白藜芦醇通过调控CCL3/CCR5信号通路减轻脑缺血再灌注大鼠模型的血脑屏障损伤

doi:10.12300/j.issn.1674-5817.2025.180

摘要/Abstract

摘要：

目的探讨白藜芦醇对脑缺血再灌注大鼠模型血脑屏障损伤的保护作用，并明确该作用是否由C-C基序趋化因子配体3（C-C motif chemokine ligand 3，CCL3）/C-C趋化因子受体5（C-C chemokine receptor type 5，CCR5）信号通路介导。方法将100只6～8周龄SPF级雄性SD大鼠随机分为假手术（Sham）组、模型（MCAO）组、白藜芦醇治疗（Res）组、白藜芦醇+CCR5激动剂（Res+Agonist）组和白藜芦醇+CCR5抑制剂（Res+Antagonist）组。Sham组只暴露颈动脉不做缺血处理；MCAO组、Res组、Res+Agonist组和Res+Antagonist组均采用大脑中动脉闭塞法构建大鼠脑缺血模型，缺血45 min后进行再灌注。完成再灌注后，Res组、Res+Agonist组和Res+Antagonist组即刻腹腔注射白藜芦醇（60 mg/kg），Sham组和MCAO组注射等体积生理盐水；30 min后，Res+Agonist组尾静脉注射CCR5激动剂（20 μg/kg），Res+Antagonist组尾静脉注射CCR5抑制剂（1 mg/kg），Res组、Sham组及MCAO组尾静脉注射等体积生理盐水。此后，每24 h给药（包括白藜芦醇、CCR5激动剂、CCR5抑制剂和生理盐水）1次，连续3 d。造模完成后，通过2，3，5-氯化三苯基四氮唑（2，3，5-triphenyltetrazolium chloride，TTC）染色法评估脑梗死体积占比；采用伊文思蓝染色法检测血脑屏障通透性；采用ELISA检测血清中促炎性细胞因子白细胞介素-1β（interleukin-1β，IL-1β）、肿瘤坏死因子-α（tumor necrosis factor alpha，TNF-α）与CCL3水平；通过HE染色和尼氏染色观察脑组织病理变化；采用免疫荧光染色法检测脑组织中小胶质细胞标志物离子钙结合适配器分子1（ionized calcium-binding adapter molecule 1，Iba-1）与CCR5的荧光强度；采用蛋白质印迹法检测脑组织中CCL3/CCR5信号通路相关蛋白（CCL3、CCR5）、血清促炎性细胞因子［磷酸化核因子-κB（phosphorylated nuclear factor-kappa B，p-NF-κB）］和血脑屏障相关蛋白［基质金属蛋白酶9（matrix metalloproteinase 9，MMP9）及闭合蛋白（occludin）］的表达水平。结果与Sham组相比，MCAO组大鼠大脑的各项损伤指标［脑梗死体积占比、脑组织伊文思蓝渗出率、血清促炎性细胞因子（IL-1β、TNF-α、CCL3）含量、脑组织Iba-1和CCR5的阳性表达情况、脑组织CCL3/CCR5信号通路相关蛋白（CCL3、CCR5）表达水平和血脑屏障相关蛋白（p-NF-κB和MMP9）表达水平］均显著升高（P<0.001），occludin表达水平显著降低（P<0.001），HE染色和尼氏染色结果显示其脑组织发生明显损伤。与MCAO组相比，Res组大鼠的上述各项损伤指标均显著下降（P<0.01），occludin表达水平显著升高（P<0.01），HE染色和尼氏染色结果显示其脑损伤得到一定的改善。与Res组相比，Res+Agonist组大鼠的上述损伤指标均显著升高（P<0.05），occludin表达水平显著降低（P<0.05），脑组织病理损伤显著加重；而Res+Antagonist组大鼠的上述损伤指标均显著下降（P<0.05），occludin表达水平显著升高（P<0.05），脑组织病理损伤进一步缓解。结论白藜芦醇通过靶向调控CCL3/CCR5信号通路减轻缺血再灌注损伤中的外周炎症反应，从而发挥血脑屏障保护作用。

关键词: 白藜芦醇, 大脑中动脉闭塞, 脑缺血再灌注, CCR5抑制剂, CCR5激动剂, 血脑屏障, 大鼠

Abstract:

