新型细胞膜穿透肽增强Nrf2抗氧化能力在减轻慢性低灌注所致的白质脑网络及认知功能损害中的研究

Recent studies have demonstrated that "asymptomatic" carotid artery stenosis patients have cognitive impairment in tests of executive function, psychomotor speed and memory. Although hypoperfusion has been established as the most important causal factor leading to cognitive impairment, the pathophysiological mechanism has not been understood thoroughly. Deep white matter is the most important site of injury in the early stage of chronic hypoperfusion and oxidative stress is one of the main mechanisms. Hannink14 is a short peptide that can enhance the antioxidant capacity of nuclear factor E2 correlation factor 2 (Nrf2) by interfering the interaction between Nrf2 and Keap1. The new membrane-penetrating peptide, hCPP10, can provides a new pathway for Hannink14 to function. We hypothesize that hCPP10 could bring Hannink14 into the cell to promote the dissociation of Nrf2, further enhance the anti-oxidative effect of Nrf2 and finally alleviate the impairment of white matter brainnet and cognition in the early stage of chronic hypoperfusion. Based on our previous work, we plan to prepare a fusion peptide of hCPP10 and Hannink14 and then inject the fusion peptide into the lateral ventricle in a rat model of chronic hypoperfusion. Then utilize water maze and eight-arm maze to evaluate cognitive function, conduct brain networks based on diffusion tensor images (DTI) and carry on graph theoretical analysis at global and regional levels, utilize transmission electron microscopy and LFB staining to detect deep white matter ultrastructure damage, utilize laser confocal to observe the positioning of Nrf2, utilize ELISA to detect oxidative stress damage. By comparing these indicators in different time series, the present research will try to explore the feasibility of utilizing penetrating peptide to reduce the impairment of white matter brainnet and cognition in a rat model of chronic hypoperfusion.

慢性脑低灌注是“无症状”性颈动脉狭窄患者认知功能受损的主要原因。深部白质是慢性脑低灌注早期最主要的损伤部位，氧化应激为其主要受损机制之一。Hannink14为一种能够促进胞质内核因子E2相关性因子2(Nrf2)解离的短肽(氨基酸序列：LQLDEETGEFLPIQ)。新型细胞膜穿透肽为促使Hannink14发挥作用提供了新的途径。我们提出假设：穿膜肽可携带Hannink14入胞促进Nrf2的解离，进而增强Nrf2的抗氧化能力，进一步可减轻慢性低灌注早期的白质脑网络及认知功能受损。我们拟制备新型穿膜肽与Hannink14的融合肽，然后在大鼠慢性脑低灌注模型上进行侧脑室注射。通过术后不同时间点对氧化应激损伤程度、深部白质的超微损伤、白质脑网络的拓扑属性及认知功能的检测，从分子、超微病理、网络和整体水平探讨利用穿膜肽增强Nrf2抗氧化能力在减轻慢性脑低灌注早期白质脑网络及认知功能受损中的可行性。

本项目共包括三个子课题：.一，可靶向降解Keap1的多肽KKP1的研究。.基于细胞膜穿透肽和PROTAC技术，合成了多肽KKP1。细胞水平实验显示KKP1可有效进入HSC-T6细胞，并且对细胞活性没有明显影响。Western Blot实验表明KKP1可有效降解Keap1，并且可被蛋白酶体抑制剂抑制。进一步研究表明KKP1既可增强Nrf2/ARE信号通路促进GCLC和HO-1的表达，亦可抑制NF-κB信号通路，下调TNF-α、IL-1β和IL-6的表达。.二，RP101075减轻创伤性认知障碍机制的研究。.RP101075是1-磷酸鞘氨醇受体调节剂，能够抑制多种炎症反应。首先采用控制性皮层撞击损伤（CCI）模型法建立小鼠创伤性颅脑损伤模型，实验组接受RP101075治疗。mNSS试验和脚踏故障试验结果表明RP101075可减轻创伤性颅脑损伤的神经功能缺损。Morris水迷宫试验结果表明RP101075可改善创伤性颅脑损伤认知功能受损。湿脑重/干脑重法和HE染色数据显示RP101075可显著减少脑水肿和脑损伤体积百分比。流式细胞仪对免疫细胞的定量分析结果表明RP101075可显著减少T细胞、B细胞和NK细胞在损伤脑内的浸润。Western blot结果表明RP101075可显著降低脑损伤后MCP1和IL-1β的过度表达。.三，无症状性颈动脉狭窄患者认知功能受损的功能影像学研究。.无症状性颈动脉狭窄患者认知功能受损的机制尚不明确，我们纳入了24名无症状性重度颈动脉狭窄患者、24名共患病匹配对照以及39名健康对照进行研究。首先运用复合量表评价无症状性颈动脉狭窄患者认知功能受损的功能域，发现无症状性颈动脉狭窄患者存在多维度认知功能受损。然后运用复合磁共振技术评价灌注受损、微出血、脑白质病变及镜像同伦功能连接等，最后证实了外侧裂周区镜像同伦功能连接降低和延迟回忆等认知功能受损密切相关。.总之，我们成功合成了可靶向降解Keap1的多肽KKP1，阐明了1-磷酸鞘氨醇受体调节剂RP101075对创伤性认知障碍保护的机制，阐明了无症状性颈动脉狭窄患者认知功能受损的功能影像学机制。