瑞马唑仑通过AMPK/PGC-1α/SIRT3改善老年小鼠胫骨骨折术后神经认知障碍的研究

doi:10.3877/cma.j.issn.2096-0263.2026.03.002

目的

基于AMPK/PGC-1α/SIRT3信号通路，探究瑞马唑仑改善老年小鼠胫骨骨折术后神经认知障碍的效果及作用机制。

方法

将老年小鼠随机分为对照组、模型组、瑞马唑仑组、瑞马唑仑+AMPK抑制剂Compound C组，每组8只。Morris水迷宫检测各组小鼠的学习记忆能力，HE染色检测海马组织的病理学损伤，Western blot检测海马组织中PINK1、Parkin、p62、Beclin-1、LC3Ⅱ/Ⅰ、p-AMPK/AMPK、PGC-1α、SIRT3蛋白的表达，试剂盒检测海马组织中丙二醛（MDA）、超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）水平。

结果

对于逃避潜伏期，对照组、模型组、瑞马唑仑组、瑞马唑仑+AMPK抑制剂Compound C组小鼠末次给药第1~5 d进行重复测量设计的方差分析显示各组间差异有统计学意义，组别与时间点间存在交互作用，各时间点除第一时间点外两组差别均有统计学意义，对照组、模型组、瑞马唑仑组、瑞马唑仑+AMPK抑制剂Compound C组在各时间点间差异均有统计学意义。相对于对照组，模型组小鼠穿越原平台的次数减少、在目标象限停留的时间缩短（P<0.05），海马组织呈现明显的病理学损伤，海马组织中PINK1、Parkin、Beclin-1、LC3Ⅱ/Ⅰ、p-AMPK/AMPK、PGC-1α、SIRT3、SOD、GSH-Px降低（P<0.05），p62蛋白、MDA的表达水平升高（P<0.05）；相对于模型组，瑞马唑仑组小鼠穿越原平台的次数、在目标象限停留的时间延长（P<0.05），海马组织的病理学损伤减轻，海马组织中PINK1、Parkin、Beclin-1、LC3Ⅱ/Ⅰ、p-AMPK/AMPK、PGC-1α、SIRT3、SOD、GSH-Px升高（P<0.05），p62、MDA蛋白的表达水平降低（P<0.05）；Compound C能够一定程度上逆转瑞马唑仑的作用（P<0.05）。

结论

瑞马唑仑能够通过促进线粒体自噬、抑制氧化应激来减轻海马组织的病理学损伤，从而改善骨折术后小鼠的认知障碍，其机制可能与激活AMPK/PGC-1α/SIRT3信号通路相关。

关键词: 瑞马唑仑, 胫骨骨折, 术后认知功能障碍, AMPK/PGC-1α/SIRT3

Objective

Based on the AMPK/PGC-1α/SIRT3 signaling pathway, the effect and mechanism of remimazolam in improving neurocognitive impairment after tibial fracture in elderly mice was investigated.

Methods

The elderly mice were randomly divided into control group, model group, remimazolam group, remimazolam + AMPK inhibitor Compound C group, with 8 mice in each group. The Morris water maze was used to detect the learning and memory ability of mice in each group. HE staining was used to detect pathological damage in hippocampal tissue. The expression of PINK1, Parkin, p62, Beclin-1, LC3Ⅱ/Ⅰ, p-AMPK/AMPK, PGC-1α, SIRT3 proteins in hippocampal tissue were detected by Western blot. The levels of malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in hippocampal tissues were detected by kits.

Results

For the escape latency period, the analysis of variance of the control group, model group, remimazolam group, and remimazolam + AMPK inhibitor Compound C group on the 1st-5th day of the last dose showed that the differences between the groups were statistically significant, and there was an interaction between groups and time points, and except for the first time point, there were statistically significant differences between the two groups, and there were significant differences between the control group, the model group, the remimazolam group, and the remimazolam + AMPK inhibitor Compound C group at all time points. Compared with the control group, the traversed original platform numbers and dwell time in the target quadrant shortened (P<0.05), and hippocampal tissue presented significant pathological damage, and the PINK1, Parkin, Beclin-1, LC3Ⅱ/Ⅰ, p-AMPK/AMPK, PGC-1α, SIRT3, SOD, GSH-Px in hippocampal tissue decreased whereas p62, MDA increased (P<0.05). Compared with the model group, the traversed original platform numbers and dwell time in the target quadrant prolonged (P<0.05), and pathological damage in hippocampal tissue alleviated, and the PINK1, Parkin, Beclin-1, LC3Ⅱ/Ⅰ, p-AMPK/AMPK, PGC-1α, SIRT3, SOD, GSH-Px in hippocampal tissue increased whereas p62, MDA decreased (P<0.05). Compound C partly reversed the effect of remimazolam (P<0.05).

