| Stroke is a common clinical disease with high incidence,high morbidity and high mortality.In all of stroke happens,ischemic stroke accounts for 80-85%,which belongs to Traditional Chinese Medicine “Zhongfeng” disease.The neurovascular unit(NVU)is a conceptual structure including neurons,microvessels and supporting cells.The components are interrelated each other to maintain the homeostasis of the brain microenvironment together.Houshiheisan(HSHS),a classic traditional compound prescription,was first documented in Synopsis of Golden Chamber(Jingui Yaolue)as the first decoction in stroke treatment.It was invented by Zhang Zhongjing,which based on Huangdineijing and under the guidance of etiology and pathogenesis of stroke,syndrome classification theory.The compatibility character of Houshiheisan is to use wind-dispelling drugs to assist deficiency-nourishing drugs.“Tonifying Qi and warming Yang to consolidate fundation,wind-dispelling drugs affect the liver via regulating Qi and blood”,which fully embodies the thinking “replenishing the deficient,reducing the excessive”.In this study,we aimed to investigate the protective effects of Houshiheisan on neurovascular unit damage after cerebral ischemia and try to explore its mode of action in vivo and in vitro.Objective 1.Neurological function evaluation and neuropathological morphology observation were used to assess the effect of Houshiheisan on neurorepairation after ischemic stroke;2.Cell culture,cell morphology observation,cell activity analysis and associated molecular biology detection technology were used to explore the effect of containing serum of Houshiheisan on ERK/CREB pathway and its downstream apoptotic associated molecules such as Bad,Bax,Bcl-2 and Caspase-3 in oxygen glucose deprivation(OGD)-induced SY5 Y cells;3.Molecular biology detection technologies were used to the explore the influence of chlorogenic acid,cynaroside,luteolin,ginsenoside Rg1 on expression of inflammatory cytokines like NO,TNF-?,IL-6,IL-10,TGF-?1 and NF-?B signaling pathway.Methods 1.Focal cerebral ischemia model was induced by permanent middle cerebral artery occlusion(p MCAO).Male Sprague-Dawley(SD)rats were randomly divided into 5 experimental groups: sham,model,wind-dispelling drugs,deficiencynourishing drugs and Houshiheisan groups;Zea Longa 5 grade neurological score was assessed after surgery;brain tissue pathological changes were evaluated by hematoxylin and eosin(HE)staining analysis;ultrastructure changes of neurovascular unit and mitochondrion were observed by transmission electron microscope;2.Using transwell to build neurons,glial cells and vascular endothelial cells co-culture system in vitro;cultured without oxygen or glucose to establish the neurovascular unit injury model;co-cultured cells were divided into 9 experimental groups: control,model,wind-dispelling drugs,deficiency-nourishing drugs,Houshiheisan,wind-dispelling drugs+U0126,deficiency-nourishing drugs+U0126,Houshiheisan+U0126 and U0126 groups;SY5Y cell morphology was observed;SY5Y cell activation was detected by CCK-8 detection;cell transendothelial electrical resistance(TEER)was detected via cell voltage resistance meter;the expression levels of ERK,p-ERK,CREB,p-CREB,Bad,Bax and Bcl-2 were detected by western blot(WB);the activity of Caspase-3 was detected by fluorescence colorimetric method;3.The mimic ischemia injured microglia model was induced by lipopolysaccharide(LPS);BV2 cells were randomly divided into 6 experimental groups: control,model,chlorogenic acid,luteolin,cynaroside,ginsenoside Rg1 treatment groups;cell morphology was observed by microscope;cell activity was detected by CCK-8 detection;the cyto-activity was detected using cell count kit.The NO content was determined by Griess Reagent;contents of TNF-? ? IL-6 ? IL-10 and TGF-?1 were detected by enzyme-linked immunosorbent assay(ELISA);The expression levels of p-p65 and p-I?B? were detected by western blot.Results 1.Compared with model group,Houshiheisan group markedly improved neurological function from 3-7 days after Pmcao(P < 0.05,P < 0.01);wind-dispelling drugs and deficiency-nourishing drugs groups improved neurological function 4-5 days after p MCAO(P < 0.05);Houshiheisan,wind-dispelling drugs and deficiencynourishing drugs groups alleviated pathological damage,reduced inflammatory cell infiltration and proliferation of glial cells degree,promoted the neuronal survival(P < 0.05,P < 0.05,P < 0.01);compared with the model group,Houshiheisan,winddispelling drugs and deficiency-nourishing drugs groups significantly reduced ultrastructural injury.The structure of neuronal,vascular and astrocyte remained intact,edema,nuclear pyknosis slightly,vacuole decreased,mitochondrial morphology is basically complete,swelling and vacuolization decreased,cristae number and morphology