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中华脑血管病杂志(电子版)

中华脑血管病杂志(电子版) ›› 2021, Vol. 15 ›› Issue (06) : 373 -381. doi: 10.11817/j.issn.1673-9248.2021.06.004

论著

PPAR-γ/Nrf2介导Heme-Hx-LRP1通路在脑出血后血肿清除中的作用机制
董梁1, 王改青2,†(), 段淑娜1, 柏琴琴1, 刘丽荣1   
  1. 1. 030001　太原，山西医科大学第二临床医学院神经内科
    2. 030001　太原，山西医科大学第二临床医学院神经内科；572000　三亚，三亚中心医院（海南省第三人民医院）神经内科
  • 收稿日期:2021-08-04 出版日期:2021-12-01
  • 通信作者: 王改青
  • 基金资助:
    国家自然科学基金面上项目(81771294); 深圳市科创委资助项目(JCYJ20190808161013492); 海南省临床; 医学中心建设项目资助

The mechanism of hematoma clearance followed intracerebral hemorrhage via PPAR-γ/Nrf2 mediated Hx-heme-LRP1 pathway

Liang Dong1, Gaiqing Wang2,†(), Shuna Duan1, Qinqin Bai1, Lirong Liu1   

  1. 1. Department of Neurology, the Second Clinical Medical College of Shanxi Medical University, Taiyuan 030001, China
    2. Department of Neurology, the Second Clinical Medical College of Shanxi Medical University, Taiyuan 030001, China; Department of Neurology, Sanya Central Hospital (Hainan Third People's Hospital), Sanya 572000, China
  • Received:2021-08-04 Published:2021-12-01
  • Corresponding author: Gaiqing Wang
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董梁, 王改青, 段淑娜, 柏琴琴, 刘丽荣. PPAR-γ/Nrf2介导Heme-Hx-LRP1通路在脑出血后血肿清除中的作用机制[J]. 中华脑血管病杂志(电子版), 2021, 15(06): 373-381.

Liang Dong, Gaiqing Wang, Shuna Duan, Qinqin Bai, Lirong Liu. The mechanism of hematoma clearance followed intracerebral hemorrhage via PPAR-γ/Nrf2 mediated Hx-heme-LRP1 pathway[J]. Chinese Journal of Cerebrovascular Diseases(Electronic Edition), 2021, 15(06): 373-381.

目的

探讨血红素（Heme）-血红素结合蛋白（Hx）-低密度脂蛋白受体相关蛋白1（LRP1）通路在脑出血后清除血肿中的作用，以及过氧化物酶体增殖物激活受体γ（PPAR-γ）/核因子E2相关因子2（Nrf2）对该通路的调控机制。

方法

采用Ⅳ型胶原酶大鼠脑出血模型；SD大鼠86只，随机分为手术对照组（n=20）、脑出血组（n=22）、PPAR-γ/Nrf2激动剂（红曲素）+脑出血组（n=21）、PPAR-γ抑制剂（格列卫）+脑出血组（n=23）。术后3 d应用头颅MRI不同序列测定血肿及水肿体积，同期测定血肿周围脑水含量及血红蛋白含量，应用免疫荧光双标法观察LRP1及Hx的表达部位，免疫印迹法测定PPAR-γ、Nrf2、LRP1、Hx的表达量。多组间血肿、水肿体积、血红蛋白含量、脑水含量、PPAR-γ、Nrf2、LRP1、Hx蛋白量比较采用单因素方差分析，组间多重比较采用Tukey's检验；血肿与血红蛋白含量、水肿与脑水含量相关关系采用Pearson相关分析。

结果

血肿、水肿体积、脑组织脑水含量及血红蛋白含量结果：格列卫+脑出血组＞脑出血组＞红曲素+脑出血组＞手术对照组；LRP1及Hx的表达量：红曲素+脑出血组＞脑出血组＞格列卫+脑出血组＞手术对照组；红曲素+脑出血组的LRP1及Hx在小胶质细胞的表达显著增高，且以血管周围更明显。相关分析显示MRI测量的血肿与脑组织血红蛋白含量呈显著正相关（r=0.956，P=0.005）；头颅MRI测量的水肿体积与脑水含量呈显著正相关（r=0.924，P=0.010）。

结论

红曲素作为PPAR-γ/Nrf2双靶点激动剂，可能通过强化血管周围小胶质细胞Hx及LRP1的表达促进血肿清除，同时减轻脑出血后水肿形成。提示PPAR-γ/Nrf2通过调控Heme-Hx-LRP1通路参与脑出血后血肿清除；头颅MRI测定脑组织血肿及水肿与脑组织检测血红蛋白及脑水含量所得出的结论一致。

关键词: 脑出血, 血肿清除, 血红素, 血红素结合蛋白, 低密度脂蛋白受体相关蛋白1
Objective

To explore the role of heme-hemopexin-low density lipoprotein receptor related protein 1 (LRP1) pathway in hematoma clearance followed intracerebral hemorrhage (ICH) as well as its regulatory mechanism of peroxisome proliferator-activated receptor γ (PPAR-γ)/nuclear factor-erythroid 2 related factor-2 (Nrf2).

