瑞马唑仑调节ROS/RAGE/NF-κB信号通路对LPS诱导的小胶质细胞炎症的影响

doi:10.3969/j.issn.1006-5725.2025.02.001

Abstract

Abstract:

Objective To investigate the anti?inflammatory protective effects of remimazolam on microglial cells and elucidates the potential molecular mechanisms underlying these effects. Methods The mouse microglial cell line （BV2） was selected as the research object. The following groups were set up： the control group （complete medium）， the Rema group （200 μg/mL remimazolam）， the model group （1 μg/mL lipopolysaccharide， LPS）， and different?concentration administration groups （1 μg/mL LPS + 50， 100， 200 μg/mL remimazolam）. In the Rema group， cells were treated with 200 μg/mL remimazolam alone for 26 h. In the model group， cells were treated with LPS for 24 h. In the different?concentration administration groups， cells were pre?treated with different concentrations of remimazolam for 2 h， and then treated with LPS for 24 h. The effects of LPS and remimazolam on the morphology of BV2 cells were observed and evaluated using an optical microscope. Cell viability was determined using the CCK?8 assay， while the expression and secretion of inflammatory cytokines were quantified by quantitative real?time PCR and ELISA. Reactive oxygen species （ROS） levels were measured using a fluorescent probe. Additionally， malondialdehyde （MDA） content， superoxide dismutase （SOD） activity， and glutathione peroxidase （GSH） activity were evaluated using respective assay kits. Western blot analysis was conducted to examine the protein expression levels of Bax， Bcl?2， IL?1β， RAGE， NF?κB， p?NF?κB， IκBα， p?IκBα， iNOS， and Arg?1. Immunofluorescence staining was employed to visualize NF?κB nuclear translocation and M1/M2 polarization in the cells. Results Compared to the control group， LPS?treated BV2 cells demonstrated significantly reduced cell viability， elevated expression and secretion of inflammatory cytokines （TNF?α， IL?6， IL?1β）， decreased activities of SOD and GSH， and increased intracellular levels of MDA and ROS. Additionally， RAGE protein levels were upregulated， along with enhanced phosphorylation of IκBα and NF?κB， leading to observable NF?κB nuclear translocation. The expression of the M1 marker iNOS was upregulated， while that of the M2 marker Arg?1 was downregulated. In contrast， in the LPS+Rema group， cell viability was restored， expression and secretion of inflammatory cytokines were attenuated， SOD and GSH activities were improved， and levels of MDA and ROS were reduced compared to the LPS group. Furthermore， RAGE protein expression and phosphorylation levels of IκBα and NF?κB were diminished， inhibiting NF?κB nuclear translocation. The expression of the M1 marker iNOS was downregulated， while that of the M2 marker Arg?1 was upregulated. Conclusion Remimazolam mitigates LPS?induced inflammation by facilitating the transition of microglial cells from the M1 to the M2 phenotype via modulation of the NF?κB pathway and reduction of ROS production.

Key words: neuroinflammation, remimazolam, NF-κB pathway, oxidative stress, microglial polarization

CLC Number:

R364.5

Xingwei WU,Jianying WANG,Chengxiao GUO,Ziyi LIU,Chao SUN,Fei. YU. The effect of remimazolam on modulating the ROS/RAGE/NF-κB signaling pathway in LPS-induced microglial inflammation[J]. The Journal of Practical Medicine, 2025, 41(2): 153-161.

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References 24

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组别	细胞活性
对照组	100.00 ± 1.46
Rema（10 μg/mL）	100.56 ± 1.19
Rema（20 μg/mL）	99.03 ± 1.21
Rema（50 μg/mL）	98.59 ± 1.60
Rema（100 μg/mL）	99.27 ± 1.53
Rema（200 μg/mL）	98.70 ± 0.13
Rema（500 μg/mL）	87.45 ± 2.10^#
F值	31.29
P值	< 0.001

组别	细胞活力
对照组	100 ± 7.71
Rema组	98.97 ± 9.42
模型组	49.63 ± 10.01^#
LPS + Rema（50 μg/mL）组	74.94 ± 5.04^*
LPS + Rema（100 μg/mL）组	85.38 ± 3.79^*
LPS + Rema（200 μg/mL）组	83.63 ± 9.76^*
F值	20.95
P值	< 0.001

