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 expression, reduces nuclear factor-κB p65 and
vascular endothelial growth factor expression, and reduces
I kappa B alpha, extracellular signal-regulated
protein kinase 1/2, and
I kappa B kinase phosphorylation levels. Overall, these findings suggest that
Shuxuetong 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 endothelial growth factor, extracellular signal-regulated
protein kinase 1/2, and the nuclear factor-κB p65 signaling pathway.