Objective To investigate the protective effect of resveratrol against blood-brain barrier damage in a rat model of cerebral ischemia-reperfusion and to determine whether this effect is mediated by the C-C motif chemokine ligand 3 (CCL3)/C-C chemokine receptor type 5 (CCR5) signaling pathway　Methods　A total of 100 specific pathogen-free (SPF) male Sprague-Dawley (SD) rats, aged 6-8 weeks, were randomly divided into five groups: sham-operated (Sham), model (MCAO), resveratrol-treated (Res), resveratrol+CCR5 agonist (Res+Agonist), and resveratrol+CCR5 antagonist (Res+Antagonist) groups. The MCAO, Res, Res+Agonist, and Res+Antagonist groups were subjected to middle cerebral artery occlusion (MCAO) for 45 min followed by reperfusion to establish the rat model of cerebral ischemia, while the Sham group underwent carotid artery exposure without ischemia. After reperfusion, resveratrol (60 mg/kg) was intraperitoneally injected in Res, Res+Agonist and Res+Antagonist groups, and an equal volume of normal saline was injected in Sham and MCAO groups. Thirty minutes later, CCR5 agonist (20 μg/kg) was injected via the tail vein in the Res+Agonist group, CCR5 inhibitor (1 mg/kg) was injected via the tail vein in the Res+Antagonist group, and the equal volume of normal saline was injected via the tail vein in the Res, Sham and MCAO groups. Thereafter, resveratrol, CCR5 agonist, CCR5 inhibitor, or saline were administered every 24 hours for 3 consecutive days. After modeling, cerebral infarct volume was evaluated by 2,3,5?triphenyltetrazolium chloride (TTC) staining. Blood?brain barrier (BBB) permeability was assessed by Evans blue extravasation. Serum levels of the pro?inflammatory cytokines interleukin?1β (IL?1β), tumor necrosis factor?α (TNF?α), and CCL3 were measured by enzyme?linked immunosorbent assay (ELISA). Histopathological changes in brain tissue were observed by hematoxylin and eosin (HE) staining and Nissl staining. Immunofluorescence staining was used to detect the fluorescence intensity of the microglial marker ionized calcium?binding adapter molecule 1 (Iba?1) and CCR5. Western blotting analysis was used to detect proteins associated with the CCL3/CCR5 signaling pathway (CCL3, CCR5) in brain tissue, serum pro-inflammatory cytokines [phosphorylated nuclear factor-kappa B (p-NF-κB)] and blood-brain barrier-related proteins [matrix metalloproteinase 9 (MMP9) and occludin]. Results Compared with the Sham group, the MCAO group exhibited significantly increased levels of all injury parameters, including cerebral infarct volume, Evans blue extravasation rate in brain tissue, serum levels of the pro?inflammatory cytokines (IL-1β, TNF-α, and CCL3), the rate of Iba-1- and CCR5-positive immunofluorescence signals in brain tissue, and the protein expression levels of CCL3, CCR5, p-NF-κB, and MMP9 (all P<0.001), accompanied by markedly decreased expression of occludin (P<0.001). HE and Nissl staining revealed pronounced histopathological damage in the MCAO group.Compared with the MCAO group, the Res group showed significant reductions in all the aforementioned injury indicators and a significant increase in occludin expression (P<0.01), together with ameliorated histopathological changes. Compared with the Res group, the Res+Agonist group displayed significantly elevated injury indicators and decreased occludin expression (P<0.05), with aggravated brain tissue damage. In contrast, the Res+Antagonist group exhibited significant decreases in injury indicators and a significant increase in occludin expression (P<0.05), along with further alleviation of histopathological injury. Conclusion Resveratrol attenuates peripheral inflammation in cerebral ischemia?reperfusion injury by targeting the CCL3/CCR5 signaling pathway, thereby exerting a protective effect on the blood?brain barrier.

Key words: Resveratrol, Middle cerebral artery occlusion, Cerebral ischemia-reperfusion, CCR5 inhibitor, CCR5 agonist, Blood-brain barrier, Rats

中图分类号:

Q95-33

雷建军,索晨浩,张贺. 白藜芦醇通过调控CCL3/CCR5信号通路减轻脑缺血再灌注大鼠模型的血脑屏障损伤[J]. 实验动物与比较医学, 2026, 46(4): 487-497. DOI: 10.12300/j.issn.1674-5817.2025.180.

LEI Jianjun,SUO Chenhao,ZHANG He. Resveratrol Alleviates Blood‑Brain Barrier Damage in Cerebral Ischemia‑Reperfusion Rat Models through Modulation of the CCL3/CCR5 Signaling Pathway[J]. Laboratory Animal and Comparative Medicine, 2026, 46(4): 487-497. DOI: 10.12300/j.issn.1674-5817.2025.180.