组别	TNF-α	IL-6	IL-1β
对照组	1.01 ± 0.01	1.08 ± 0.09	1.01 ± 0.01
Rema组	2.28 ± 0.35	1.21 ± 0.26	0.27 ± 0.06
模型组	50.77 ± 10.81^#	6 578.87 ± 808.00^#	866.69 ± 158.07^#
LPS + Rema（50 μg/mL）组	33.99 ± 7.75^*	4 479.15 ± 920.42^*	601.24 ± 148.29^*
LPS + Rema（100 μg/mL）组	31.30 ± 3.22^*	3 320.10 ± 693.78^*	479.18 ± 87.37^*
LPS + Rema（200 μg/mL）组	26.49 ± 9.56^*	2 129.11 ± 410.63^*	299.96 ± 50.57^*
F值	24.23	37.14	56.05
P值	< 0.001	<0.001	< 0.001

组别	TNF-α	IL-6	IL-1β
对照组	31.33 ± 2.06	17.74 ± 4.84	0.22 ± 0.11
Rema组	32.92 ± 1.72	8.06 ± 5.35	0.16 ± 0.04
模型组	2 335.67 ± 119.6^#	4 046.77 ± 142.68^#	1.25 ± 0.05^#
LPS + Rema（50 μg/mL）组	1 572.65 ± 75.04^*	3 664.52 ± 133.06^*	0.96 ± 0.08^*
LPS + Rema（100 μg/mL）组	1 458.66 ± 33.74^*	3 520.97 ± 127.87^*	0.87 ± 0.12^*
LPS + Rema（200 μg/mL）组	1 113.46 ± 31.19^*	3 372.58 ± 64.86^*	0.73 ± 0.08^*
F值	678.9	1 098	50.68
P值	< 0.001	< 0.001	< 0.001

组别	MDA含量	SOD活性	GSH活性
Control组	1.54 ± 0.29	10.85 ± 0.65	70.67 ± 10.10
Rema组	1.61 ± 0.28	10.46 ± 1.03	69.15 ± 5.57
模型组	5.39 ± 1.00^#	7.36 ± 0.72^#	41.19 ± 3.26^#
LPS + Rema（50 μg/mL）组	2.32 ± 0.57^*	9.15 ± 1.01	59.69 ± 3.30^*
LPS + Rema（100 μg/mL）组	1.66 ± 0.13^*	9.92 ± 1.60^*	70.02 ± 7.88^*
LPS + Rema（200 μg/mL）组	1.51 ± 0.39^*	9.93 ± 0.89^*	81.72 ± 7.67^*
F值	25.17	4.375	12.39
P值	< 0.001	0.008 8	< 0.001

The effect of remimazolam on modulating the ROS/RAGE/NF-κB signaling pathway in LPS-induced microglial inflammation

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组别	Bax	Bcl-2	RAGE	p-IκBα/IκBα	p-NF-κB/NF-κB
Control组	0.86 ± 0.02	0.96 ± 0.12	0.22 ± 0.11	0.68 ± 0.26	0.73 ± 0.21
Rema组	1.03 ± 0.20	1.09 ± 0.04	0.16 ± 0.04	0.97 ± 0.49	0.94 ± 0.21
模型组	1.99 ± 0.38^#	0.45 ± 0.10^#	1.25 ± 0.05^#	2.36 ± 0.32^#	1.50 ± 0.04^#
LPS + Rema（50 μg/mL）组	1.39 ± 0.15^*	0.84 ± 0.04^*	0.96 ± 0.08^*	1.37 ± 0.23^*	0.88 ± 0.25^*
LPS + Rema（100 μg/mL）组	1.12 ± 0.25^*	1.07 ± 0.07^*	0.87 ± 0.12^*	1.18 ± 0.27^*	0.75 ± 0.23^*
LPS + Rema（200 μg/mL）组	0.90 ± 0.36^*	1.21 ± 0.27^*	0.73 ± 0.08^*	0.59 ± 0.39^*	0.74 ± 0.17^*
F值	5.355	8.212	7.353	7.224	4.462
P值	0.008 1	0.001 4	0.002 3	0.002 5	0.015 8

组别	INOS		Arg-1
组别	mRNA	蛋白表达量	mRNA	蛋白表达量
Control组	1.13 ± 0.38^*	0.33 ± 0.03^*	1.01 ± 0.10^*	0.26 ± 0.09
模型组	2041.3 ± 356.19	1.33 ± 0.08	0.62 ± 0.07	0.21 ± 0.12
LPS + rema（200 μg/mL）组	849.01 ± 111.64^*	1.08 ± 0.09^*	1.67 ± 0.17^*	1.25 ± 0.28^*
F值	45.23	107.3	39.43	20.28
P值	< 0.001	< 0.001	< 0.001	0.002 1

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