清热利胆解毒方通过调控线粒体自噬改善高铜负荷大鼠的认知功能障碍

doi:10.12122/j.issn.1673-4254.2025.11.16

摘要/Abstract

摘要：

目的探讨清热利胆解毒方改善高铜负荷大鼠认知功能障碍的可能机制。方法将75只SD大鼠（雄）随机分为空白组（NG）、模型组（MG）、清热利胆解毒方组（TCM）、青霉胺组（PCA）和清热利胆解毒方加青霉胺组（TCM+PCA），15只/组。巴恩斯迷宫实验和避暗实验测试大鼠学习记忆能力，Western blotting和免疫荧光检测海马区NIX、FUNDC1、LC3的表达，用透射电镜观察海马区线粒体形态和结构。结果行为学测试结果显示，MG组较NG组大鼠目标洞潜伏期明显升高（P<0.05），进入暗室的潜伏期缩短（P<0.01），错误次数增加（P<0.01）；TCM+PCA组较MG组第4天、测试期目标洞潜伏期时间明显缩短（P<0.05），进入暗室的潜伏期延长（P<0.01），错误次数减少（P<0.01），TCM+PCA组较TCM组、PCA组错误次数减少（P<0.01）。Western blotting结果显示，NG组及治疗各组较MG组大鼠海马区NIX、FUNDC1的表达水平增高（P<0.01），LC3Ⅰ、LC3Ⅱ的表达水平明显减少（P<0.05）；TCM+PCA组较TCM组、PCA组NIX、FUNDC1的表达水平明显增高（P<0.05），LC3Ⅰ的表达水平明显减少（P<0.01）。免疫荧光检测结果显示MG组较NG组、TCM+PCA组大鼠海马区NIX、FUNDC1蛋白总荧光强度减弱（P<0.01），LC3蛋白总荧光强度增强（P<0.01）；TCM组较PCA组各蛋白总荧光强度差异列统计学意义（P>0.05）。透射电镜可见NG组细胞内线粒体大多形态完整，MG组线粒体结构紊乱且边缘模糊，治疗各组线粒体形态结构均有不同程度改善。结论清热利胆解毒方可改善高铜负荷WD模型大鼠认知功能障碍，调控线粒体自噬可能是其作用机制之一。

关键词: 清热利胆解毒方, 肝豆状核变性, 认知障碍, NIX, FUNDC1

Abstract:

Objective To explore the mechanisms of Qingre Lidan Jiedu Recipe (QLJR) for improving cognitive dysfunction in rats with high copper load. Methods Seventy-five male SD rats were randomized into normal control group, model group, QLJR group, penicillamine (PCA) group, and QLJR+ PCA group. Except for those in the control group, all the rats were fed a high-copper diet for 12 weeks. The effects of the treatments on cognitive function of the rats were assessed using the Barnes maze and passive avoidance tests. Hippocampal expressions of NIX, FUNDC1 and LC3 of the rats were detected using Western blotting and immunofluorescence staining, and changes in mitochondrial morphology were observed with transmission electron microscopy. Results Behavioral tests showed prolonged target hole latency, shortened latency to enter the dark chamber, and increased error counts of the rats in the model group, which were significantly improved in QLJR+PCA group; the error counts were significantly lower in QLJR+PCA group than in either QLJR or PCA group. Among all the groups, the hippocampal expressions of NIX and FUNDC1 were the lowest and LC3 I/II expression the highest in the model group; NIX and FUNDC1 expressions were significantly higher and LC3 I expression was lower in QLJR+PCA group than in QLJR group and PCA group. Immunofluorescence staining revealed weakened NIX and FUNDC1 expressions and enhanced LC3 expression in the hippocampus of the rats in the model group as compared with those in the normal control and QLJR+PCA groups, but their expressions did not differ significantly between QLJR and PCA groups. The rats in the model group showed obvious structural disarray of the mitochondria, which were improved in all the treatment groups. Conclusion QLJR improves cognitive dysfunction in rats with high copper load possibly by regulating mitophagy.

Key words: Qingre Lidan Jiedu Recipe, Wilson's disease, cognitive impairment, NIX, FUNDC1

王钰岚, 方向, 陈泽铭, 阮炳坤, 韩欣礼, 唐雨婕, 朱璐瑶. 清热利胆解毒方通过调控线粒体自噬改善高铜负荷大鼠的认知功能障碍[J]. 南方医科大学学报, 2025, 45(11): 2437-2443.

Yulan WANG, Xiang FANG, Zeming CHEN, Bingkun RUAN, Xinli HAN, Yujie TANG, Luyao ZHU. Qingre Lidan Jiedu Recipe improves high copper load-induced cognitive dysfunction in rats by regulating mitophagy[J]. Journal of Southern Medical University, 2025, 45(11): 2437-2443.