图/表 5

注：A，各组大鼠脑中伊文思蓝（EB）含量；B，各组大鼠脑TTC染色结果；C，各组大鼠脑梗死体积占比计算结果。Sham组只暴露颈动脉不做缺血处理；MCAO组、Res组、Res+Agonist组和Res+Antagonist组均采用大脑中动脉闭塞法构建大鼠脑缺血模型，缺血45 min后进行再灌注。完成再灌注后，Res组、Res+Agonist组和Res+Antagonist组即刻腹腔注射白藜芦醇（60 mg/kg），Sham组和MCAO组注射等体积生理盐水；30 min后，Res+Agonist组尾静脉注射CCR5激动剂（20 μg/kg），Res+Antagonist组尾静脉注射CCR5抑制剂（1 mg/kg），Res组、Sham组及MCAO组尾静脉注射等体积生理盐水。此后，每24 h给药（包括白藜芦醇、CCR5激动剂、CCR5抑制剂或生理盐水）1次，连续3 d。n=5；与Sham组相比，^***P<0.001；与MCAO组相比，^##P<0.01；与Res组相比，^&P<0.05。
">

图1 白藜芦醇对大脑中动脉闭塞大鼠脑损伤的影响
注：A，各组大鼠脑中伊文思蓝（EB）含量；B，各组大鼠脑TTC染色结果；C，各组大鼠脑梗死体积占比计算结果。Sham组只暴露颈动脉不做缺血处理；MCAO组、Res组、Res+Agonist组和Res+Antagonist组均采用大脑中动脉闭塞法构建大鼠脑缺血模型，缺血45 min后进行再灌注。完成再灌注后，Res组、Res+Agonist组和Res+Antagonist组即刻腹腔注射白藜芦醇（60 mg/kg），Sham组和MCAO组注射等体积生理盐水；30 min后，Res+Agonist组尾静脉注射CCR5激动剂（20 μg/kg），Res+Antagonist组尾静脉注射CCR5抑制剂（1 mg/kg），Res组、Sham组及MCAO组尾静脉注射等体积生理盐水。此后，每24 h给药（包括白藜芦醇、CCR5激动剂、CCR5抑制剂或生理盐水）1次，连续3 d。n=5；与Sham组相比，^***P<0.001；与MCAO组相比，^##P<0.01；与Res组相比，^&P<0.05。

Figure 1 Effects of resveratrol on brain injury in middle cerebral artery occlusion rats
Note：A， Evans blue （EB） content of rat brains in each group； B， TTC staining results of rat brains in each group； C， Calculation of the proportion of cerebral infarction volume in each group of rat brains. The MCAO, Res, Res+Agonist, and Res+Antagonist groups were subjected to middle cerebral artery occlusion (MCAO) for 45 min followed by reperfusion to establish the rat model of cerebral ischemia, while the Sham group underwent carotid artery exposure without ischemia. After reperfusion, resveratrol (60 mg/kg) was intraperitoneally injected in Res, Res+Agonist and Res+Antagonist groups, and an equal volume of normal saline was injected in Sham and MCAO groups. Thirty minutes later, CCR5 agonist (20 μg/kg) was injected via the tail vein in the Res+Agonist group, CCR5 inhibitor (1 mg/kg) was injected via the tail vein in the Res+Antagonist group, and the equal volume of normal saline was injected via the tail vein in the Res, Sham and MCAO groups. Thereafter, resveratrol, CCR5 agonist, CCR5 inhibitor, or saline were administered every 24 hours for 3 consecutive days. n=5； compared with Sham group， ^***P<0.001； compared with MCAO group， ^##P<0.01； compared with Res group， ^&P<0.05.

注：各组大鼠脑组织的HE染色（A）和尼氏染色（B）结果。Sham组只暴露颈动脉不做缺血处理；MCAO组、Res组、Res+Agonist组和Res+Antagonist组均采用大脑中动脉闭塞法构建大鼠脑缺血模型，缺血45 min后进行再灌注。完成再灌注后，Res组、Res+Agonist组和Res+Antagonist组即刻腹腔注射白藜芦醇（60 mg/kg），Sham组和MCAO组注射等体积生理盐水；30 min后，Res+Agonist组尾静脉注射CCR5激动剂（20 μg/kg），Res+Antagonist组尾静脉注射CCR5抑制剂（1 mg/kg），Res组、Sham组及MCAO组尾静脉注射等体积生理盐水。此后，每24 h给药（包括白藜芦醇、CCR5激动剂、CCR5抑制剂和生理盐水）1次，连续3 d。n=5，标尺为20 μm。
">

