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 neuroprotect...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% N_2/5% CO_2 for 6 hours, followed by high-glucose medium containing 95% O_2 and 5% CO_2 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, reduce展开更多
基金supported in part by the National Natural Science Foundation of China,No.81573644(to LMH),81573733(to SWX)the Tianjin 131 Innovative Team Project,China(to HW)+5 种基金the National Major Science and Technology Project of China,No.2012ZX09101201-004(to SWX)the Science and Technology Plan Project of Tianjin of China,No.16PTSYJC00120(to LMH)the Applied Foundation and Frontier Technology Research Program of Tianjin of China(General Project),No.14JCYBJC28900(to SXW)the National International Science and Technology Cooperation Project of China,No.2015DFA30430(to HW)the Key Program of the Natural Science Foundation of Tianjin of China,No.16ICZDJC36300(to HW)the Scientific Research and Technology Development Plan Project of Guangxi Zhuang Autonomous Region of China,No.14125008-2-5(to SXW)
文摘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% N_2/5% CO_2 for 6 hours, followed by high-glucose medium containing 95% O_2 and 5% CO_2 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, reduce