图/表 8

表1 各组大鼠巴恩斯迷宫实验训练期的目标洞潜伏期时间比较

Tab.1 Comparison of the target hole latency time in the Barnes maze experiment during the training period among the groups (Mean±SD, n=8)

Group	1 day	2 days	3 days	4 days
NG	203.12±26.11	157.55±30.61	79.14±17.49	50.01±15.38
MG	230.58±21.85¹⁾	204.83±38.17²⁾	142.24±15.02²⁾	134.99±32.44²⁾
TCM	215.44±16.70	190.55±29.41	124.26±28.70²⁾	117.23±14.60²⁾
PCA	212.14±16.81	187.89±25.33	125.87±30.75²⁾	120.49±15.22²⁾
TCM+PCA	209.21±10.49	186.68±19.86	117.89±26.67¹⁾	106.59±12.45²⁾³⁾

表 2 各组大鼠巴恩斯迷宫实验测试期的目标洞潜伏期时间比较

Tab.2 Comparison of target hole latency time during the test period of the Barnes maze experiment among the groups (Mean±SD, n=8)

Group	5 days (Short-term memory)	12 days (Long-term memory)
NG	16.02±5.54	17.88±10.21
MG	49.08±13.54²⁾	50.32±23.64²⁾
TCM	32.99±10.91¹⁾³⁾	31.26±19.93⁴⁾
PCA	35.51±11.99²⁾	33.11±20.10¹⁾⁴⁾
TCM+PCA	29.21±10.25⁴⁾	28.55±18.92⁴⁾

表3 各组大鼠避暗实验潜伏期及错误次数比较

Tab.3 Comparison of latency and error counts in dark avoidance test among the groups (Mean±SD, n=8)

Group	Latency (s)	Error count
NG	189.02±58.94	1.75±1.40
MG	64.07±40.38²⁾	7.75±1.04²⁾
TCM	155.22±40.82⁴⁾	4.75±1.28²⁾⁴⁾⁶⁾
PCA	152.01±48.44⁴⁾	5.00±1.69²⁾⁴⁾⁶⁾
TCM+PCA	179.73±38.11⁴⁾	2.13±1.96⁴⁾

表4 各组大鼠海马区NIX、FUNDC1、LC3I、LC3Ⅱ蛋白的相对表达量对比

Tab.4 Comparison of relative expression levels of NIX, FUNDC1, LC3I, and LC3II proteins in the hippocampus among the groups (Mean±SD, n=3)

Group	NIX	FUNDC1	LC3I	LC3Ⅱ
NG	0.99±0.12	1.07±0.15	0.32±0.05	0.36±0.08
MG	0.28±0.05²⁾	0.26±0.10²⁾	0.91±0.05²⁾	0.95±0.05²⁾
TCM	0.62±0.06²⁾⁴⁾⁵⁾	0.59±0.05²⁾⁴⁾⁵⁾	0.67±0.06²⁾⁴⁾⁶⁾	0.66±0.13²⁾⁴⁾
PCA	0.58±0.04²⁾⁴⁾⁵⁾	0.59±0.03²⁾⁴⁾⁵⁾	0.66±0.11²⁾³⁾⁶⁾	0.65±0.10²⁾⁴⁾
TCM+PCA	0.81±0.08⁴⁾	0.80±0.07¹⁾⁴⁾	0.44±0.04⁴⁾	0.47±0.05⁴⁾

图 1 清热利胆解毒方对各组大鼠海马区NIX、FUNDC1、LC3I、LC3Ⅱ蛋白表达的影响

Fig.1 Effect of Qingre Lidan Jiedu Recipe on protein expression of NIX, FUNDC1, LC3I, and LC3II in the hippocampus of the rats in each group. A: NG group; B: MG group; C: TCM group; D: PCA group; E: TCM+PCA group.

图2 清热利胆解毒方对各组大鼠海马区NIX、FUNDC1、LC3荧光强度的影响

Fig.2 Effects of Qingre Lidan Jiedu Recipe on the fluorescence intensities of NIX, FUNDC1 and LC3 in the hippocampus of the rats in each group (scale bar=20 μm). A: NG group; B: MG group; C: TCM group; D: PCA group; E: TCM+PCA group.

表5 各组大鼠海马区NIX、FUNDC1、LC3的总荧光强度对比

Tab.5 Total fluorescence intensities of NIX, FUNDC1, and LC3 in the hippocampus of the rats in each group (Mean±SD, n=3)

Group	NIX(×10⁷)	FUNDC1 (×10⁷⁾	LC3(×10⁷⁾
NG	16.35±0.24	14.44±1.79	1.80±0.42
MG	3.24±0.78²⁾	1.17±0.12²⁾	19.42±3.14²⁾
TCM	4.27±1.11²⁾⁶⁾	3.32±0.39²⁾⁵⁾	8.14±1.85²⁾⁴⁾⁶⁾
PCA	4.75±1.41²⁾⁶⁾	3.27±1.68²⁾⁵⁾	5.20±0.95⁴⁾
TCM+PCA	8.34±0.67²⁾⁴⁾	6.26±0.62⁴⁾	1.37±0.11⁴⁾

图 3 清热利胆解毒方对各组大鼠海马区线粒体形态影响

Fig.3 Effect of Qingre Lidan Jiedu Recipe on mitochondrial morphology in the hippocampus of the rats in each group (scale bar=2 μm/500 nm).

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