Chen Hansen, Luo Yunxia, Tsoi Bun, Gu Bing, Qi Suhua, Shen Jiangang
School of Chinese Medicine, The University of Hong Kong, 10 Sassoon Road, Pokfulam, Hong Kong, SAR, People's Republic of China.
School of Medical Technology, Xuzhou Medical University, Xuzhou, 221002, China.
Chin Med. 2022 Apr 27;17(1):51. doi: 10.1186/s13020-022-00595-7.
Hemorrhagic transformation (HT) is a common complication of delayed tissue plasminogen activator (t-PA) treatment for ischemic stroke. Peroxynitrite plays an important role in the breakdown of blood-brain barrier (BBB) and the development of HT. We tested the hypothesis that Angong Niuhuang Wan (AGNHW), a traditional Chinese medicinal formula, could be used in conjunction with t-PA to protect the BBB, minimize HT, and improve neurological function by suppressing peroxynitrite-mediated matrix metalloproteinase-9 (MMP-9) activation.
We first performed quality control study and chemical identification of AGNHW by using UPLC. In animal experiments, male Sprague-Dawley rats were subjected to 5 h of middle cerebral artery occlusion (MCAO) followed by 19 h of reperfusion plus t-PA infusion (10 mg/kg) at 5 h of cerebral ischemia. AGNHW (257 mg/kg) was given orally at 2 h after MCAO. Hemorrhagic transformation was measured using hemorrhagic scores and hemoglobin levels in ischemic brains. Evans blue leakage was utilized to assess the severity of the blood-brain barrier (BBB) damage. The modified neurologic severity score (mNSS) test was used to assess neurological functions. Peroxynitrite and superoxide was detected by using fluorescent probes. MMP-9 activity and expression were examined by gelatin zymography and immunostaining. The antioxidant effects were also studied by using brain microvascular endothelial b.End3 cells exposed to 5 h of oxygen and glucose deprivation (OGD) plus 5 h of reoxygenation with t-PA treatment (20 µg/ml).
AGNHW significantly reduced the BBB damage, brain edema, reduced hemorrhagic transformation, enhanced neurological function, and reduced mortality rate in the ischemic stroke rats with t-PA treatment. AGNHW reduced peroxynitrite and superoxide in vivo and in vitro and six active chemical compounds were identified from AGNHW with peroxynitrite scavenging activity. Furthermore, AGNHW inhibited MMP-9 activity, and preserved tight junction protein claudin-5 and collagen IV in the ischemic brains.
AGNHW could be a potential adjuvant therapy with t-PA to protect the BBB integrity, reduce HT, and improve therapeutic outcome in ischemic stroke treatment via inhibiting peroxynitrite-mediated MMP-9 activation.
出血性转化（HT）是缺血性卒中延迟组织型纤溶酶原激活剂（t-PA）治疗的常见并发症。过氧亚硝酸盐在血脑屏障（BBB）破坏和HT发生中起重要作用。我们检验了如下假设：中药方剂安宫牛黄丸（AGNHW）可与t-PA联合使用，通过抑制过氧亚硝酸盐介导的基质金属蛋白酶-9（MMP-9）激活来保护血脑屏障、使HT最小化并改善神经功能。
我们首先通过超高效液相色谱法（UPLC）对AGNHW进行质量控制研究和化学鉴定。在动物实验中，雄性Sprague-Dawley大鼠接受5小时大脑中动脉闭塞（MCAO），随后在脑缺血5小时时再灌注19小时并输注t-PA（10毫克/千克）。MCAO后2小时口服AGNHW（257毫克/千克）。使用缺血脑的出血评分和血红蛋白水平测量出血性转化。利用伊文思蓝渗漏评估血脑屏障（BBB）损伤的严重程度。使用改良神经功能缺损评分（mNSS）测试评估神经功能。使用荧光探针检测过氧亚硝酸盐和超氧化物。通过明胶酶谱法和免疫染色检测MMP-9活性和表达。还使用暴露于5小时氧糖剥夺（OGD）加5小时再氧合并接受t-PA治疗（20微克/毫升）的脑微血管内皮b.End3细胞研究抗氧化作用。
在接受t-PA治疗的缺血性卒中大鼠中，AGNHW显著减轻血脑屏障损伤、脑水肿，减少出血性转化，增强神经功能并降低死亡率。AGNHW在体内和体外均降低过氧亚硝酸盐和超氧化物水平，并且从AGNHW中鉴定出六种具有过氧亚硝酸盐清除活性的活性化合物。此外，AGNHW抑制MMP-9活性，并在缺血脑中保留紧密连接蛋白claudin-5和胶原蛋白IV。
AGNHW可能是一种与t-PA联合使用的潜在辅助疗法，通过抑制过氧亚硝酸盐介导的MMP-9激活来保护血脑屏障完整性、减少HT并改善缺血性卒中治疗的疗效。