tended to be normal,the matrix density increased,high electron density granules and vacuoles reduced;2.Morphological observation showed that the model group cells were swelling and out of shape,the outline was indistinct,diopter degraded,granular degeneration can be seen in the cytoplasm,the intercellular space increased;TEER results showed that compared with the model group,each containing serum and containing serum+U0126 groups were improved markedly(P < 0.05,P < 0.01,P < 0.001),compared with wind-dispelling drugs group,Houshiheisan group were increased significantly(P < 0.05);CCK-8 detection showed that compared with model group,wind-dispelling drugs,deficiency-nourishing drugs,Houshiheisan and Houshiheisan+U0126 groups cell viability were increased significantly(P < 0.01,P < 0.001,P < 0.001,P < 0.05),compared with wind-dispelling drugs group,deficiencynourishing drugs group were increased significantly(P < 0.01);protein detection results showed that compared with model group,wind-dispelling drugs,deficiencynourishing and Houshiheisan drugs groups significant improved p-ERK protein expression(P < 0.05),compared with the wind-dispelling drugs group,wind-dispelling drugs+U0126 expression of p-ERK protein was significant decreased(P < 0.05),compared with the Houshiheisan group,Houshiheisan+U0126 group p-ERK protein expression was significant decreased(P < 0.01);compared with model group,deficiency-nourishing drugs and Houshiheisan groups p-CREB protein expression increased significantly(P < 0.001),compared with the deficiency-nourishing drugs,deficiency-nourishing drugs+U0126 group p-CREB protein expression decreased significantly(P < 0.001),compared with Houshiheisan group,Houshiheisan+U0126 group p-CREB protein expression significantly decreased(P < 0.001);compared with model group,deficiency-nourishing drugs and Houshiheisan groups Bad protein expression were significant decreased(P < 0.01,P < 0.05),compared with the deficiency-nourishing drugs,deficiency-nourishing drugs+U0126 group the expression of Bad protein increased obviously(P < 0.05),compared with the Houshiheisan group,Houshiheisan+U0126 group Bad protein expression increased significantly(P < 0.05);compared with model group,deficiency-nourishing drugs and Houshiheisan groups Bax protein expression were significant decreased(P < 0.05,P < 0.01),compared with the deficiency-nourishing drugs,deficiency-nourishing drugs+U0126 Bax protein expression increased significantly(P < 0.01);compared with model group,winddispelling drugs,deficiency-nourishing drugs,Houshiheisan and wind-dispelling drugs+U0126 groups Bcl-2 protein expression increased significantly(P < 0.001,P < 0.01,P < 0.01,P < 0.05);compared with model group,each containing serum and containing serum+U0126 groups significantly decreased the activity of Caspase-3(P < 0.001,P < 0.05),compared with wind-dispelling drugs group,Houshiheisan group Caspase-3 activity decreased significantly(P < 0.001),wind-dispelling drugs+U0126 Caspase-3 activity increased significantly(P < 0.01);3.Inflammatory factors results showed that chlorogenic acid,luteolin,cynaroside,ginsenoside Rg1 can significantly decrease the BV2 cell supernatant pro-inflammatory factor NO,TNF-?,IL-6 level(P < 0.001);chlorogenic acid,cynaroside,ginsenoside Rg1 can increase the antiinflammatory factor IL-10 level(P < 0.001),ginsenoside Rg1 can increase the antiinflammatory factor TGF-?1 level(P < 0.001);chlorogenic acid,luteolin,cynaroside,ginsenoside Rg1 can significantly decrease the p-p65(P < 0.01,P < 0.05,P < 0.05,P < 0.01)and p-I?B? protein expression(P < 0.05,P < 0.05,P < 0.05,P < 0.01).Conclusion 1.Wind-dispelling drugs,deficiency-nourishing drugs and Houshiheisan groups markly reduced neurological deficit after cerebral ischemia;reduced brain tissue pathological changes,promoted neuronal survival;reduced the neuronal,vascular,astrocyte and mitochondrial ultrastructure to protect the pathological injury of the neurovascular unit;2.Wind-dispelling drugs,deficiencynourishing drugs and Houshiheisan containing serums groups significant reduced the damage of SY5 Y cells in neurovascular unit after OGD injury,which may worked through ERK/CREB signaling pathway and its downstream apoptosis molecules,including Bcl-2,Bad,Bax expression and Caspase-3 activity.According to the results,Houshiheisan has the best effect,the two parts of whih wind-dispelling drugs and deficiency-nourishing drugs have their own emphasis;3.Chlorogenic acid,cynaroside,luteolin and ginsenoside Rg1 had regulating roles in cytokines NO,TNF-?,IL-6,IL-10 and TGF-?1 after ischemia injured via anti-inflammatory and inflammatory aspects,reduced the excessive activation of BV2 cells,and the mechanism of which may associated with NF-?B signaling pathway. |
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