中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2020, Vol. 15 ›› Issue (10): 1920-1930.doi: 10.4103/1673-5374.280321

• 原著:脑损伤修复保护与再生 • 上一篇    下一篇

L-半胱氨酸对蛛网膜下腔出血的神经保护机制

  

  • 出版日期:2020-10-15 发布日期:2020-08-19
  • 基金资助:
    国家自然科学基金（81873768，81671213，81571284，81874083）；山东省自然科学基金面上项目（2017GSF218091）；山东省自然科学基金项目（ZR2016HM33）；中国山东省医疗卫生科技发展计划项目（2017WS068）；山东省泰山学者项目（ts201511093）。

Neuroprotective mechanism of L-cysteine after subarachnoid hemorrhage

Ye Xiong1, 2, Dan-Qing Xin1, Quan Hu1, 3, Ling-Xiao Wang1, 2, Jie Qiu1, Hong-Tao Yuan1, Xi-Li Chu1, De-Xiang Liu4, Gang Li2, Zhen Wang1   

  1. 1 Department of Physiology, School of Basic Medical Sciences, Shandong University, Jinan, Shandong Province, China
    2 Department of Neurosurgery, Qilu Hospital of Shandong University and Brain Science Research Institute, Shandong University, Jinan, Shandong Province, China
    3 Department of Neurosurgery, Taian Central Hospital, Taian, Shandong Province, China
    4 Department of Medical Psychology and Ethics, School of Basic Medicine Sciences, Shandong University, Jinan, Shandong Province, China
  • Online:2020-10-15 Published:2020-08-19
  • Contact: Zhen Wang, PhD,wangzhen@sdu.edu.cn; Gang Li, ligangqiluhospital@163.com.
  • Supported by:
    The study was supported by the National Natural Science Foundation of China, Nos. 81873768 and 81671213 (to ZW), 81571284 and 81874083 (to GL); the Key Research and Development Foundation of Shandong Province of China, No. 2017GSF218091 (to ZW); the Natural Science Foundation of Shandong Province of China, No. ZR2016HM33 (to DXL); the Shandong Medical and Health Science and Technology Development Plan Project of China, No. 2017WS068 (to QH); the Taishan Scholars of Shandong Province of China, No. ts201511093 (to GL).

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摘要：

研究已表明，中枢神经系统中胱硫醚β合成酶催化左旋半胱氨酸(L-Cysteine，L-Cys)产生的硫化氢可能对实验性蛛网膜下腔出血有保护作用，但具体的作用机制未知。实验采用血管内穿孔法建立实验性蛛网膜下腔出血大鼠模型，造模后30 min腹腔注射30 μL 100 mM L-Cys。造模后48 h，(1)用HE染色测定前额叶皮质细胞变化发现，L-Cys 能明显减轻细胞水肿；(2)用改良Garcia评分评估神经功能缺损，用干湿法测定脑含水量发现，L-Cys能明显减轻蛛网膜下腔出血损伤后的神经功能缺损和脑水肿；(3)用免疫荧光技术检测激活的小胶质细胞的数量，用RT-PCR检测前额叶皮质白细胞介素1β和CD86 mRNA水平发现，L-Cys可抑制前额叶皮质中小胶质细胞的激活并降低白细胞介素1β和CD86的mRNA水平；(4)补体蛋白的RT-PCR和Western blot检测显示，L-Cys可降低前额叶皮质中补体系统C1q、C3α及其受体C3aR1蛋白的表达和C1q的沉积；(5)用DHE染色检测活性氧的变化，用免疫组织化学技术检测Nrf2和HO-1的阳性细胞数发现，L-Cys可降低前额叶皮质活性氧水平，Nrf2和HO-1的阳性细胞数；(6)用Western blot和免疫组织化学染色检测前额叶皮质中Chop和GRP78的阳性细胞数及蛋白表达发现，L-Cys可降低Chop和GRP78的蛋白表达以及Chop和GRP78的阳性细胞数；（7）以上全部结果同时还表明，采用CBS抑制剂氨基氧乙酸可显著逆转L-Cys的神经保护作用；（8）作者认为，L-Cys通过调节神经炎性反应，补体沉积，氧化应激和内质网应激，发挥了神经保护作用。

orcid: 0000-0003-3173-6961 (Zhen Wang) 

         0000-0003-3184-1032 (Gang Li)

关键词: 氨基氧乙酸, 中枢神经系统, 补体沉积, 胱硫醚-β-合成酶, 早期脑损伤, 内质网应激, 硫化氢, 神经炎症, 氧化应激, 蛛网膜下腔出血

Abstract: Hydrogen sulfide, which can be generated in the central nervous system from the sulfhydryl-containing amino acid, L-cysteine, by cystathionine- β-synthase, may exert protective effects in experimental subarachnoid hemorrhage; however, the mechanism underlying this effect is unknown. This study explored the mechanism using a subarachnoid hemorrhage rat model induced by an endovascular perforation technique. Rats were treated with an intraperitoneal injection of 100 mM L-cysteine (30 μL) 30 minutes after subarachnoid hemorrhage. At 48 hours after subarachnoid hemorrhage, hematoxylin-eosin staining was used to detect changes in prefrontal cortex cells. L-cysteine significantly reduced cell edema. Neurological function was assessed using a modified Garcia score. Brain water content was measured by the wet-dry method. L-cysteine significantly reduced neurological deficits and cerebral edema after subarachnoid hemorrhage. Immunofluorescence was used to detect the number of activated microglia. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the levels of interleukin 1β and CD86 mRNA in the prefrontal cortex. L-cysteine inhibited microglial activation in the prefrontal cortex and reduced the mRNA levels of interleukin 1β and CD86. RT-PCR and western blot analysis of the complement system showed that L-cysteine reduced expression of the complement factors, C1q, C3α and its receptor C3aR1, and the deposition of C1q in the prefrontal cortex. Dihydroethidium staining was applied to detect changes in reactive oxygen species, and immunohistochemistry was used to detect the number of NRF2- and HO-1-positive cells. L-cysteine reduced the level of reactive oxygen species in the prefrontal cortex and the number of NRF2- and HO-1-positive cells. Western blot assays and immunohistochemistry were used to detect the protein levels of CHOP and GRP78 in the prefrontal cortex and the number of CHOP- and GRP78-positive cells. L-cysteine reduced CHOP and GRP78 levels and the number of CHOP- and GRP78-positive cells. The cystathionine-β-synthase inhibitor, aminooxyacetic acid, significantly reversed the above neuroprotective effects of L-cysteine. Taken together, L-cysteine can play a neuroprotective role by regulating neuroinflammation, complement deposition, oxidative stress and endoplasmic reticulum stress. The study was approved by the Animals Ethics Committee of Shandong University, China on February 22, 2016 (approval No. LL-201602022).

Key words: aminooxyacetic acid, central nervous system, complement deposition, cystathionine-β-synthase, early brain injury, endoplasmic reticulum stress, hydrogen sulfide, neuroinflammation, oxidative stress, subarachnoid hemorrhage