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, 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.展开更多
Objective To study the protective effect and mechanism of Shuxuetong on gerbil brain tissue from the area of ischemia-reperfusion. Methods Cerebral ischemia-reperfusion animal model by transient clipping bilateral com...Objective To study the protective effect and mechanism of Shuxuetong on gerbil brain tissue from the area of ischemia-reperfusion. Methods Cerebral ischemia-reperfusion animal model by transient clipping bilateral common carotid arteries in gerbils. Pathological changes in the hippocampal tissue were observed at different reperfusion time （12 h, 3 d, 7 d）. The expression levels of GABA and TNF-α in the hippocampal CA1 subfield were observed using immunohistochemitry at 12 h, 3 d after reperfusion. The difference of above indices among false operation group, ischemia-reperfusion group and treatment group were compared. Results The injuries of pathology to hippocampal area in ischemia-reperfusion group were more serious than treatment group. The expression levels of GABA in treatment group were significantly increased compared with ischemia-reperfusion group, but the expression levels of TNF-α between the two groups have no difference. Conclusion Shuxuetong has protective effect on brain tissue of ischemia-reperfusion by enhancing the expression of GABA in the hippocampal tissue.展开更多
Objective:To investigate the effect of Shuxuetong injection on platelet function, hemorheology and cerebral blood flow in patients with ischemic stroke.Methods: One hundred cases of ischemic stroke admitted in our hos...Objective:To investigate the effect of Shuxuetong injection on platelet function, hemorheology and cerebral blood flow in patients with ischemic stroke.Methods: One hundred cases of ischemic stroke admitted in our hospital from February 2015 to January 2017 were randomly divided into control group and observation group. The control group was given routine treatment. The observation group was given Shuxuetong injection on the basis of the control group. The changes of platelet function, hemorheology and cerebral blood flow before and after treatment were detected in both groups.Results: After treatment, the whole blood viscosity, plasma viscosity and fibrinogen level in both groups were significantly lower than those before treatment. The whole blood viscosity, plasma viscosity and fibrinogen level in the observation group were (4.18±0.05) mPa?s, (1.66±0.12) mPa?s and (3.45±0.08) g/L, respectively, which were significantly lower than the control group;After treatment, the levels of MCA, ACA and PCA in both groups were significantly increased. MCA, ACA and PCA levels in the observation group were (70.82±4.13) cm/s, (60.62±3.55) cm/s and (54.11±2.36) cm/s, which were significantly higher than those in the control group;After treatment, the maximum platelet aggregation rate, PLT, MPV and PDW levels in the two groups significantly decreased. The maximum platelet aggregation rate, PLT, MPV and PDW levels in the observation group were (27.93±1.44)% and (155.32±13.46)×109/L, (9.42±0.32) fL and (9.12±0.24) fL, respectively, which were significantly lower than those in the control group. Conclusions: Shuxuetong injection can effectively improve the patient's hemorheology and platelet function, improve the level of cerebral blood flow in patients with significant effect, it is worth further clinical application.展开更多
Objective：To explore the influence of the combination of Shuxuetong（SXT）and aspirin on coagulation and fibrinolytic system of rats.Methods：Suture method was applied to establish focal cerebral ischemia-reperfusion...Objective：To explore the influence of the combination of Shuxuetong（SXT）and aspirin on coagulation and fibrinolytic system of rats.Methods：Suture method was applied to establish focal cerebral ischemia-reperfusion injury models in rats.SD rats were randomly divided into sham group,middle cerebral artery occlusion/reperfusion（MCAO/R）group,aspirin group,SXT group,and SXT＋aspirin group（S＆A）.The neurological deficits were assessed according to Longa’s grade 5 scoring method.The cerebral edema was detected by measuring the content of water in brain tissue.The volume of cerebral infarction was observed by 2,3,5-Triphenyltetrazolium chloride（TTC）staining.Blood plasma was collected by abdominal aortic method to test maximum platelet aggregation rate and four blood coagulation.CD61,CD62p,6-keto prostaglandin F1αantithrombinⅢ（AT-Ⅲ）,D-dimer,plasminogen activator inhibitor-1（PAI-1）,tissue factor（TF）,tissue plasminogen activator（t-PA）,platelet thromboxaneand von Willebrand factor（v WF）content in rat plasma were detected by ELISA.Results：SXT combined with aspirin could improve the neurological deficits,alleviate cerebral edema,and decrease the cerebral infarct value.Compared with the sham operation group,fibrinogen（FIB）,6-AT-III,and t-PA in model group were significantly decreased;Compared with the model group,the above-mentioned indexes in SXT and aspirin treatment group were significantly increased.The prothrombin time（PT）,activated partial thromboplastin time（APTT）,thrombin time（TT）,D-dimer,PAI-1,TF,TXB2,and v WF of the model group were significantly increased;The above-mentioned indexes in blood SXT＋aspirin treated group were significantly decreased.There was a significant difference between the combined group and SXT group.The maximum concentration of plateletsin aspirin treated rats was significantly decreased,however,MPAR was reversed in SXT＋aspirin treated group.Conclusion：SXT combined with aspirin can effectively inhibit platelet activation,regulate the maximum concentration of platelets,and improve coagulation function and fibrinolysis system.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, 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.
