目的 基于Kelch樣環(huán)氧氯丙烷相關(guān)蛋白1（Keap1）/核因子E2相關(guān)因子2（Nrf2）/抗氧化反應(yīng)元件（ARE）信號通路，探討同仁牛黃清心丸治療輕度認(rèn)知障礙（MCI）大鼠的作用及潛在機(jī)制。方法 采用D-半乳糖聯(lián)合亞硝酸鈉（NaNO₂）ip構(gòu)建MCI大鼠模型。將SD大鼠隨機(jī)分為對照組、模型組、吡拉西坦組（陽性藥，0.48 g·kg^-1）及同仁牛黃清心丸高、中、低劑量組（2.4、1.2、0.6 g·kg^-1），每組10只。除對照組ip等量0.9%氯化鈉溶液外，其余各組均每日ip D-半乳糖120 mg·kg^-1與NaNO₂ 90 mg·kg^-1，連續(xù)40 d完成造模。自造模第11天起，各給藥組ig給予對應(yīng)藥物，連續(xù)干預(yù)30 d；對照組與模型組則ig等體積純化水。藥物干預(yù)結(jié)束后，通過Morris水迷宮檢測大鼠認(rèn)知能力；蘇木素-伊紅（HE）染色觀察大腦海馬組織病理變化；免疫組化法檢測腦組織中β-淀粉樣蛋白（Aβ）表達(dá)； ELISA法檢測腦脊液中神經(jīng)絲蛋白（AD7c-NTP）及腦組織中乙酰膽堿酯酶（AChE）、乙酰膽堿轉(zhuǎn)移酶（ChAT）、丙二醛（MDA）水平； WST-1法檢測腦組織中超氧化物歧化酶（SOD）活性； Western blotting法檢測腦組織中Keap1、Nrf2、NADPH氧化還原酶1（NQO1）、血紅素加氧酶1（HO-1）、過氧化物酶6（PRDX6）和谷胱甘肽過氧化物酶1（GSH-Px1）蛋白表達(dá)。結(jié)果 與模型組相比，同仁牛黃清心丸各劑量組大鼠在測試象限的游泳路徑長度百分比顯著提高（P＜0.05、0.01），低劑量組測試象限游泳時間百分比顯著提高（P＜0.01）；大腦皮質(zhì)及海馬區(qū)神經(jīng)元減少、變性壞死等病理損傷明顯改善；腦脊液中AD7c-NTP水平顯著降低（P＜0.05、0.01），腦組織中Aβ表達(dá)顯著降低（P＜0.01）；腦組織中ChAT含量顯著升高、AChE含量顯著降低（P＜0.01），SOD活性顯著升高、MDA含量顯著降低（P＜0.01）；中、低劑量組腦組織中Keap1蛋白表達(dá)顯著降低（P＜0.05），高、中劑量組Nrf2蛋白表達(dá)顯著升高（P＜0.01）；各劑量組NQO1、HO-1、GSH-Px1蛋白表達(dá)顯著升高（P＜0.05、0.01），中、低劑量組PRDX6蛋白表達(dá)顯著升高（P＜0.05、0.01）。結(jié)論 同仁牛黃清心丸可不同程度改善MCI大鼠認(rèn)知功能，發(fā)揮神經(jīng)保護(hù)作用，其核心機(jī)制可能在于調(diào)控Keap1/Nrf2/ARE信號通路活化，抑制氧化應(yīng)激損傷。

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

北京市中醫(yī)藥科技發(fā)展資金資助項目（ BJZYYB-2023-51）