings over the original platform (P < 0.05). (3) Compared with the normal group, the hippocampal tissue of rats in the model group showed disrupted neural structure, disorganized cell arrangement, and a significant increase in the number of TUNEL-positive apoptotic neurons. Compared with the model group, the structural integrity of hippocampal neurons in the Huangpu Tongqiao Capsule group and the positive control group was significantly improved, cell arrangement became more orderly, and the number of TUNEL-positive apoptotic neurons was also significantly reduced. (4) Compared with the normal group, the protein expression of glucose-regulated protein 78, inositol-requiring enzyme 1α, tumor necrosis factor receptor-associated factor 2, phosphorylated c-Jun N-terminal kinase, caspase-12, and Bax in hippocampal tissue was increased in the model group, while Bcl-2 protein expression in hippocampal tissue was decreased (P < 0.05). Compared with the model group, the Huangpu Tongqiao Capsule group and the positive control group showed opposite changes in the above indicators (P < 0.05). In conclusion, Huangpu Tongqiao Capsule can improve spatial learning and memory ability, reduce neuronal damage in the hippocampal tissue, and exert a neuroprotective effect in the rat model of Wilson’s disease. Its mechanism of action may be related to inhibiting the glucose-regulated protein 78/inositol-requiring enzyme 1α/tumor necrosis factor receptor-associated factor 2/c-Jun N-terminal kinase signaling pathway, thereby alleviating endoplasmic reticulum stress.

Key words: Huangpu Tongqiao Capsule, Wilson’s disease, cognitive function, endoplasmic reticulum stress, signaling pathway