图2 白藜芦醇对大脑中动脉闭塞大鼠脑组织神经元损伤的影响
注：各组大鼠脑组织的HE染色（A）和尼氏染色（B）结果。Sham组只暴露颈动脉不做缺血处理；MCAO组、Res组、Res+Agonist组和Res+Antagonist组均采用大脑中动脉闭塞法构建大鼠脑缺血模型，缺血45 min后进行再灌注。完成再灌注后，Res组、Res+Agonist组和Res+Antagonist组即刻腹腔注射白藜芦醇（60 mg/kg），Sham组和MCAO组注射等体积生理盐水；30 min后，Res+Agonist组尾静脉注射CCR5激动剂（20 μg/kg），Res+Antagonist组尾静脉注射CCR5抑制剂（1 mg/kg），Res组、Sham组及MCAO组尾静脉注射等体积生理盐水。此后，每24 h给药（包括白藜芦醇、CCR5激动剂、CCR5抑制剂和生理盐水）1次，连续3 d。n=5，标尺为20 μm。

Figure 2 Effects of resveratrol on neuronal damage in the brain tissue of middle cerebral artery occlusion rats
Note： Results of HE staining （A） and Nissl staining （B） of rat brain tissues in each group. The MCAO, Res, Res+Agonist, and Res+Antagonist groups were subjected to middle cerebral artery occlusion (MCAO) for 45 min followed by reperfusion to establish the rat model of cerebral ischemia, while the Sham group underwent carotid artery exposure without ischemia. After reperfusion, resveratrol (60 mg/kg) was intraperitoneally injected in Res, Res+Agonist and Res+Antagonist groups, and an equal volume of normal saline was injected in Sham and MCAO groups. Thirty minutes later, CCR5 agonist (20 μg/kg) was injected via the tail vein in the Res+Agonist group, CCR5 inhibitor (1 mg/kg) was injected via the tail vein in the Res+Antagonist group, and the equal volume of normal saline was injected via the tail vein in the Res, Sham and MCAO groups. Thereafter, resveratrol, CCR5 agonist, CCR5 inhibitor, or saline were administered every 24 hours for 3 consecutive days. n=5， scale bar = 20 μm.

注：大鼠血清中IL-1β（A）、TNF-α（B）和CCL3（C）的ELISA检测结果。Sham组只暴露颈动脉不做缺血处理；MCAO组、Res组、Res+Agonist组和Res+Antagonist组均采用大脑中动脉闭塞法构建大鼠脑缺血模型，缺血45 min后进行再灌注。完成再灌注后，Res组、Res+Agonist组和Res+Antagonist组即刻腹腔注射白藜芦醇（60 mg/kg），Sham组和MCAO组注射等体积生理盐水；30 min后，Res+Agonist组尾静脉注射CCR5激动剂（20 μg/kg），Res+Antagonist组尾静脉注射CCR5抑制剂（1 mg/kg），Res组、Sham组及MCAO组尾静脉注射等体积生理盐水。此后，每24 h给药（包括白藜芦醇、CCR5激动剂、CCR5抑制剂和生理盐水）1次，连续3 d。IL-1β，白细胞介素1β；TNF-α，肿瘤坏死因子-α；CCL3，C-C基序趋化因子配体3。n=20；与Sham组相比，^*P<0.05，^***P<0.001；与MCAO组相比，^#P<0.05，^##P<0.01；与Res组相比，^&P<0.05。
">

图3 白藜芦醇对大脑中动脉闭塞大鼠外周血促炎性细胞因子的影响
注：大鼠血清中IL-1β（A）、TNF-α（B）和CCL3（C）的ELISA检测结果。Sham组只暴露颈动脉不做缺血处理；MCAO组、Res组、Res+Agonist组和Res+Antagonist组均采用大脑中动脉闭塞法构建大鼠脑缺血模型，缺血45 min后进行再灌注。完成再灌注后，Res组、Res+Agonist组和Res+Antagonist组即刻腹腔注射白藜芦醇（60 mg/kg），Sham组和MCAO组注射等体积生理盐水；30 min后，Res+Agonist组尾静脉注射CCR5激动剂（20 μg/kg），Res+Antagonist组尾静脉注射CCR5抑制剂（1 mg/kg），Res组、Sham组及MCAO组尾静脉注射等体积生理盐水。此后，每24 h给药（包括白藜芦醇、CCR5激动剂、CCR5抑制剂和生理盐水）1次，连续3 d。IL-1β，白细胞介素1β；TNF-α，肿瘤坏死因子-α；CCL3，C-C基序趋化因子配体3。n=20；与Sham组相比，^*P<0.05，^***P<0.001；与MCAO组相比，^#P<0.05，^##P<0.01；与Res组相比，^&P<0.05。

