南方医科大学学报

南方医科大学学报 ›› 2022, Vol. 42 ›› Issue (7): 1050-1056.doi: 10.12122/j.issn.1673-4254.2022.07.13

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HMGB1下调的作用：通过抑制神经元细胞自噬和凋亡减轻大鼠脑出血后的神经元损伤

张 列，苗树船，杨中鑫，李宗喜，范英俊，余 凯，黄可阳，黄庆喜，夏 勋   

  1. 成都医学院四川养老与老年健康协同创新中心，四川 成都 610500；成都医学院第一附属医院神经外科，信息科，四川 成都 610500
  • 出版日期:2022-07-20 发布日期:2022-07-15

Suppression of HMGB1 inhibits neuronal autophagy and apoptosis to improve neurological deficits in rats following intracerebral hemorrhage

ZHANG Lie, MIAO Shuchuan, YANG Zhongxin, LI Zongxi, FAN Yingjun, YU Kai, HUANG Keyang, HUANG Qingxi, XIA Xun   

  1. Collaborative Innovation Center of Sichuan for Elderly Care and Health, Chengdu Medical College, Chengdu 610500, China; Department of Neurosurgery, Department of Information, First Affiliated Hospital of Chengdu Medical College, Chengdu 610500, China
  • Online:2022-07-20 Published:2022-07-15

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摘要： 目的 探讨高迁移率族蛋白1（HMGB1）在大鼠脑出血后对神经元细胞自噬和凋亡的作用机制。方法 将20只Wistar大鼠随机分为4组（5只/组）：假手术组（Sham组）、脑出血组、HMGB1中和抗体治疗（anti-HMGB1）组、control IgG组。模型组选择颅内纹状体注射0.2 U/mL胶原酶Ⅳ建立脑出血大鼠模型。Sham组颅内注射2 μL生理盐水，术后尾静脉注射PBS，6 h后再次注入等量PBS；anti-HMGB1组术后尾静脉注射1 mg/kg anti-HMGB1单克隆抗体，6 h后再次注入等量抗体；control IgG组术后尾静脉1 mg/kg anti-IgG单克隆抗体，6 h后再次注入等量抗体。采用改良神经损害程度（mNSS）评分评估大鼠神经功能和损伤程度。TUNEL染色检测纹状体神经元凋亡。Western blot检测血肿周围脑组织神经元细胞HMGB1、自噬蛋白（Beclin-1、LC3-II和LC3-I）和凋亡相关蛋白（Bcl-2、Bax和cleaved caspase-3）的表达。免疫组化检测纹状体组织中HMGB1阳性表达。ELISA检测血清HMGB1含量变化。结果 造模成功后，脑出血组中mNSS评分增加（P<0.05），给予anti-HMGB1单克隆抗体后，mNSS评分减少（P<0.05）。与 Sham 组相比，脑出血组大鼠纹状体神经元凋亡率增加（P<0.001），Beclin-1、LC3-II、Bax 和 cleavedcaspase-3的表达增加（P<0.05），而LC3-I和Bcl-2的表达减少（P<0.05），HMGB1含量增加（P<0.05）。给予anti-HMGB1单克隆抗体后，大鼠纹状体神经元凋亡率减少（P<0.05），Beclin-1、LC3-II、Bax和cleaved caspase-3的表达减少（P<0.05），而LC3-I和Bcl-2的表达增加（P<0.05），HMGB1含量减少（P<0.05）。结论 anti-HMGB1单克隆抗体下调HMGB1水平改善大鼠脑出血后的神经功能，其机制可能是抑制大鼠脑出血后的神经元细胞自噬和凋亡。

关键词: 高迁移率族蛋白1；脑出血；神经元细胞；自噬；凋亡

Abstract: Objective To investigate the effect of suppressing high-mobility group box 1 (HMGB1) on neuronal autophagy and apoptosis in rats after intracerebral hemorrhage (ICH) in rats. Methods Rat models of ICH induced by intracerebral striatum injection of 0.2 U/mL collagenase IV were treated with 1 mg/kg anti-HMGB1 mAb or a control anti-IgG mAb injected via the tail immediately and at 6 h after the operation (n=5). The rats in the sham-operated group (with intracranial injection of 2 μL normal saline) and ICH model group (n=5) were treated with PBS in the same manner after the operation. The neurological deficits of the rats were evaluated using modified neurological severity score (mNSS). TUNEL staining was used to detect apoptosis of the striatal neurons, and the expressions of HMGB1, autophagy-related proteins (Beclin-1, LC3-II and LC3-I) and apoptosis-related proteins (Bcl-2, Bax and cleaved caspase- 3) in the brain tissues surrounding the hematoma were detected using Western blotting. The expression of HMGB1 in the striatum was detected by immunohistochemistry, and serum level of HMGB1 was detected with ELISA. Results The rat models of ICH showed significantly increased mNSS (P<0.05), which was markedly lowered after treatment with anti- HMGB1 mAb (P<0.05). ICH caused a significant increase of apoptosis of the striatal neurons (P<0.05), enhanced the expressions of beclin-1, LC3-II, Bax and cleaved caspase-3 (P<0.05), lowered the expressions of LC3-I and Bcl-2 (P<0.05), and increased the content of HMGB1 (P<0.05). Treatment with anti-HMGB1 mAb obviously lowered the apoptosis rate of the striatal neurons (P<0.05), decreased the expressions of Beclin-1, LC3-II, Bax and cleaved caspase-3 (P<0.05), increased the expressions of LC3-I and Bcl-2 (P<0.05), and reduced the content of HMGB1 in ICH rats (P<0.05). Conclusion Down- regulation of HMGB1 by anti-HMGB1 improves neurological functions of rats after ICH possibly by inhibiting autophagy and apoptosis of the neurons.

Key words: high-mobility group box1; intracerebral hemorrhage; neuronal cells; autophagy; apoptosis