Bendamustine improves brain edema after cerebral ischemia-reperfusion in mice by inhibiting NETs formation
10.3760/cma.j.cn115354-20250822-00500
- VernacularTitle:苄达明通过抑制NETs形成改善小鼠脑缺血再灌注后脑水肿
- Author:
Yang GENG
1
;
Ningning ZONG
1
;
Lixuan YANG
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:
Benzydamine;
Cerebral infarction;
Cerebral edema;
Blood brain barrier;
Neutrophil extracellular traps;
Matrix metalloproteinase-9
- From:
Chinese Journal of Neuromedicine
2025;24(10):986-996
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To explore how benzydamine (BA) improves brain edema in mice after cerebral ischemia-reperfusion.Methods:(1) One hundred and twenty 8 week-old male C57BL/6 mice were randomly divided into a sham-operated group, a middle cerebral artery occlusion (MCAO) group, a MCAO+low-dose BA group (L-BA group), and a MCAO+high-dose BA group (H-BA group), with 30 mice in each group. MCAO models in mice of the later 3 groups were established by suture method, while mice in the sham-operated group underwent the same surgical procedure without MCAO. At 6 hours after modeling, mice in the L-BA group and H-BA group were intraperitoneally injected with 5 mg/kg or 10 mg/kg BA, respectively, once daily for 3 days, while mice in the shamoperated group and MCAO group were intraperitoneally injected with same volume of normal saline instead. Dynamics of cerebral perfusion were monitored by laser speckle imaging in MCAO model mice at baseline, during occlusion, and following reperfusion. Three days after modeling, neurological deficits were assessed by modified neurological severity score (mNSS), neurological function was evaluated by forelimb grip strength and rotarod tests; cerebral infarct size was detected by 2,3,5-triphenyltetrazolium chloride (TTC) staining, and neuronal death was assessed by Fluoro-Jade B staining; cerebral edema was quantified by dry-wet weight method, blood-brain barrier (BBB) permeability was assessed by Evans blue dye extravasation, and expressions of tight junction proteins (Claudin-5, zonula occludens-1 [ZO-1]) were detected by immunofluorescent staining; expressions of neutrophil extracellular traps (NETs)-related proteins (citrullinated histone H3 [citH3], myeloperoxidase [MPO] and matrix metalloproteinase 9 [MMP-9]) were determined by Western blotting. (2) Bone marrow neutrophils were extracted from male C57BL/6 mice and randomly divided into a control group, a phorbol 12-myristate 13-acetate (PMA) group, and a PMA+BA group; neutrophils in the PMA group were stimulated with PMA (50 nmol/L), while neutrophils in the PMA+BA group were co-treated with 50 nmol/L PMA and 50 μmol/L BA; and those in the control group were given an equal amount of dimethyl sulfoxide. Sytox Green staining was used to detect the NETs proportion in neutrophils.Results:(1) Baseline cerebral perfusion was robust (1 237.75±98.16 PU), which was markedly reduced during occlusion (297.36±77.63 PU) in the ipsilateral middle cerebral artery territory, and improved following reperfusion (939.21±73.63 PU). Compared with the MCAO group, mice in the L-BA group and H-BA group had lower mNSS score, increased paw grip strength, prolonged rotarod retention time, reduced infarct size, fewer neuronal death, lower brain tissue water content, reduced blood-brain barrier permeability, increased fluorescent intensities of Claudin-5 (0.51±0.11, 0.71±0.04, and 0.83±0.05) and ZO-1 (0.43±0.09, 0.65±0.05, and 0.81±0.03), and decreased protein expressions of citH3 (2.33±0.15, 1.92±0.03, and 1.42±0.04), MPO (2.14±0.08, 1.71±0.06, and 1.37±0.03) and MMP-9 (2.62±0.09, 1.83±0.06, and 1.41±0.05), with significant differences ( P<0.05). All the above changes in the H-BA group were more significant than those in the L-BA group ( P<0.05). (2) Compared with that in the control group (10.00%±8.00%), the proportion of NETs formation per field in both PMA group (85.33%±2.08%) and PMA+BA group (58.46%±5.29%) was significantly increased ( P<0.05); the PMA+BA group showed a significant reduction in NETs formation compared with the PMA group ( P<0.05). Conclusion:BA can alleviate cerebral edema in mice after cerebral ischemia-reperfusion, and its mechanism may be involved in inhibiting NETs formation.
