松弛素-3调控NLRP3炎症小体抑制小鼠脑缺血再灌注损伤的机制研究

Mechanism of Relaxin-3 alleviating cerebral ischemia/reperfusion injury in mice by regulating the nucleotide-binding domain leucine-rich repeat and pyrin domain-containing receptor 3 inflammasome

摘要:

目的 探讨松弛素-3（Relaxin-3）调控核苷酸结合结构域富含亮氨酸重复序列和含热蛋白结构域受体3（NLRP3）炎症小体抑制脑缺血再灌注损伤的分子机制。方法 80只雄性C57BL/6小鼠随机分为空白对照组（假手术组）、脑缺血再灌注组（模型组）、Relaxin-3抑制剂组（抑制剂组）和Relaxin-3激动剂组（激动剂组），每组20只。采用线栓法建立小鼠大脑中动脉闭塞（MCAO）脑缺血再灌注模型（缺血90 min，再灌注24 h）。抑制剂组和激动剂组于造模前30 min分别于腹腔内注射Relaxin-3抑制剂（10 μg/kg）和Relaxin-3激动剂（1 mg/kg）。分别检测各组神经细胞凋亡［原位末端转移酶标记法（TUNEL法）］、NLRP3 mRNA［实时荧光定量逆转录聚合酶链式反应（qRT-PCR）］及Relaxin-3蛋白表达（蛋白质印迹法）。结果 与假手术组相比，模型组及抑制剂组的TUNEL阳性细胞率、NLRP3 mRNA表达水平均显著升高（P<0.05），Relaxin-3蛋白表达水平显著降低（P<0.05）。与模型组相比，激动剂组TUNEL阳性细胞率显著降低（P<0.01），NLRP3 mRNA表达水平下调（P<0.01），Relaxin-3蛋白表达水平上调（P<0.05）。抑制剂组上述各项指标的恶化程度较模型组更为显著（P<0.05）。结论 Relaxin-3可通过抑制NLRP3炎症小体的表达，减少神经细胞凋亡，发挥对脑缺血再灌注损伤的保护作用。

Abstract:

Objective To investigate the molecular mechanism of Relaxin-3 alleviating cerebral ischemia/reperfusion injury by regulating the nucleotide-binding domain leucine-rich repeat and pyrin domain-containing receptor 3 （NLRP3） inflammasome.Methods A total of 80 male C57BL/6 mice were randomly divided into blank control group （sham-operation group）， cerebral ischemia/reperfusion group （model group）， Relaxin-3 inhibitor group （inhibitor group）， and Relaxin-3 agonist group （agonist group）， with 20 mice in each group. The suture method was used to establish a mouse model of middle cerebral artery occlusion （MCAO） and cerebral ischemia/reperfusion （ischemia for 90 minutes and reperfusion for 24 hours）. The mice in the inhibitor group and the agonist group were given intraperitoneal injection of Relaxin-3 inhibitor （10 μg/kg） and Relaxin-3 agonist （1 mg/kg）， respectively， at 30 minutes before modeling. TUNEL assay was used to observe neuronal apoptosis， qRT-PCR was used to measure the mRNA expression level of NLRP3， and Western blot was used to measure the protein expression level of Relaxin-3.Results Compared with the sham-operation group， both the model group and the inhibitor group had significant increases in the percentage of TUNEL-positive cells and the mRNA expression level of NLRP3 and a significant reduction in the protein expression level of Relaxin-3 （P<0.05）. Compared with the model group， the agonist group had a significant reduction in the percentage of TUNEL-positive cells （P<0.01）， a significant reduction in the mRNA expression level of NLRP3 （P<0.01）， and a significant increase in the protein expression level of Relaxin-3 （P<0.05）. Compared with the model group， the inhibitor group had significantly greater deterioration of the above indicators （P<0.05）.Conclusions Relaxin-3 exerts a neuroprotective effect against cerebral ischemia/reperfusion injury by inhibiting the expression of NLRP3 inflammasome and reducing neuronal apoptosis.