和厚朴酚衍生物HH-A通过与血红蛋白相互作用缓解动物模型脑缺血缺氧损伤

doi:10.5246/jcps.2024.02.009

摘要/Abstract

摘要：

本研究旨在探讨一种新合成的和厚朴酚衍生小分子化合物HH-A在缺氧和永久性大脑中动脉闭塞条件下的抗缺氧以及减轻缺血缺氧所致脑损伤作用。利用大鼠低氧(11%氧含量)模型, 给予HH-A 1 mg/kg (i.v.)和40 mg/kg (i.g.)以及和厚朴酚0.55 mg/kg, 采用血气分析动脉血氧饱和度。利用大鼠永久性脑缺血模型, 给予HH-A 0.25和1 mg/kg (i.v.), 进行神经行为学评估, 手术7天后取脑组织进行脑梗死体积测量。使用表面等离子共振技术进行HH-A靶蛋白钓取; 利用缺氧探针评估脑组织缺氧程度; 采用NeuN免疫荧光染色和H&E染色评估脑组织缺血缺氧损伤程度; 分子对接试验分析预测HH-A与候选靶蛋白血红蛋白之间结合位点。结果显示, HH-A显著增加低氧状态下的血氧饱和度, 并且显著减少脑组织的缺氧程度; 在大鼠永久性脑缺血模型中, 1 mg/kg HH-A显著减少神经行为评分, 显著降低脑组织梗死体积, 缓解脑组织的损伤; 分子钓靶试验获得9个候选靶蛋白, 分子对接试验进一步验证HH-A与血红蛋白之间具有很高的亲和力。研究表明, HH-A可能通过血红蛋白发挥抗缺氧作用, 并且可以提高缺氧耐受性, 减轻脑缺血缺氧损伤。

关键词: 低氧, 脑卒中, 脑缺血损伤, 永久性大脑中动脉闭塞

Abstract:

Exposure to hypoxic conditions can result in significant brain damage, such as that experienced during an ischemic stroke. Thus, finding ways to mitigate ischemia/hypoxia-induced brain tissue damage is a critical issue that needs addressing. HH-A, a derivative of honokiol, has demonstrated potent pharmacological activities and medicinal properties in treating brain ischemia/reperfusion injury. However, its effect on anti-hypoxic responses during stroke remains largely unexplored. In the present study, we subjected male Sprague-Dawley rats to 24 h of hypoxia (oxygen content at 11%) or to 7 d of permanent middle cerebral artery occlusion. We discovered that both 1 mg/kg (i.v.) and 40 mg/kg (i.g.) of HH-A elevated arterial oxygen saturation after 24 h of hypoxia and significantly reduced the infarct volume after 7 d of ischemia. Furthermore, fluorescence staining with hypoxyprobe-1 indicated that HH-A significantly mitigated the severity of hypoxia in brain tissue. Hematoxylin and eosin staining, along with neuronal nuclei immunofluorescent staining, further revealed that HH-A curbed the death of brain cells. To identify potential protein partners of HH-A, we used a molecular fishing approach based on surface plasmon resonance technology. A high binding affinity was detected between HH-A and the hemoglobin subunit beta, with an estimated binding free energy of –8.7 kcal/mol. These findings suggested that HH-A was capable of enhancing hypoxia tolerance and alleviating brain injury caused by ischemia.

Key words: Hypoxia, Stroke, Brain ischemic injury, Permanent middle cerebral artery occlusion

Supporting:

张玉英, 张平平, 丁汉鑫, 张欣, 刘晔. 和厚朴酚衍生物HH-A通过与血红蛋白相互作用缓解动物模型脑缺血缺氧损伤[J]. 中国药学(英文版), 2024, 33(2): 98-109.

Yuying Zhang, Pingping Zhang, Hanxin Ding, Xin Zhang, Ye Liu. HH-A, a honokiol derivative, alleviates hypoxic brain injury in the animal ischemic model by interacting with hemoglobin[J]. Journal of Chinese Pharmaceutical Sciences, 2024, 33(2): 98-109.

图/表 6

Figure 1. Anti-hypoxia effect of HH-A in rats under hypoxic conditions. SaO2 was detected at 24 h before hypoxia treatment and 24 h after under hypoxic conditions (oxygen content, 11%). HH-A was administered at 1 mg/kg (i.v.) and 40 mg/kg (i.g.) before hypoxia treatment. Honokiol was administered at 0.55 mg/kg (i.v.) before hypoxia treatment. Data are reported as the mean (SD). Student t-test: *P < 0.05, **P < 0.01, ***P < 0.001 versus control group after hypoxia; #P < 0.05, ###P < 0.001 versus normoxia group; &P < 0.05 versus honokiol group after hypoxia.

Figure 2. Long-term effects of HH-A on pMCAO in rats. (A) Representative of TTC-staining of brain sections collected at 7 d after surgery. The white region shows the infarct area, while the red region shows the viable tissue; (B) Effect of HH-A on cerebral infarct volume at 7 d after surgery. Data are reported as the mean (SD). Student t-test: ***P < 0.001 versus vehicle group; (C) Effect of HH-A on Neurologic deficit scores at 1, 3 and 7 d after surgery. Data are reported as the mean (SD). Student t-test: **P < 0.01 HH-A (1 mg/kg) versus vehicle group.

Figure 3. Anti-hypoxia effect of HH-A in a cerebral ischemic mouse model. (A) FITC conjugated mouse IgG1 monoclonal antibody against hypoxyprobe-1 antibody (green) was used for immunofluorescence staining of the brain after 1.5 h of cerebral ischemic injury in mice (scale bar, 2 mm; nuclei are stained by DAPI in blue); (B) The integrated density of green. Data are reported as the mean (SD). One-way ANOVA: ***P < 0.001 versus vehicle group.

Figure 4. HH-A alleviates brain injury induced by ischemia. (A) Representative pictures of immunofluorescent staining of NeuN (red) in brain 7 d after cerebral ischemia in rats (scale bar, 2 mm; nuclei are stained by DAPI in blue); (B) Representative pictures of H&E staining in brain 7 d after cerebral ischemia in rats (scale bar, 200 µm). Arrows indicate the nucleus. NeuN, neuronal nuclei.

Table 1. Potential target proteins of HH-A.

Figure 5. Three-dimensional structure of the HBB complexed with HH-A. (A) The chemical structure of HH-A; (B) Protein crystal structure of HBB; (C) The binding pattern between HH-A and HBB; (D) HH-A and relative amino acids residues in HBB. HBB, hemoglobin subunit beta.

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