Figure 3 Effects of resveratrol on pro-inflammatory cytokines in peripheral blood of rats with middle cerebral artery occlusion
Note： ELISA test results of IL-1β（A）， TNF-α（B） and CCL3（C） in rat serum. The MCAO, Res, Res+Agonist, and Res+Antagonist groups were subjected to middle cerebral artery occlusion (MCAO) for 45 min followed by reperfusion to establish the rat model of cerebral ischemia, while the Sham group underwent carotid artery exposure without ischemia. After reperfusion, resveratrol (60 mg/kg) was intraperitoneally injected in Res, Res+Agonist and Res+Antagonist groups, and an equal volume of normal saline was injected in Sham and MCAO groups. Thirty minutes later, CCR5 agonist (20 μg/kg) was injected via the tail vein in the Res+Agonist group, CCR5 inhibitor (1 mg/kg) was injected via the tail vein in the Res+Antagonist group, and the equal volume of normal saline was injected via the tail vein in the Res, Sham and MCAO groups. Thereafter, resveratrol, CCR5 agonist, CCR5 inhibitor, or saline were administered every 24 hours for 3 consecutive days. IL-1β， interleukin 1β； TNF-α， tumor necrosis factor-α； CCL3， C-C motif chemokine ligand 3. n=20； compared with Sham group， ^*P<0.05， ^***P<0.001； compared with MCAO group， ^#P<0.05， ^##P<0.01； compared with Res group， ^&P<0.05.

注：大鼠脑组织中Iba-1和CCR5的免疫荧光检测（A）和荧光强度定量分析（B～C）结果。Sham组只暴露颈动脉不做缺血处理；MCAO组、Res组、Res+Agonist组和Res+Antagonist组均采用大脑中动脉闭塞法构建大鼠脑缺血模型，缺血45 min后进行再灌注。完成再灌注后，Res组、Res+Agonist组和Res+Antagonist组即刻腹腔注射白藜芦醇（60 mg/kg），Sham组和MCAO组注射等体积生理盐水；30 min后，Res+Agonist组尾静脉注射CCR5激动剂（20 μg/kg），Res+Antagonist组尾静脉注射CCR5抑制剂（1 mg/kg），Res组、Sham组及MCAO组尾静脉注射等体积生理盐水。此后，每24 h给药（包括白藜芦醇、CCR5激动剂、CCR5抑制剂和生理盐水）1次，连续3 d。Iba-1，离子钙结合适配器分子1；CCR5，C-C基序趋化因子受体5。标尺为50 μm；n=5；与Sham组相比，^***P<0.001；与MCAO组相比，^##P<0.01；与Res组相比，^&P<0.05，^&&P<0.01。
">

图4 白藜芦醇对大脑中动脉闭塞大鼠小胶质细胞活化的影响
注：大鼠脑组织中Iba-1和CCR5的免疫荧光检测（A）和荧光强度定量分析（B～C）结果。Sham组只暴露颈动脉不做缺血处理；MCAO组、Res组、Res+Agonist组和Res+Antagonist组均采用大脑中动脉闭塞法构建大鼠脑缺血模型，缺血45 min后进行再灌注。完成再灌注后，Res组、Res+Agonist组和Res+Antagonist组即刻腹腔注射白藜芦醇（60 mg/kg），Sham组和MCAO组注射等体积生理盐水；30 min后，Res+Agonist组尾静脉注射CCR5激动剂（20 μg/kg），Res+Antagonist组尾静脉注射CCR5抑制剂（1 mg/kg），Res组、Sham组及MCAO组尾静脉注射等体积生理盐水。此后，每24 h给药（包括白藜芦醇、CCR5激动剂、CCR5抑制剂和生理盐水）1次，连续3 d。Iba-1，离子钙结合适配器分子1；CCR5，C-C基序趋化因子受体5。标尺为50 μm；n=5；与Sham组相比，^***P<0.001；与MCAO组相比，^##P<0.01；与Res组相比，^&P<0.05，^&&P<0.01。

Figure 4 Effects of resveratrol on microglial activation in rats with middle cerebral artery occlusion
Note： Immunofluorescence detection （A） and quantitative analysis of fluorescence intensity （B-C） of Iba-1 and CCR5 in rat brain tissue. The MCAO, Res, Res+Agonist, and Res+Antagonist groups were subjected to middle cerebral artery occlusion (MCAO) for 45 min followed by reperfusion to establish the rat model of cerebral ischemia, while the Sham group underwent carotid artery exposure without ischemia. After reperfusion, resveratrol (60 mg/kg) was intraperitoneally injected in Res, Res+Agonist and Res+Antagonist groups, and an equal volume of normal saline was injected in Sham and MCAO groups. Thirty minutes later, CCR5 agonist (20 μg/kg) was injected via the tail vein in the Res+Agonist group, CCR5 inhibitor (1 mg/kg) was injected via the tail vein in the Res+Antagonist group, and the equal volume of normal saline was injected via the tail vein in the Res, Sham and MCAO groups. Thereafter, resveratrol, CCR5 agonist, CCR5 inhibitor, or saline were administered every 24 hours for 3 consecutive days. Iba-1， ionized calcium-binding adapter molecule 1； CCR5， C-C motif chemokine receptor 5. Scale bar=50 μm； n=5； compared with Sham group， ^***P<0.001； compared with MCAO group， ^##P<0.01； compared with Res group， ^&P<0.05， ^&&P<0.01.

