目的 探讨青蒿琥酯联合血必净注射液对实验性脑型疟小鼠的保护作用及机制探讨。方法 取 4~5 周龄 的雌性 C57BL/6 小鼠，随机分为空白对照组（0.9% NaCl）、感染对照组（0.9% NaCl）、血必净注射液组（血必净， 8 mL·kg-1 ，5 d）、青蒿琥酯组（青蒿琥酯，20 mg·kg-1 ，首剂加倍，5 d）、青蒿琥酯联合血必净注射液组（简称 联合用药组；血必净 8 mL·kg-1 +青蒿琥酯 20 mg·kg-1 ，青蒿琥酯首剂加倍，5 d），每组 20 只。按照上述分组腹 腔注射给药，每日 1 次。记录小鼠体质量、体温、外周血感染率及生存情况；伊文思蓝染色检测小鼠脑组织血 脑屏障通透性；苏木素-伊红（HE）染色检测小鼠脑组织病理变化；ELISA 法检测小鼠血清中肿瘤坏死因子 α （TNF-α）、干扰素 γ（IFN-γ）水平；Western Blot 法检测小鼠脑组织中紧密连接蛋白（Occludin）、闭锁小带蛋白 1（ZO-1）、血管内皮钙黏蛋白（VE-Cadherin）、血管生成素 1（Ang-1）、Ang-2、细胞间黏附分子 1（ICAM-1）、血管 细胞黏附分子 1（VCAM-1）、血管内皮生长因子（VEGF）、磷脂酰肌醇-3-激酶（PI3K）、蛋白激酶 B（Akt）、 p-Akt、内皮型一氧化氮合酶（eNOS）、p-eNOS 蛋白水平；免疫荧光法检测小鼠脑组织中 ICAM-1 蛋白水平； 总一氧化氮（NO）检测试剂盒检测小鼠脑组织中一氧化氮（NO）水平。结果 与空白对照组比，感染对照组小鼠 体温、体质量下降（P＜0.01），给药结束时感染率达到 30.99 %，生存率为 0，小鼠出现跛行、反射消失等神经 症状，小鼠昏迷及行为量表评分（RMCBS）评分下降（P＜0.01），血脑屏障通透性增大（P＜0.05），脑微血管中有 受感染红细胞和白细胞黏附，存在出血点，血清中 TNF-α、IFN-γ 水平上升（P＜0.05，P＜0.01），脑组织中 Occludin、ZO-1、VE-Cadherin、Ang-1、p-Akt/Akt 蛋白表达下降（P＜0.05，P＜0.01），ICAM-1、VCAM-1 蛋 白表达上升（P＜0.05，P＜0.01），NO 表达下降（但 P＞0.05）；与感染对照组比，联合用药组小鼠体温、体质量 上升（P＜0.01），给药结束时感染率为 0.67%，抑制率达到 97.84%，生存率为 20%，小鼠神经症状改善， RMCBS 评分上升（P＜0.01），血脑屏障通透性减小（但 P＞0.05），脑微血管无明显细胞黏附，血清中 TNF-α、 IFN- γ 水平下降（P＜0.05，P＜0.01），脑组织中 Occludin、ZO-1、VE-Cadherin、Ang-1、VEGF、PI3K、 p-Akt/Akt、p-eNOS/eNOS 蛋白表达上升（P＜0.05，P＜0.01），ICAM-1、VCAM-1 蛋白表达下降（P＜0.01）， NO 表达上升（P＜0.01）。结论 青蒿琥酯联合血必净注射液能有效改善脑型疟小鼠的症状体征，减少脑微血管 中的黏附，减轻过度炎症反应以及降低血脑屏障的通透性，其作用机制可能与激活 VEGF/Akt/eNOS 信号途径， 提高 NO 的表达有关。

Objective To investigate the protective effect and mechanism of artesunate combined with Xuebijing Injection on experimental cerebral malaria （ECM） mice. Methods Female C57BL/6 mice aged 4-5 weeks were randomly divided into blank control group （0.9% NaCl），infection control group （0.9% NaCl），Xuebijing Injection group （Xuebijing，8 mL·kg-1 ，5 days），artesunate group （artesunate，20 mg·kg-1 ，double the first dose，5 days）， artesunate combined with Xuebijing Injection group （“combination group”）. Xuebijing 8 mL·kg-1 +artesunate 20 mg·kg-1 ， double the first dose of artesunate，5 days），20 rats in each group. According to the above groups，intraperitoneal injection was given once a day. The body mass，body temperature，peripheral blood infection rate and survival of mice were recorded. Evans blue staining was used to detect the permeability of blood-brain barrier in mouse brain tissue；the pathological changes of brain tissue in mice were detected by hematoxylin-eosin （HE） staining. The levels of tumor necrosis factor-α （TNF-α） and interferon-γ （IFN-γ） in serum of mice were detected by ELISA. Western Blot was used to detect the protein levels of Occludin，ZO-1，VE-Cadherin，Ang-1，Ang-2，ICAM-1，VCAM-1，VEGF， PI3 K，Akt，p-Akt，eNOS and p-eNOS in brain tissue of mice. The level of ICAM-1 protein in brain tissue of mice was detected by immunofluorescence. The level of nitric oxide （NO） in brain tissue of mice was detected by total nitric oxide detection kit. Results Compared with the blank control group，the body temperature and body mass of the mice in the infection control group decreased （P＜0.01）. At the end of administration，the infection rate reached 30.99%，and the survival rate was 0. The mice showed neurological symptoms such as limp and loss of reflex. The Rapid Murine Coma and Behavioral Scale（RMCBS） score decreased （P＜0.01），and the permeability of the blood-brain barrier increased （P＜0.05）. There were infected red blood cells and white blood cells adhered to the brain microvessels，and there were bleeding points. The levels of TNF-α and IFN-γ in serum increased （P＜0.05，P＜0.01）. The protein expressions of Occludin，ZO-1，VE-Cadherin，Ang-1 and p-Akt/Akt in brain tissue decreased （P＜0.05，P＜0.01），the protein expressions of ICAM-1 and VCAM-1 increased （P＜0.05， P＜0.01）， and the expression of NO decreased （P＞ 0.05）；compared with the infection control group，the body temperature and body mass of the mice in the combined treatment group increased （P＜0.01）. At the end of administration，the infection rate was 0.67%，the inhibition rate reached 97.84%，the survival rate was 20%，the neurological symptoms of the mice were improved，the RMCBS score increased （P＜0.01），the permeability of the blood-brain barrier decreased （P＞0.05），there was no obvious cell adhesion in the brain microvessels，and the levels of TNF-α and IFN-γ in the serum decreased （P＜0.05，P＜0.01）. The protein expressions of Occludin，ZO-1，VE-Cadherin，Ang-1，VEGF，PI3K，p-Akt/Akt and p-eNOS/eNOS in brain tissue increased （P＜0.05，P＜0.01），the expressions of ICAM-1 and VCAM-1 decreased （P＜0.01），and the expression of NO increased （P＜0.01）. Conclusion Artesunate combined with Xuebijing Injection can effectively improve the symptoms and signs of ECM mice， reduce the adhesion in brain microvessels， alleviate excessive inflammatory reaction and reduce the permeability of blood-brain barrier. The mechanism may be related to the activation of VEGF/Akt/eNOS signaling pathway and the increase of NO expression.

R285.5

国家中医药管理局中医药国际合作专项（XDZYJZC-001）；国家自然科学基金项目（82374315）。