Methods

ICH was induced by stereotactic, intrastriatal injection of type Ⅳ collagenase. 86 male SD rats were randomly divided into sham, ICH+vehicle (normal saline), ICH+monascin (dual PPAR-γ/Nrf2 agonist, 10 mg/kg, twice daily), ICH+gleevec (PPAR-γ antagonist, 100 mg/kg, once a day). Monascin and gleevec were administered by gastric perfusion for 3 days after ICH induction. Hematoma volume and brain edema were assayed by brain magnetic resonance imaging (MRI), and hemoglobin levels and brain water content were evaluated at 3 days after ICH. Brain expression of Hx/LRP1 with microglia were analyzed by double-labelling immunofluorescence and western blot. One-way analysis of variance (ANOVA) was performed for comparisons among multiple groups, and Tukey's test was used for pairwise comparison between groups. The correlation between hematoma and hemoglobin content, edema and cerebral water content were analyzed by Pearson correlation.

Results

Hematoma volume and brain edema assessed by MRI as well as hemoglobin levels and brain water content showed which were reduced in monascin-treated ICH while increased in gleevec-treated ICH; Expression of LRP1 and Hx on microglia were significantly increased after ICH, especially expressed around vessels in monascin-treated ICH. Pearson correlation analysis revealed that there was a positive correlation between edema volume shown by MRI and brain water content (r=0.956, P=0.005), hematoma volume as shown by MRI and brain hemoglobin levels (r=0.924，P=0.010).

Conclusion

As a PPAR-γ/Nrf2 dual-target agonist，monascin promoted hematoma removal through inducing LRP1 and Hx expressed on microglia around vessels, while alleviating secondary edema formation followed ICH; It showed that PPAR-γ/Nrf2 participates in hematoma removal after ICH by regulating Heme-Hx-LRP1 pathway; The determination of hematoma and edema by MRI was consistent with the detection of hemoglobin and brain water content in brain tissue.

Key words: Intracerebral hemorrhage, Hematoma clearance, Heme, Hemopexin, lipoprotein receptor related protein 1
图1 各组大鼠头颅MRI检查各序列图像（箭头所示为血肿核心）
图2 各组大鼠头颅MRI检查各序列血肿、水肿大小结果及分析。图a为T2*加权梯度回波成像（T2*WI）序列各组血肿体积比较；图b为磁敏感加权成像（SWI）序列各组血肿体积比较；图c为T2加权成像（T2WI）序列各组水肿体积比较；图d为T2液体衰减反转恢复（T2Flair）序列各组水肿体积比较；各组与脑出血组相比，差异具有统计学意义（*P＜0.05）
图3 各组大鼠脑水含量及血红蛋白含量比较。图a为各组大鼠干湿重法测定脑水含量结果：脑出血后各组脑水含量较手术对照组明显增加，差异具有统计学意义（*P＜0.05）；各组与脑出血组相比，红曲素+脑出血组脑水含量降低，格列卫+脑出血组脑水含量增加，差异具有统计学意义（#P＜0.05）；图b为各组血红蛋白含量结果：与手术对照组相比，脑出血后各组血红蛋白含量明显增加，差异具有统计学意义（*P＜0.05）；与脑出血组相比，红曲素+脑出血组血红蛋白含量降低，格列卫+脑出血组血红蛋白含量增加，差异具有统计学意义（#P＜0.05）
图4 各组LRP1表达量的免疫荧光染色。LRP1/Iba-1免疫荧光双标图片，可见Iba-1在脑出血后各组表达明显增多；LRP1表达在脑出血后数量略增多；红曲素+脑出血组明显增多，且以血管周围Iba-1增多明显；格列卫+脑出血组表达明显降低；且红曲素+脑出血组LRP1在血管周围表达增多
图5 各组Western blot LRP1/β-actin的增强化学发光法条带图像
图6 各组LRP1/β-actin的Western blot结果分析。各组与手术对照组相比，差异具有统计学意义（*P＜0.05）；各组与脑出血组相比，差异具有统计学意义（#P＜0.05）
图7 各组Hx免疫荧光染色及Western blot结果。各组Hx/Iba-1免疫荧光双标图片，可见Iba-1在脑出血后各组表达明显增多；Hx表达在脑出血后数量明显增多；红曲素+脑出血组较脑出血组明显增多；格列卫+脑出血组几乎无阳性表达；且红曲素+脑出血组Hx在血管周围表达增多明显
图8 各组Western blot Hx/β-actin的增强化学发光法条带图像
图9 各组大鼠Hx/β-actin的Western blot结果分析。各组与手术对照组相比，差异具有统计学意义（*P＜0.05）；各组与脑出血组相比，差异具有统计学意义（#P＜0.05）
图10 各组Western blot PPAR-γ、Nrf2、β-actin的增强化学发光法条带图像
图11 各组PPAR-γ/β-actin的Western blot结果分析。各组与手术对照组相比，差异具有统计学意义（*P＜0.05）；各组与脑出血组相比，差异具有统计学意义（#P＜0.05）
图12 各组Nrf2/β-actin的Western blot结果分析。各组与手术对照组相比，差异具有统计学意义（*P＜0.05）；各组与脑出血组相比，差异具有统计学意义（#P＜0.05）
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