文摘Objective To study the protective effect and mechanism of Shuxuetong on gerbil brain tissue from the area of ischemia-reperfusion. Methods Cerebral ischemia-reperfusion animal model by transient clipping bilateral common carotid arteries in gerbils. Pathological changes in the hippocampal tissue were observed at different reperfusion time （12 h, 3 d, 7 d）. The expression levels of GABA and TNF-α in the hippocampal CA1 subfield were observed using immunohistochemitry at 12 h, 3 d after reperfusion. The difference of above indices among false operation group, ischemia-reperfusion group and treatment group were compared. Results The injuries of pathology to hippocampal area in ischemia-reperfusion group were more serious than treatment group. The expression levels of GABA in treatment group were significantly increased compared with ischemia-reperfusion group, but the expression levels of TNF-α between the two groups have no difference. Conclusion Shuxuetong has protective effect on brain tissue of ischemia-reperfusion by enhancing the expression of GABA in the hippocampal tissue.
文摘Objective:To investigate the effect of Shuxuetong injection on platelet function, hemorheology and cerebral blood flow in patients with ischemic stroke.Methods: One hundred cases of ischemic stroke admitted in our hospital from February 2015 to January 2017 were randomly divided into control group and observation group. The control group was given routine treatment. The observation group was given Shuxuetong injection on the basis of the control group. The changes of platelet function, hemorheology and cerebral blood flow before and after treatment were detected in both groups.Results: After treatment, the whole blood viscosity, plasma viscosity and fibrinogen level in both groups were significantly lower than those before treatment. The whole blood viscosity, plasma viscosity and fibrinogen level in the observation group were (4.18±0.05) mPa?s, (1.66±0.12) mPa?s and (3.45±0.08) g/L, respectively, which were significantly lower than the control group;After treatment, the levels of MCA, ACA and PCA in both groups were significantly increased. MCA, ACA and PCA levels in the observation group were (70.82±4.13) cm/s, (60.62±3.55) cm/s and (54.11±2.36) cm/s, which were significantly higher than those in the control group;After treatment, the maximum platelet aggregation rate, PLT, MPV and PDW levels in the two groups significantly decreased. The maximum platelet aggregation rate, PLT, MPV and PDW levels in the observation group were (27.93±1.44)% and (155.32±13.46)×109/L, (9.42±0.32) fL and (9.12±0.24) fL, respectively, which were significantly lower than those in the control group. Conclusions: Shuxuetong injection can effectively improve the patient's hemorheology and platelet function, improve the level of cerebral blood flow in patients with significant effect, it is worth further clinical application.
基金supported by Natural Sciences Foundation of China(81303257)
文摘Objective：To explore the influence of the combination of Shuxuetong（SXT）and aspirin on coagulation and fibrinolytic system of rats.Methods：Suture method was applied to establish focal cerebral ischemia-reperfusion injury models in rats.SD rats were randomly divided into sham group,middle cerebral artery occlusion/reperfusion（MCAO/R）group,aspirin group,SXT group,and SXT＋aspirin group（S＆A）.The neurological deficits were assessed according to Longa’s grade 5 scoring method.The cerebral edema was detected by measuring the content of water in brain tissue.The volume of cerebral infarction was observed by 2,3,5-Triphenyltetrazolium chloride（TTC）staining.Blood plasma was collected by abdominal aortic method to test maximum platelet aggregation rate and four blood coagulation.CD61,CD62p,6-keto prostaglandin F1αantithrombinⅢ（AT-Ⅲ）,D-dimer,plasminogen activator inhibitor-1（PAI-1）,tissue factor（TF）,tissue plasminogen activator（t-PA）,platelet thromboxaneand von Willebrand factor（v WF）content in rat plasma were detected by ELISA.Results：SXT combined with aspirin could improve the neurological deficits,alleviate cerebral edema,and decrease the cerebral infarct value.Compared with the sham operation group,fibrinogen（FIB）,6-AT-III,and t-PA in model group were significantly decreased;Compared with the model group,the above-mentioned indexes in SXT and aspirin treatment group were significantly increased.The prothrombin time（PT）,activated partial thromboplastin time（APTT）,thrombin time（TT）,D-dimer,PAI-1,TF,TXB2,and v WF of the model group were significantly increased;The above-mentioned indexes in blood SXT＋aspirin treated group were significantly decreased.There was a significant difference between the combined group and SXT group.The maximum concentration of plateletsin aspirin treated rats was significantly decreased,however,MPAR was reversed in SXT＋aspirin treated group.Conclusion：SXT combined with aspirin can effectively inhibit platelet activation,regulate the maximum concentration of platelets,and improve coagulation function and fibrinolysis system.
