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中华介入放射学电子杂志 ›› 2024, Vol. 12 ›› Issue (03) : 221 -227. doi: 10.3877/cma.j.issn.2095-5782.2024.03.006

基础研究

熊果酸通过抑制NLRP3介导的小胶质细胞焦亡减轻脑缺血再灌注损伤的研究
刘俊彬1, 张晓婷1, 郭镜培1, 刘佳妮2, 于本帅1,(), 张可1,(), 周斌1,()   
  1. 1. 519000 广东珠海,中山大学附属第五医院介入医学中心;519000 广东珠海,中山大学附属第五医院脑血管病中心;519000 广东珠海,中山大学附属第五医院广东省介入医学粤港澳高校联合实验室
    2. 519000 广东珠海,中山大学附属第五医院广东省介入医学粤港澳高校联合实验室;519000 广东珠海,中山大学附属第五医院肿瘤中心头颈部肿瘤科
  • 收稿日期:2024-05-24 出版日期:2024-08-25
  • 通信作者: 于本帅, 张可, 周斌
  • 基金资助:
    国家自然科学基金(82102164); 北京科创医学发展基金(KC2021-JX-0186-4)

Study on mechanism of ursolic acid ameliorating cerebral ischemia-reperfusion injury by inhibiting NLRP3-mediated microglia pyroptosis

Junbin Liu1, Xiaoting Zhang1, Jingpei Guo1, Jiani Liu2, Benshuai Yu1,(), Ke Zhang1,(), Bin Zhou1,()   

  1. 1. Department of Interventional Medicine; Center of Cerebrovascular Disease; Guangdong Provincial Engineering Research Center of Molecular Imaging
    2. Guangdong Provincial Engineering Research Center of Molecular Imaging; Department of Head and Neck Oncology, Cancer Center, the Fifth Affiliated Hospital of Sun Yat-sen University, Guangdong Zhuhai 519000, China
  • Received:2024-05-24 Published:2024-08-25
  • Corresponding author: Benshuai Yu, Ke Zhang, Bin Zhou
引用本文:

刘俊彬, 张晓婷, 郭镜培, 刘佳妮, 于本帅, 张可, 周斌. 熊果酸通过抑制NLRP3介导的小胶质细胞焦亡减轻脑缺血再灌注损伤的研究[J]. 中华介入放射学电子杂志, 2024, 12(03): 221-227.

Junbin Liu, Xiaoting Zhang, Jingpei Guo, Jiani Liu, Benshuai Yu, Ke Zhang, Bin Zhou. Study on mechanism of ursolic acid ameliorating cerebral ischemia-reperfusion injury by inhibiting NLRP3-mediated microglia pyroptosis[J]. Chinese Journal of Interventional Radiology(Electronic Edition), 2024, 12(03): 221-227.

目的

探索熊果酸(ursolic acid,UA)治疗缺血性脑卒中(ischemic stroke,IS)的潜在机制。

方法

使用细胞计数试剂(cell counting kit-8,CCK-8)检测UA对BV-2细胞存活率的影响,建立氧-葡萄糖剥夺/再灌注模型,使用蛋白免疫印迹法、免疫荧光、实时荧光定量聚合酶链式反应和酶联免疫吸附测定检测UA治疗后焦亡相关分子的表达水平。同时建立小鼠短暂性大脑中动脉闭塞模型,UA灌胃治疗后做死亡病例、神经功能评分统计和凋亡细胞计数,评估UA的神经保护作用。

结果

CCK-8实验结果表明,UA促进缺氧细胞存活。蛋白免疫印迹和免疫荧光结果表明,UA抑制缺氧细胞中焦亡蛋白的表达。实时荧光定量聚合酶链式反应和酶联免疫吸附测定结果表明,UA抑制缺氧引起的焦亡相关炎症因子表达。动物实验结果表明,UA降低IS小鼠的死亡率、Zea-Longa评分及凋亡细胞数量。

结论

本研究明确IS后UA可能通过抑制NLRP3炎症小体介导的小胶质细胞焦亡发挥神经保护作用。

Objective

To elucidate the mechanism of ursolic acid (UA) ameliorating cerebral ischemia-reperfusion injury.

Methods

An oxygen-glucose deprivation/reoxygenation (OGD/R) model in BV-2 microglia cells was established. Cell Counting Kit-8 (CCK-8) assay was used to measure cell viability. Western blot and immunofluorescence were conducted to measure pyroptotic protein levels. RT-qPCR was used to measure NOD-like receptor thermal protein domain associated protein 3 (NLRP3), Interleukin (IL)-1β, and IL-18 mRNA levels. ELISA was utilized to measure IL-1β and IL-18 concentration. The mice transient middle cerebral artery occlusion (tMCAO) model was established. UA was administered intragastrically after reperfusion. Neurological deficits were evaluated by Zea-Longa scoring criteria. Neuronal damage was assessed through TUNEL staining.

Results

UA promoted cell survival in OGD/R-challenged BV-2 cells. UA inhibited OGD/R-induced NLRP3 inflammasome activation and pyroptosis-related cytokines release in BV-2 cells. UA reduced mortality rates, Zea-Longa score, and neuronal damage in MCAO mice.

Conclusion

UA may exert neuroprotective effects by inhibiting NLRP3 inflammasome-mediated microglia pyroptosis.

图1 UA在缺氧条件下恢复BV-2细胞活力(n = 5)1A、1B:CCK-8检测不同浓度UA处理BV-2细胞后的细胞活力。OGD/R,氧糖剥夺/复氧;UA,熊果酸。*P < 0.05,**P < 0.01,***P < 0.001。
图2 UA在缺氧条件下抑制BV-2细胞焦亡蛋白表达(n = 3)注:蛋白免疫印迹法检测药物处理24 h后焦亡蛋白水平。GSDMD-FL,全长消皮素D;GSDMD-NT,N端消皮素D;NLRP3,NOD样受体热蛋白结构域相关蛋白3;OGD/R,氧糖剥夺/复氧;UA,熊果酸。*P < 0.05,**P < 0.01,***P < 0.001。
图3 UA在缺氧条件下抑制BV-2细胞焦亡蛋白表达(n = 3,× 25 μm)注:免疫荧光检测药物处理BV-2细胞后焦亡蛋白表达水平。GSDMD,消皮素D;NLRP3,NOD样受体热蛋白结构域相关蛋白3;OGD/R,氧糖剥夺/复氧;UA,熊果酸。
图4 UA在缺氧条件下抑制BV-2细胞焦亡蛋白表达4A:qPCR检测BV-2细胞中焦亡蛋白表达水平(n = 3);4B:ELISA检测BV-2细胞中焦亡蛋白表达水平(n = 5)。IL,白介素;NLRP3,NOD样受体热蛋白结构域相关蛋白3;OGD/R,氧糖剥夺/复氧;UA,熊果酸。*P < 0.05,**P < 0.01,***P < 0.001。
图5 UA的体内治疗效果5A:动物实验生存曲线(n = 10);5B:不同剂量UA治疗MCAO小鼠后用Zea-Longa评分标准评估小鼠神经功能缺损情况(n = 8);5C:大脑皮层缺血半暗带TUNEL阳性细胞统计(n = 6);5D:TUNEL染色代表图(×50 μm)。Sham,假手术组;tMCAO,短暂大脑中动脉栓塞;L-UA,低剂量熊果酸;H-UA,高剂量熊果酸。*P < 0.05,*** P <0.001。
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