Nefazodone improves neurological function impairment in MCAO mice by inhibiting excessive activation of microglia
10.3760/cma.j.cn115354-20250817-00485
- VernacularTitle:奈法唑酮通过抑制小胶质细胞过度激活改善MCAO小鼠神经功能损伤
- Author:
Lixuan YANG
1
;
Ningning ZONG
1
;
Yang GENG
1
;
Jian CHEN
1
;
Yun XU
1
Author Information
1. 南京大学医学院附属鼓楼医院神经内科，南京 210008
- Publication Type:Journal Article
- Keywords:
Nefazodone;
Ischemic stroke;
Microglia;
Inflammatory response;
Neuroprotection
- From:
Chinese Journal of Neuromedicine
2025;24(10):1013-1024
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the effect of nefazodone on excessive activation of microglia and its regulatory mechanism, as well as its effect on neurological injury in mice subjected to middle cerebral artery occlusion (MCAO).Methods:(1) BV2 cell line was routinely cultured in vitro, and primary microglia from the cortex of neonatal C57BL/6J mice were cultured. Cell counting kit-8 (CCK-8) assay was employed to assess the effects of nefazodone (0, 10, 20, 30, 50 μmol/L) on viability of BV2 cells and primary microglia to determine the working concentration. BV2 cells and primary microglia were divided into a normal control group, a lipopolysaccharide (LPS) group, and a nefazodone group; cells in the nefazodone group were pretreated with 20 μmol/L nefazodone for 2 h; cells in the LPS group and nefazodone group were stimulated with LPS (0.5 μg/mL for BV2 cells and 0.1 μg/mL for primary microglia) for 24 h; cells in the normal control group received an equivalent volume of solvent. Immunofluorescent staining was used to detect the expressions of ionized calcium-binding adapter molecule 1 (Iba1) and CD68. Reverse transcription quantitative PCR (RT-qPCR) was performed to measure interleukin ( IL) -1β, IL-6, tumor necrosis factor-α ( TNF-α), nitric oxide synthase 2 ( Nos2), C-C motif chemokine ligand 2 ( CCL2), and β-hexosaminidase subunit β ( Hexb) mRNA expressions. ELISA was used to quantify the concentrations of supernatant IL-1β, IL-6, and TNF-α in BV2 cells. Western blotting was applied to detect the protein expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in BV2 cells. Griess reagent assay was used to measure supernatant nitric oxide (NO) level in BV2 cells. Western blotting was also used to assess the protein expressions of extracellular signal-regulated kinase (ERK), phosphorylated (p)-ERK, c-Jun N-terminal kinase (JNK), p-JNK, p38, p-p38, nuclear factor kappa B p65 and p-p65 in BV2 cells. (2) Thirty male C57BL/6J mice were randomly divided into a normal control group, a MCAO group, and a nefazodone group, with 10 mice in each group. MCAO model in the MCAO group and nefazodone group was established using suture method; the nefazodone group received an intraperitoneal injection of nefazodone (15 mg/kg) 30 min after modeling, while the normal control group received an equivalent volume of solvent. Three days after modeling, neurological deficits were evaluated using modified neurological severity score (mNSS), and behavioral changes were evaluated by paw grip strength test and foot-fault test. Cerebral infarction volume was assessed by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Iba1 protein expression in the ischemic penumbra was detected by Western blotting. Results:(1) CCK-8 assay showed no significant difference in viability of BV2 cells between the normal control group and 10 or 20 μmol/L nefazodone groups ( P>0.05), and viability of BV2 cells in 30 and 50 μmol/L nefazodone groups was statistically lower than that of normal control group ( P<0.05). Immunofluorescent staining revealed that compared with the normal control group, the LPS group had significantly increased fluorescent intensities of CD68 and Iba1; compared with the LPS group, the nefazodone group had significantly decreased fluorescent intensities of CD68 and Iba1 ( P<0.05). RT-qPCR results indicated that compared with those in the normal control group, the Nos2, CCL2, IL-1β, IL-6, and TNF-α mRNA expressions in both BV2 cells and primary microglia of the LPS group were significantly increased; compared with the LPS group, the nefazodone group had significantly decreased CCL2, IL-1β, and IL-6 mRNA expressions in BV2 cells, and significantly decreased Nos2, CCL2, IL-1β, IL-6, and TNF-α mRNA expressions in primary microglia ( P<0.05). ELISA showed that compared with those in the normal control group, the supernatant IL-1β, IL-6, and TNF-α levels significantly increased in the BV2 cells of LPS group; compared with those in the LPS group, supernatant IL-1β, IL-6 and TNF-α levels statistically decreased in the nefazodone group ( P< 0.05). Western blotting demonstrated that compared with those in the normal control group, the iNOS and COX-2 protein expressions significantly increased in BV2 cells of the LPS group ( P<0.05); compared with those in the LPS group, the iNOS and COX-2 protein expressions in BV2 cells of the nefazodone group statistically decreased ( P<0.05). Griess assay showed that compared with the normal control group, BV2 cells in the LPS group had significantly increased supernatant NO concentration ( P <0.05); compared with the LPS group, BV2 cells in the nefazodone group had significantly decreased supernatant NO concentration ( P<0.05). Western blotting revealed that compared with those in the normal control group, the p-ERK/ERK and p-JNK/JNK ratios significantly increased in BV2 cells of the LPS group ( P<0.05); compared with the LPS group, the p-p65/p65, p-ERK/ERK and p-JNK/JNK ratios significantly decreased in BV2 cells of the nefazodone group ( P<0.05). (2) Behavioral tests showed that compared with the normal control group, the MCAO group had significantly decreased forelimb grip strength and increased foot-fault rate ( P<0.05); compared with the MCAO group, the nefazodone group had significantly decreased mNSS score, increased forelimb grip strength and decreased foot-fault rate ( P<0.05). The percentage of cerebral infarction volume in the nefazodone group was significantly lower than that in the MCAO group ([9.56±1.81]% vs. [21.71±12.26]%, P< 0.05). The MCAO group had statistically higher Iba1 protein expression in ischemic penumbra (7.27±2.88) than the normal control group (1.00±0.64), and the nefazodone group had significantly lower Iba1 protein expression in ischemic penumbra (1.75±0.86) than the MCAO group ( P<0.05). Conclusion:Nefazodone can improve neurological function impairment in MCAO mice by inhibiting the excessive activation of microglia; cytological experiments suggest that its mechanism may be related to the negative regulation of ERK/JNK/NF-κB p65 signaling axis.