注：A～B，大鼠脑组织中CCR5和CCL3蛋白的蛋白质印迹和定量分析结果；C～D，大鼠脑组织中p-NF-κB、NF-κB、MMP9和occludin蛋白的蛋白质印迹和定量分析结果。CCR5，C-C基序趋化因子受体5；CCL3，C-C基序趋化因子配体3；p-NF-κB，磷酸化核因子-κB；NF-κB，核因子-κB；MMP9，基质金属蛋白酶9；occludin，闭合蛋白。Sham组只暴露颈动脉不做缺血处理；MCAO组、Res组、Res+Agonist组和Res+Antagonist组均采用大脑中动脉闭塞法构建大鼠脑缺血模型，缺血45 min后进行再灌注。完成再灌注后，Res组、Res+Agonist组和Res+Antagonist组即刻腹腔注射白藜芦醇（60 mg/kg），Sham组和MCAO组注射等体积生理盐水；30 min后，Res+Agonist组尾静脉注射CCR5激动剂（20 μg/kg），Res+Antagonist组尾静脉注射CCR5抑制剂（1 mg/kg），Res组、Sham组及MCAO组尾静脉注射等体积生理盐水。此后，每24 h给药（包括白藜芦醇、CCR5激动剂、CCR5抑制剂和生理盐水）1次，连续3 d。n=5；与Sham组相比，^***P<0.001；与MCAO组相比，^#P<0.05，^##P<0.01；与Res组相比，^&P<0.05。
">

图5 白藜芦醇治疗对大脑中动脉闭塞大鼠脑组织中CCL3/CCR5信号通路和血脑屏障相关蛋白的影响
注：A～B，大鼠脑组织中CCR5和CCL3蛋白的蛋白质印迹和定量分析结果；C～D，大鼠脑组织中p-NF-κB、NF-κB、MMP9和occludin蛋白的蛋白质印迹和定量分析结果。CCR5，C-C基序趋化因子受体5；CCL3，C-C基序趋化因子配体3；p-NF-κB，磷酸化核因子-κB；NF-κB，核因子-κB；MMP9，基质金属蛋白酶9；occludin，闭合蛋白。Sham组只暴露颈动脉不做缺血处理；MCAO组、Res组、Res+Agonist组和Res+Antagonist组均采用大脑中动脉闭塞法构建大鼠脑缺血模型，缺血45 min后进行再灌注。完成再灌注后，Res组、Res+Agonist组和Res+Antagonist组即刻腹腔注射白藜芦醇（60 mg/kg），Sham组和MCAO组注射等体积生理盐水；30 min后，Res+Agonist组尾静脉注射CCR5激动剂（20 μg/kg），Res+Antagonist组尾静脉注射CCR5抑制剂（1 mg/kg），Res组、Sham组及MCAO组尾静脉注射等体积生理盐水。此后，每24 h给药（包括白藜芦醇、CCR5激动剂、CCR5抑制剂和生理盐水）1次，连续3 d。n=5；与Sham组相比，^***P<0.001；与MCAO组相比，^#P<0.05，^##P<0.01；与Res组相比，^&P<0.05。