Conclusion

Remimazolam alleviates the pathological damage of hippocampal tissue by promoting mitophagy and inhibiting oxidative stress, thereby improving the cognitive impairment of mice after fracture surgery, and its mechanism may be related to the activation of AMPK/PGC-1α/SIRT3 signaling pathway.

Key words: Remimazolam, Tibial fracture, Postoperative cognitive dysfunction, AMPK/PGC-1α/SIRT3

表1 在Morris水迷宫实验1~5 d中，各组小鼠逃避潜伏期的比较（s，

±s）

组别	样本量	逃避潜伏期					F值	P值
组别	样本量	第1 d	第2 d	第3 d	第4 d	第5 d	F值	P值
对照组	8	50.25±2.18	40.00±2.62^a	34.00±2.00^b	23.37±1.69^c	14.38±1.92^d	354.345	<0.001
模型组	8	57.88±1.64*	53.38±2.00*^,a	50.00±1.85*^,b	44.50±1.41*^,c	40.00±2.33*^,d	114.195	<0.001
瑞马唑仑组	8	52.75±1.58^#	46.00±1.85^#,a	38.00±1.60^#,b	30.75±2.19^#,c	22.63±1.69^#,d	353.577	<0.001
瑞马唑仑+AMPK抑制剂组	8	56.87±2.10^&	51.25±1.75^&,a	46.88±1.96^&,b	41.00±1.85^&,c	36.50±1.51^&,d	153.081	<0.001
F值		28.361	65.808	129.336	228.258	321.853
P值		<0.001	<0.001	<0.001	<0.001	<0.001

表1 在Morris水迷宫实验1~5 d中，各组小鼠逃避潜伏期的比较（s，

±s）

组别	样本量	逃避潜伏期					F值	P值
组别	样本量	第1 d	第2 d	第3 d	第4 d	第5 d	F值	P值
对照组	8	50.25±2.18	40.00±2.62^a	34.00±2.00^b	23.37±1.69^c	14.38±1.92^d	354.345	<0.001
模型组	8	57.88±1.64*	53.38±2.00*^,a	50.00±1.85*^,b	44.50±1.41*^,c	40.00±2.33*^,d	114.195	<0.001
瑞马唑仑组	8	52.75±1.58^#	46.00±1.85^#,a	38.00±1.60^#,b	30.75±2.19^#,c	22.63±1.69^#,d	353.577	<0.001
瑞马唑仑+AMPK抑制剂组	8	56.87±2.10^&	51.25±1.75^&,a	46.88±1.96^&,b	41.00±1.85^&,c	36.50±1.51^&,d	153.081	<0.001
F值		28.361	65.808	129.336	228.258	321.853
P值		<0.001	<0.001	<0.001	<0.001	<0.001

表2 各组小鼠穿越原平台次数、目标象限停留时间的比较（

±s）

组别	样本量	穿越原平台次数（次）	目标象限停留时间（s）
对照组	8	8.25±0.88	36.00±1.41
模型组	8	2.12±0.64*	16.25±1.83*
瑞马唑仑组	8	5.37±1.18^#	27.50±2.13^#
瑞马唑仑+AMPK抑制剂组	8	3.37±0.91^&	20.87±1.88^&
F值		67.214	175.631
P值		<0.001	<0.001

表2 各组小鼠穿越原平台次数、目标象限停留时间的比较（

±s）

组别	样本量	穿越原平台次数（次）	目标象限停留时间（s）
对照组	8	8.25±0.88	36.00±1.41
模型组	8	2.12±0.64*	16.25±1.83*
瑞马唑仑组	8	5.37±1.18^#	27.50±2.13^#
瑞马唑仑+AMPK抑制剂组	8	3.37±0.91^&	20.87±1.88^&
F值		67.214	175.631
P值		<0.001	<0.001

图1 HE染色检测各组小鼠海马组织的病理学损伤（400×，20 μm）

图2 Western blot检测各组小鼠海马组织中PINK1、Parkin、p62、Beclin-1、LC3Ⅱ/Ⅰ蛋白的表达注：A-对照组；B-模型组；C-瑞马唑仑组；D-瑞马唑仑+AMPK抑制剂组

表3 各组小鼠海马组织中PINK1、Parkin、p62、Beclin-1、LC3Ⅱ/Ⅰ蛋白表达的比较（

±s）

组别	例数	PINK1	Parkin	p62	Beclin-1	LC3Ⅱ/Ⅰ
对照组	8	0.73±0.06	0.84±0.07	0.23±0.02	0.82±0.08	0.89±0.09
模型组	8	0.21±0.02*	0.25±0.04*	0.96±0.08*	0.18±0.03*	0.17±0.02*
瑞马唑仑组	8	0.62±0.07^#	0.70±0.06^#	0.47±0.06^#	0.51±0.06^#	0.46±0.04^#
瑞马唑仑+AMPK抑制剂组	8	0.39±0.05^&	0.49±0.04^&	0.68±0.07^&	0.27±0.04^&	0.24±0.04^&
F值		152.398	181.060	201.412	209.664	287.909
P值		<0.001	<0.001	<0.001	<0.001	<0.001

