Shuxuetong injection protects cerebral microvascular endothelial cells against oxygen-glucose deprivation reperfusion

• Published in: Neural regeneration research 2019-05, Vol.14 (5), p.783-793
• Main Authors: Sun, Zuo-Yan, Wang, Fu-Jiang, Guo, Hong, Chen, Lu, Chai, Li-Juan, Li, Rui-Lin, Hu, Li-Min, Wang, Hong, Wang, Shao-Xia
• Format: Article
• Language: English
• Subjects: Care and treatment; Cell culture; Cellular signal transduction; Dehydrogenases; Endothelium; Glucose; Kinases; Laboratories; nerve regeneration; Shuxuetong injection; brain microvascular endothelial cells; oxygen-glucose deprivation/reperfusion; tight junction proteins; mitochondrial function; inflammatory factors; blood-brain barrier; neuroprotection; neural regeneration; Permeability; Reactive oxygen species; Reperfusion injury; Stroke; Thermal cycling; Traditional Chinese medicine
• ISSN: 1673-5374; 1876-7958
• DOI: 10.4103/1673-5374.249226

Shuxuetong injection composed of leech (Hirudo nipponica Whitman) and earthworm (Pheretima aspergillum) has been used for the clinical treatment of acute stroke for many years in China. However, the precise neuroprotective mechanism of Shuxuetong injection remains poorly understood. Here, cerebral microvascular endothelial cells (bEnd.3) were incubated in glucose-free Dulbecco's modified Eagle's medium containing 95% N2/5% CO2 for 6 hours, followed by high-glucose medium containing 95% O2 and 5% CO2 for 18 hours to establish an oxygen-glucose deprivation/reperfusion model. This in vitro cell model was administered Shuxuetong injection at 1/32, 1/64, and 1/128 concentrations (diluted 32-, 64-, and 128-times). Cell Counting Kit-8 assay was used to evaluate cell viability. A fluorescence method was used to measure lactate dehydrogenase, and a fluorescence microplate reader used to detect intracellular reactive oxygen species. A fluorescent probe was also used to measure mitochondrial superoxide production. A cell resistance meter was used to measure transepithelial resistance and examine integrity of monolayer cells. The fluorescein isothiocyanate-dextran test was performed to examine blood-brain barrier permeability. Real-time reverse transcription polymerase chain reaction was performed to analyze mRNA expression levels of tumor necrosis factor alpha, interleukin-1β, interleukin-6, and inducible nitric oxide synthase. Western blot assay was performed to analyze expression of caspase-3, intercellular adhesion molecule 1, vascular cell adhesion molecule 1, occludin, vascular endothelial growth factor, cleaved caspase-3, B-cell lymphoma 2, phosphorylated extracellular signal-regulated protein kinase, extracellular signal-regulated protein kinase, nuclear factor-κB p65, I kappa B alpha, phosphorylated I kappa B alpha, I kappa B kinase, phosphorylated I kappa B kinase, claudin-5, and zonula occludens-1. Our results show that Shuxuetong injection increases bEnd.3 cell viability and B-cell lymphoma 2 expression, reduces cleaved caspase-3 expression, inhibits production of reactive oxygen species and mitochondrial superoxide, suppresses expression of tumor necrosis factor alpha, interleukin-1β, interleukin-6, inducible nitric oxide synthase mRNA, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1, markedly increases transepithelial resistance, decreases blood-brain barrier permeability, upregulates claudin-5, occludin, and zonula occludens-1 e