Figure 5 Effects of resveratrol treatment on CCL3/CCR5 signaling pathway and blood-brain barrier-related proteins in brain tissues of rats with middle cerebral artery occlusion
Note： A-B， Western blotting results and quantitative analysis of CCR5 and CCL3 proteins in rat brain tissue； C-D， Western blotting results and quantitative analysis of p-NF-κB， NF-κB， MMP9， and occludin proteins in rat brain tissue. CCR5， C-C motif chemokine receptor 5； CCL3， C-C motif chemokine ligand 3； p-NF-κB， phosphorylated nuclear factor-κB； NF-κB， nuclear factor-κB； MMP9， matrix metalloproteinase 9； occludin， tight junction protein. The MCAO, Res, Res+Agonist, and Res+Antagonist groups were subjected to middle cerebral artery occlusion (MCAO) for 45 min followed by reperfusion to establish the rat model of cerebral ischemia, while the Sham group underwent carotid artery exposure without ischemia. After reperfusion, resveratrol (60 mg/kg) was intraperitoneally injected in Res, Res+Agonist and Res+Antagonist groups, and an equal volume of normal saline was injected in Sham and MCAO groups. Thirty minutes later, CCR5 agonist (20 μg/kg) was injected via the tail vein in the Res+Agonist group, CCR5 inhibitor (1 mg/kg) was injected via the tail vein in the Res+Antagonist group, and the equal volume of normal saline was injected via the tail vein in the Res, Sham and MCAO groups. Thereafter, resveratrol, CCR5 agonist, CCR5 inhibitor, or saline were administered every 24 hours for 3 consecutive days. n=5； compared with Sham group， ^***P<0.001； compared with MCAO group， ^#P<0.05， ^##P<0.01； compared with Res group， ^&P<0.05.