表3 各组小鼠海马组织中PINK1、Parkin、p62、Beclin-1、LC3Ⅱ/Ⅰ蛋白表达的比较（

±s）

组别	例数	PINK1	Parkin	p62	Beclin-1	LC3Ⅱ/Ⅰ
对照组	8	0.73±0.06	0.84±0.07	0.23±0.02	0.82±0.08	0.89±0.09
模型组	8	0.21±0.02*	0.25±0.04*	0.96±0.08*	0.18±0.03*	0.17±0.02*
瑞马唑仑组	8	0.62±0.07^#	0.70±0.06^#	0.47±0.06^#	0.51±0.06^#	0.46±0.04^#
瑞马唑仑+AMPK抑制剂组	8	0.39±0.05^&	0.49±0.04^&	0.68±0.07^&	0.27±0.04^&	0.24±0.04^&
F值		152.398	181.060	201.412	209.664	287.909
P值		<0.001	<0.001	<0.001	<0.001	<0.001

表4 各组小鼠海马组织中MDA、SOD、GSH-Px水平的比较（

±s）

组别	例数	MDA（nmol/mg）	SOD（U/mg）	GSH-Px（U/mg）
对照组	8	8.74±0.51	186.20±9.45	6042.62±44.37
模型组	8	19.31±0.94*	107.56±11.37*	4357.07±52.61*
瑞马唑仑组	8	10.59±1.28^#	168.23±12.53^#	5744.31±69.79^#
瑞马唑仑+AMPK抑制剂组	8	17.65±1.37^&	127.66±10.63^&	4652.60±40.63^&
F值		184.974	85.499	1910.550
P值		<0.001	<0.001	<0.001

表4 各组小鼠海马组织中MDA、SOD、GSH-Px水平的比较（

±s）

组别	例数	MDA（nmol/mg）	SOD（U/mg）	GSH-Px（U/mg）
对照组	8	8.74±0.51	186.20±9.45	6042.62±44.37
模型组	8	19.31±0.94*	107.56±11.37*	4357.07±52.61*
瑞马唑仑组	8	10.59±1.28^#	168.23±12.53^#	5744.31±69.79^#
瑞马唑仑+AMPK抑制剂组	8	17.65±1.37^&	127.66±10.63^&	4652.60±40.63^&
F值		184.974	85.499	1910.550
P值		<0.001	<0.001	<0.001

图3 Western blot检测各组小鼠海马组织中p-AMPK/AMPK、PGC-1α、SIRT3蛋白的表达注：A-对照组；B-模型组；C-瑞马唑仑组；D-瑞马唑仑+AMPK抑制剂组

表5 各组小鼠海马组织中p-AMPK/AMPK、PGC-1α、SIRT3蛋白表达的比较（

±s）

组别	例数	p-AMPK/AMPK	PGC-1α	SIRT3
对照组	8	0.91±0.08	0.87±0.09	0.93±0.08
模型组	8	0.13±0.02*	0.16±0.02*	0.14±0.02*
瑞马唑仑组	8	0.53±0.06^#	0.64±0.07^#	0.54±0.06^#
瑞马唑仑+AMPK抑制剂组	8	0.27±0.04^&	0.37±0.05^&	0.29±0.04^&
F值		313.244	193.610	317.956
P值		<0.001	<0.001	<0.001

表5 各组小鼠海马组织中p-AMPK/AMPK、PGC-1α、SIRT3蛋白表达的比较（

±s）

组别	例数	p-AMPK/AMPK	PGC-1α	SIRT3
对照组	8	0.91±0.08	0.87±0.09	0.93±0.08
模型组	8	0.13±0.02*	0.16±0.02*	0.14±0.02*
瑞马唑仑组	8	0.53±0.06^#	0.64±0.07^#	0.54±0.06^#
瑞马唑仑+AMPK抑制剂组	8	0.27±0.04^&	0.37±0.05^&	0.29±0.04^&
F值		313.244	193.610	317.956
P值		<0.001	<0.001	<0.001

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Remimazolam ameliorates neurocognitive impairment after tibial fracture in elderly mice by AMPK/PGC-1α/SIRT3

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Remimazolam ameliorates neurocognitive impairment after tibial fracture in elderly mice by AMPK/PGC-1α/SIRT3