Zhou Huifen, He Yu, Zhu Jiaqi, Lin Xiaojie, Chen Juan, Shao Chongyu, Wan Haitong, Yang Jiehong
Institute of Cardiovascular-Cranial Disease, School of Life Sciences, Zhejiang Chinese Medical University, Hangzhou, China.
College of Pharmacy, Zhejiang Chinese Medical University, Hangzhou, China.
Front Pharmacol. 2021 May 13;12:650983. doi: 10.3389/fphar.2021.650983. eCollection 2021.
Guhong injection (GHI) can be used for the treatment of ischemic stroke. We investigated the antiapoptotic activity of GHI, its ability to repair the cerebral microvessels and mitochondria, and the PI3K/AKT signaling pathway of GHI against cerebral ischemia. Western blot and immunohistochemical analyses were used to determine the expression of cleaved caspase-3, B-cell lymphoma-2 (Bcl-2), cytochrome c (Cyt-c), basic fibroblast growth factor (BFGF), vascular endothelial growth factor (VEGF), transforming growth factor-β1 (TGF-β1), and proteins in the PI3K/AKT signaling pathway. Transmission electron microscopy and scanning electron microscopy were used to evaluate the structures of the cerebral microvasculature and cells. Hoechst 33342 staining was used to evaluate the nuclear morphology. FITC-AV/PI double staining was used to measure the antiapoptotic effects. The fluorescent dye JC-1 was used to measure mitochondrial membrane potential. The enzyme-linked immunosorbent assay (ELISA) was used to detect the activities of matrix metalloproteinase-9 (MMP-9). Biochemical assay kits were used to detect the activities of lactate dehydrogenase (LDH), superoxide dismutase (SOD), and malondialdehyde (MDA). Compared with the middle cerebral artery occlusion (MCAO) group, there was decreased infarct volume and significantly improved neurological deficits in the GHI group. In addition, the expression of Bcl-2 was significantly upregulated, while the expression of Cyt-c, Bax, and cleaved caspase-3 was notably downregulated. GHI administration attenuated the pathological change and morphology of the cerebral microvasculature, and immunohistochemical staining indicated that the expressions of BFGF, VEGF, and TGF-β1 were significantly increased. The cell morphology, cell viability, cell nuclei characteristics, and mitochondrial morphology normalized following GHI treatment, which decreased the release of Cyt-c and the mitochondrial membrane potential. The levels of LDH, MMP-9, and MDA decreased, while SOD increased. Moreover, GHI administration inhibited the activation of the PI3K/AKT signaling pathway in rat brain microvascular endothelial cells (rBMECs) following oxygen/glucose deprivation (OGD) injury. Therefore, our results show that GHI administration resulted in antiapoptosis of cerebral cells and repair of cerebral microvessels and mitochondria the PI3K/AKT signaling pathway.
骨红注射液（GHI）可用于治疗缺血性中风。我们研究了GHI的抗凋亡活性、修复脑微血管和线粒体的能力，以及GHI针对脑缺血的PI3K/AKT信号通路。采用蛋白质印迹法和免疫组织化学分析法测定裂解的半胱天冬酶-3、B细胞淋巴瘤-2（Bcl-2）、细胞色素c（Cyt-c）、碱性成纤维细胞生长因子（BFGF）、血管内皮生长因子（VEGF）、转化生长因子-β1（TGF-β1）的表达，以及PI3K/AKT信号通路中的蛋白质表达。采用透射电子显微镜和扫描电子显微镜评估脑微血管和细胞的结构。采用Hoechst 33342染色评估细胞核形态。采用FITC-AV/PI双重染色测量抗凋亡作用。采用荧光染料JC-1测量线粒体膜电位。采用酶联免疫吸附测定法（ELISA）检测基质金属蛋白酶-9（MMP-9）的活性。采用生化检测试剂盒检测乳酸脱氢酶（LDH）、超氧化物歧化酶（SOD）和丙二醛（MDA）的活性。与大脑中动脉闭塞（MCAO）组相比，GHI组梗死体积减小，神经功能缺损明显改善。此外，Bcl-2的表达显著上调，而Cyt-c、Bax和裂解的半胱天冬酶-3的表达显著下调。给予GHI可减轻脑微血管的病理变化和形态，免疫组织化学染色表明BFGF、VEGF和TGF-β1的表达显著增加。GHI治疗后细胞形态、细胞活力、细胞核特征和线粒体形态恢复正常，减少了Cyt-c的释放和线粒体膜电位。LDH、MMP-9和MDA水平降低，而SOD升高。此外，给予GHI可抑制氧/葡萄糖剥夺（OGD）损伤后大鼠脑微血管内皮细胞（rBMECs）中PI3K/AKT信号通路的激活。因此，我们的结果表明，给予GHI可导致脑细胞抗凋亡以及脑微血管和线粒体的修复，其作用机制与PI3K/AKT信号通路有关。