参考文献 25

[1]	刘佳, 叶岩荣, 沈赟, 等. 银杏内酯B通过调控脑内T细胞特性及与胶质细胞间相互作用促进缺血性脑卒中小鼠的神经功能恢复[J]. 实验动物与比较医学, 2024, 44(2): 139-148. DOI: 10.12300/j.issn.1674-5817.2023.121 .
	LIU J, YE Y R, SHEN Y, et al. Ginkgolide B promotes neural function recovery of ischemic stroke mice by regulating characteristics of brain T cells and their interactions with glial cells[J]. Lab Anim Comp Med, 2024, 44(2): 139-148. DOI: 10.12300/j.issn.1674-5817.2023.121 .
[2]	RAFFAELE S, CLAUSEN B H, MANNELLA F C, et al. Characterisation of GPR17-expressing oligodendrocyte precursors in human ischaemic lesions and correlation with reactive glial responses[J]. J Pathol, 2025, 265(2): 226-243. DOI: 10.1002/path.6381 .
[3]	ALIDADI M, OMIDI N, ABDI M, et al. Melatonin ameliorates astrogliosis and microgliosis in a cuprizone demyelinating mouse model[J]. Biochem Biophys Rep, 2025, 41: 101929. DOI: 10.1016/j.bbrep.2025.101929 .
[4]	LIU X, TAO X W, XIONG Z, et al. Mechanism of HDAC2 regulating Nrf2 acetylation level in neuronal ferroptosis of neonatal rats with hypoxic-ischemic brain injury[J]. Brain Inj, 2025, 39(8): 635-645. DOI: 10.1080/02699052.2025.2468309 .
[5]	SHI K B, LI H D, CHANG T, et al. Bone marrow hematopoiesis drives multiple sclerosis progression[J]. Cell, 2022, 185(13): 2234-2247.e17. DOI: 10.1016/j.cell.2022.05.020 .
[6]	KNOX E G, ABURTO M R, CLARKE G, et al. The blood-brain barrier in aging and neurodegeneration[J]. Mol Psychiatry, 2022, 27(6): 2659-2673. DOI: 10.1038/s41380-022-01511-z .
[7]	AKINLAJA Y O, NISHIYAMA A. Glial modulation of synapse development and plasticity: oligodendrocyte precursor cells as a new player in the synaptic quintet[J]. Front Cell Dev Biol, 2024, 12: 1418100. DOI: 10.3389/fcell.2024.1418100 .
[8]	HUNTEMER-SILVEIRA A, PATIL N, BRICKNER M A, et al. Strategies for oligodendrocyte and myelin repair in traumatic CNS injury[J]. Front Cell Neurosci, 2021, 14: 619707. DOI: 10.3389/fncel.2020.619707 .
[9]	WANG F, YE Z H, YIN X J, et al. Electroacupuncture at 5/100 Hz alleviates neuropathic pain in rats by inhibiting the CCL3/CCR5 axis in the spinal cord[J]. Acupunct Med, 2025, 43(2): 95-103. DOI: 10.1177/09645284251327197 .
[10]	JAFARI M, DAMANI R. Blood pressure variability and outcome after acute intracerebral hemorrhage[J]. J Neurol Sci, 2020, 413: 116766. DOI: 10.1016/j.jns.2020.116766 .
[11]	BAI M L, SUN R Z, CAO B, et al. Monocyte-related cytokines/chemokines in cerebral ischemic stroke[J]. CNS Neurosci Ther, 2023, 29(12): 3693-3712. DOI: 10.1111/cns.14368 .
[12]	ESCOBAR-PESO A, MARTÍNEZ-ALONSO E, MASJUAN J, et al. Development of pharmacological strategies with therapeutic potential in ischemic stroke[J]. Antioxidants, 2023, 12(12): 2102. DOI: 10.3390/antiox12122102 .
[13]	牟凌梅, 李雪梅, 文君. 白藜芦醇调节TREM-1表达减轻大鼠脑缺血再灌注损伤后细胞焦亡[J]. 四川医学, 2025, 46(6): 628-634. DOI: 10.16252/j.cnki.issn1004-0501-2025.06.006 .
	MOU L M, LI X M, WEN J. Resveratrol regulates TREM-1 expression to alleviate cell pyroptosis after cerebral ischemia-reperfusion injury in rats[J]. Sichuan Med J, 2025, 46(6): 628-634. DOI: 10.16252/j.cnki.issn1004-0501-2025.06.006 .
[14]	张小良, 高赛红, 杨迎春, 等. 白藜芦醇激活自噬抑制焦亡减轻脑缺血再灌注损伤[J]. 中国临床药理学与治疗学, 2024, 29(11): 1240-1248. DOI: 10.12092/j.issn.1009-2501.2024.11.005 .
	ZHANG X L, GAO S H, YANG Y C, et al. Resveratrol activates autophagy and inhibits pyroptosis to alleviate cerebral ischemia-reperfusion injury[J]. Chin J Clin Pharmacol Ther, 2024, 29(11): 1240-1248. DOI: 10.12092/j.issn.1009-2501.2024.11.005 .
[15]	AN Y, SU G, CHEN W, et al. Research progress on the mechanisms of microglial extracellular vesicles affecting the prognosis of ischemic stroke[J]. Neurochem Int, 2025, 185: 105949. DOI: 10.1016/j.neuint.2025.105949 .
[16]	LÓPEZ-MORALES M A, CASTELLÓ-RUIZ M, BURGUETE M C, et al. Effect and mechanisms of resveratrol in animal models of ischemic stroke: a systematic review and Bayesian meta-analysis[J]. J Cereb Blood Flow Metab, 2023, 43(12): 2013-2028. DOI: 10.1177/0271678x231206236 .
[17]	WANG C X, WANG J Y, ZHU Z C, et al. Spotlight on pro-inflammatory chemokines: regulators of cellular communication in cognitive impairment[J]. Front Immunol, 2024, 15: 1421076. DOI: 10.3389/fimmu.2024.1421076 .
[18]	GUO Q X, DU A N, WANG J Y, et al. Integrated bioinformatic analyses reveal immune molecular markers and regulatory networks for cerebral ischemia-reperfusion[J]. Front Biosci (Landmark Ed), 2023, 28(8): 179. DOI: 10.31083/j.fbl2808179 .
[19]	DASDELEN M F, CAGLAYAN A B, ER S, et al. Social isolation initiated post-weaning augments ischemic brain injury by promoting pro-inflammatory responses[J]. Exp Neurol, 2024, 375: 114729. DOI: 10.1016/j.expneurol.2024.114729 .
[20]	FESTA B P, SIDDIQI F H, JIMENEZ-SANCHEZ M, et al. Microglial-to-neuronal CCR5 signaling regulates autophagy in neurodegeneration[J]. Neuron, 2023, 111(13): 2021-2037.e12. DOI: 10.1016/j.neuron.2023.04.006 .
[21]	WANG L F, LI W J, ZHANG X Y, et al. Resveratrol prevents age-related heart impairment through inhibiting the Notch/NF-κB pathway[J]. Food Sci Nutr, 2024, 12(2): 1035-1045. DOI: 10.1002/fsn3.3817 .
[22]	BASHEER N, MUHAMMADI M K, FREITES C L, et al. TLR4-mediated chronic neuroinflammation has no effect on tangle pathology in a tauopathy mouse model[J]. Front Aging Neurosci, 2024, 16: 1468602. DOI: 10.3389/fnagi.2024.1468602 .
[23]	LI W B, ZHANG Y D, YANG F, et al. Molecular mechanism by which TRPC6 regulates calcium signaling and neuroinflammation in the onset and development of ischemic stroke: a review[J]. Altern Ther Health Med, 2024, 30(8): 170-175.
[24]	DENG L, ZHANG J Y, CHEN S, et al. miR-671-5p upregulation attenuates blood–brain barrier disruption in the ischemia stroke model via the NF-кB/MMP-9 signaling pathway[J]. Mol Neurobiol, 2023, 60(7): 3824-3838. DOI: 10.1007/s12035-023-03318-7 .
[25]	DENG H J, XUE P F, ZHOU X G, et al. CCL4/CCR5 regulates chondrocyte biology and OA progression[J]. Cytokine, 2024, 183: 156746. DOI: 10.1016/j.cyto.2024.156746 .