目的 基于Kelch样环氧氯丙烷相关蛋白1（Keap1）/核因子E2相关因子2（Nrf2）/抗氧化反应元件（ARE）信号通路，探讨同仁牛黄清心丸治疗轻度认知障碍（MCI）大鼠的作用及潜在机制。方法 采用D-半乳糖联合亚硝酸钠（NaNO₂）ip构建MCI大鼠模型。将SD大鼠随机分为对照组、模型组、吡拉西坦组（阳性药，0.48 g·kg^-1）及同仁牛黄清心丸高、中、低剂量组（2.4、1.2、0.6 g·kg^-1），每组10只。除对照组ip等量0.9%氯化钠溶液外，其余各组均每日ip D-半乳糖120 mg·kg^-1与NaNO₂ 90 mg·kg^-1，连续40 d完成造模。自造模第11天起，各给药组ig给予对应药物，连续干预30 d；对照组与模型组则ig等体积纯化水。药物干预结束后，通过Morris水迷宫检测大鼠认知能力；苏木素-伊红（HE）染色观察大脑海马组织病理变化；免疫组化法检测脑组织中β-淀粉样蛋白（Aβ）表达； ELISA法检测脑脊液中神经丝蛋白（AD7c-NTP）及脑组织中乙酰胆碱酯酶（AChE）、乙酰胆碱转移酶（ChAT）、丙二醛（MDA）水平； WST-1法检测脑组织中超氧化物歧化酶（SOD）活性； Western blotting法检测脑组织中Keap1、Nrf2、NADPH氧化还原酶1（NQO1）、血红素加氧酶1（HO-1）、过氧化物酶6（PRDX6）和谷胱甘肽过氧化物酶1（GSH-Px1）蛋白表达。结果 与模型组相比，同仁牛黄清心丸各剂量组大鼠在测试象限的游泳路径长度百分比显著提高（P＜0.05、0.01），低剂量组测试象限游泳时间百分比显著提高（P＜0.01）；大脑皮质及海马区神经元减少、变性坏死等病理损伤明显改善；脑脊液中AD7c-NTP水平显著降低（P＜0.05、0.01），脑组织中Aβ表达显著降低（P＜0.01）；脑组织中ChAT含量显著升高、AChE含量显著降低（P＜0.01），SOD活性显著升高、MDA含量显著降低（P＜0.01）；中、低剂量组脑组织中Keap1蛋白表达显著降低（P＜0.05），高、中剂量组Nrf2蛋白表达显著升高（P＜0.01）；各剂量组NQO1、HO-1、GSH-Px1蛋白表达显著升高（P＜0.05、0.01），中、低剂量组PRDX6蛋白表达显著升高（P＜0.05、0.01）。结论 同仁牛黄清心丸可不同程度改善MCI大鼠认知功能，发挥神经保护作用，其核心机制可能在于调控Keap1/Nrf2/ARE信号通路活化，抑制氧化应激损伤。

Objective To Explore the mechanism of Tongren Niuhuang Qingxin Pills in treating mild cognitive impairment based on the the Kelch-like epoxychloropropane-related protein 1 (Keap1)/nuclear factor E2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway. Methods The MCI rat model was established by ip injection of D-galactose combined with sodium nitrite (NaNO?). SD rats were randomly divided into the control group, the model group, the piracetam group (positive drug, 0.48 g·kg^-1), and the high-, medium-, and low-dose groups of Tongren Niuhuang Qingxin Pills (2.4, 1.2, and 0.6 g·kg^-1), with 10 rats in each group. Except for the control group which was intraperitoneally injected with an equal volume of 0.9% sodium chloride solution, the other groups were intraperitoneally injected with 120 mg·kg^-1 D-galactose and 90 mg·kg^-1 NaNO? daily for 40 days to complete the modeling. From the 11th day of modeling, the drug administration groups were ig administered the corresponding drugs for 30 consecutive days; the control group and the model group were ig administered the same volume of purified water. After the drug intervention, the cognitive ability of the rats was detected by the Morris water maze; the pathological changes of the hippocampus were observed by hematoxylin-eosin (HE) staining; the expression of β-amyloid protein (Aβ) in brain tissue was detected by immunohistochemistry; the levels of neurofilament protein (AD7c-NTP) in cerebrospinal fluid and acetylcholinesterase (AChE), choline acetyltransferase (ChAT), and malondialdehyde (MDA) in brain tissue were detected by ELISA; the activity of superoxide dismutase (SOD) in brain tissue was detected by the WST-1 method; and the protein expressions of Keap1, Nrf2, NADPH oxidoreductase 1(NQO1), heme oxygenase 1(HO-1), peroxiredoxin 6 (PRDX6), and glutathione peroxidase 1 (GSH-Px1) in brain tissue were detected by western blotting. Results Compared with the model group, the swimming path length percentage of rats in each dose group of Tongren Niuhuang Qingxin Pills in the test quadrant was significantly increased (P<0.05, 0.01), and the swimming time percentage in the test quadrant of the low-dose group was significantly increased (P<0.01); the pathological damages such as reduction and degeneration of neurons in the cerebral cortex and hippocampus were significantly improved; the level of AD7c-NTP in cerebrospinal fluid was significantly decreased (P<0.05, 0.01), and the expression of Aβ in brain tissue was significantly reduced (P<0.01); the content of ChAT in brain tissue was significantly increased, and the content of AChE was significantly decreased (P<0.01), the activity of SOD was significantly increased, and the content of MDA was significantly decreased (P<0.01); the expression of Keap1 protein in brain tissue of the medium and low-dose groups was significantly decreased (P<0.05), and the expression of Nrf2 protein in the high and medium-dose groups was significantly increased (P<0.01); the expression of NQO1, HO-1, and GSH-Px1 proteins was significantly increased (P<0.05, 0.01) in each dose group, and the expression of PRDX6 protein was significantly increased (P<0.05, 0.01) in the medium and low-dose groups. Conclusion Tongren Niuhuang Qingxin Pills can improve the cognitive function of MCI rats to varying degrees and exert neuroprotective effects. The underlying mechanism may lie in regulating the activation of the Keap1/Nrf2/ARE signaling pathway, inhibiting oxidative stress damage.

R285.5

北京市中医药科技发展资金资助项目（ BJZYYB-2023-51）