Shuxuetong injection protects cerebral microvascular endothelial cells against oxygen-glucose deprivation reperfusion
Zuo-Yan Sun
Fu-Jiang Wang
Hong Guo
Lu Chen
Li-Juan Chai
Rui-Lin Li
Li-Min Hu
Hong Wang
Shao-Xia Wang
摘要：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 ofShuxuetong 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. Thisin vitro cell model was administeredShuxuetong injection at 1/32, 1/64, and 1/128 con-centrations (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 permeabil-ity. 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 thatShuxuetong 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 expression, reduces nuclear factor-κB p65 and vascular endothelial growth factor expression, and reduces I kappa B alpha, extracellular sig-nal-regulated protein kinase 1/2, and I kappa B kinase phosphorylation levels. Overall, these findings suggest thatShuxuetong injection has protective effects on brain microvascular endothelial cells after oxygen-glucose deprivation/reperfusion. Moreover, its protective effect is associated with reduction of mitochondrial superoxide production, inhibition of the inflammatory response, and inhibition of vascular endo-thelial growth factor, extracellular signal-regulated protein kinase 1/2, and the nuclear factor-κB p65 signaling pathway.
机标关键词：
分类号：R453(治疗学)R364(病理学)Q26(细胞生物化学)
论文发表日期：2019-01-01
页数：11( 783-793 )
英文信息