外源性激活大麻素-2 受体可调节脊髓缺血再灌注大鼠模型中 TLR4/MMP9 的表达。
Exogenous activation of cannabinoid-2 receptor modulates TLR4/MMP9 expression in a spinal cord ischemia reperfusion rat model.
机构信息
Department of Anesthesiology, First Affiliated Hospital, China Medical University, 155 North Nanjing Street, Shenyang, 110001, Liaoning, People's Republic of China.
出版信息
J Neuroinflammation. 2020 Apr 6;17(1):101. doi: 10.1186/s12974-020-01784-7.
BACKGROUND
Cannabinoid-2 receptor (CB2R) plays an important role in the cascading inflammation following ischemic injury. The toll-like receptors 4 (TLR4)/matrix metalloproteinase 9 (MMP9) signal pathway is involved in blood-brain barrier dysfunction induced by ischemia stroke. The aim of this study is to investigate the roles of exogenous activation of CB2R on attenuating neurological deficit and blood-spinal cord barrier (BSCB) disruption during rat spinal cord ischemia reperfusion (I/R) injury, through modulation of the TLR4/MMP9 axis.
METHODS
Animals were intraperitoneally pretreated with TLR4 inhibitor TAK-242, CB2R agonist JWH-133 with or without CB2R antagonist AM630, or equivalent volume of vehicle 1 h before undergoing 14-min occlusion of descending aorta or sham operation. One, two, three, and 7 days after reperfusion, hindlimb locomotor function was evaluated with Basso, Beattie, and Bresnahan (BBB) Locomotor Scale, BSCB integrity was detected by measurement of Evans blue (EB) extravasation and spinal cord edema. The protein expression levels of CB2R, tight junction protein Zonula occluden-1 (ZO-1), TLR4, MMP9, MyD88, NF-κB p65, and NF-κB p-p65 were determined by western blot. The MMP9 activity was analyzed by gelatin zymography. Double immunofluorescence staining was used to identify the perivascular localization of CB2R, TLR4, MMP9, and reactive astrocytes, as well as the colocalization of CB2R, TLR4, and MMP9 with reactive astrocytes.
RESULTS
JWH-133 pretreatment attenuated hindlimb motor functional deficit and BSCB leakage, along with preventing downregulation of ZO-1 and upregulation of TLR4/MMP9, similar to the effects of TAK-242 preconditioning. JWH-133 or TAK-242 pretreatment reduced the perivascular expression of TLR4/MMP9 and reactive astrocytes following injury. JWH-133 pretreatment also downregulated MyD88/NF-κB level, MMP9 activity, and the astrocytic TLR4/MMP9 after I/R injury.
CONCLUSIONS
Exogenous activation of CB2R by JWH-133 attenuated neurological deficit and BSCB disruption after spinal cord I/R injury via inhibition of TLR4/MMP9 expression.
背景
大麻素-2 型受体（CB2R）在缺血性损伤后级联炎症中发挥重要作用。 Toll 样受体 4（TLR4）/基质金属蛋白酶 9（MMP9）信号通路参与了缺血性脑卒中引起的血-脊髓屏障（BSCB）功能障碍。本研究旨在通过调节 TLR4/MMP9 轴，研究外源性激活 CB2R 对大鼠脊髓缺血再灌注（I/R）损伤时神经功能缺损和 BSCB 破坏的作用。
方法
动物在腹内预先用 TLR4 抑制剂 TAK-242、CB2R 激动剂 JWH-133 加或不加 CB2R 拮抗剂 AM630 或等体积的载体处理 1 小时，然后进行 14 分钟的降主动脉闭塞或假手术。再灌注后 1、2、3 和 7 天，用 Basso、Beattie 和 Bresnahan（BBB）运动评分评估后肢运动功能，通过测量 Evans 蓝（EB）渗出和脊髓水肿来检测 BSCB 完整性。通过 Western blot 测定 CB2R、紧密连接蛋白 Zonula occluden-1（ZO-1）、TLR4、MMP9、MyD88、NF-κB p65 和 NF-κB p-p65 的蛋白表达水平。通过明胶酶谱分析 MMP9 活性。通过双免疫荧光染色鉴定 CB2R、TLR4、MMP9 和反应性星形胶质细胞的血管周定位，以及 CB2R、TLR4 和 MMP9 与反应性星形胶质细胞的共定位。
结果
JWH-133 预处理可减轻后肢运动功能障碍和 BSCB 渗漏，并防止 ZO-1 下调和 TLR4/MMP9 上调，与 TAK-242 预处理的效果相似。JWH-133 或 TAK-242 预处理可减少损伤后血管周围 TLR4/MMP9 和反应性星形胶质细胞的表达。JWH-133 预处理还可下调 I/R 损伤后 MyD88/NF-κB 水平、MMP9 活性和星形胶质细胞 TLR4/MMP9。
结论
JWH-133 通过抑制 TLR4/MMP9 表达，减轻脊髓 I/R 损伤后的神经功能缺损和 BSCB 破坏。
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