水蛭-虻虫配伍对脑出血体外模型血肿清除的影响及其作用机制  

Effect of the combination of leech and tabanus on the removal of hematoma in model of intracerebral hematoma in vitro and its mechanism

作　　者：王若男 赵德喜[2] 张昕烨[1] 崔妍[3] 吴九如 WANG Ruo-Nan;ZHAO De-Xi;ZHANG Xin-Ye(Nursing School,Changchun University of Chinese Medicine,Changchun 130000,Jilin,China)

机构地区：[1]长春中医药大学护理学院,吉林长春130000 [2]长春中医药大学脑病科,吉林长春130000 [3]长春中医药大学基础学院,吉林长春130000 [4]长春中医药大学针灸推拿学院,吉林长春130000

出　　处：《中国老年学杂志》2023年第9期2156-2161,共6页Chinese Journal of Gerontology

基　　金：国家自然科学基金项目面上项目(81774224);吉林省科技厅面上项目(YDZJ202301ZYTS156);吉林省中医药科技项目(2023006)。

摘　　要：目的研究水蛭-虻虫配伍对脑出血(ICH)体外模型脑血肿内源性清除的影响,探索其促进血肿吸收的作用机制,揭示中药配伍机制。方法取状态良好的小胶质细胞BV-2制成单细胞悬液,随机分为空白组、模型组、辛伐他汀组、不同浓度水蛭水提物组(0.1075、0.2150、0.4300、0.8600、1.7200、3.4400、6.8800、13.7600μg/ml)和不同浓度虻虫水提物组(0.134、0.269、0.538、1.075、2.150、4.250、8.500、17.000μg/ml),根据不同组别加入相应药物,应用噻唑蓝(MTT)法检测各浓度的水蛭、虻虫提取液对于BV-2存活率的影响;免疫荧光法观察红细胞(RBC)诱导BV-2细胞建立的ICH体外模型;流式细胞术检测水蛭、虻虫、水蛭-虻虫对BV-2细胞清除RBC的影响;Western印迹法检测水蛭、虻虫、水蛭-虻虫对BV-2内CD36、Toll样受体(TLR)4、过氧化物酶体增殖物激活受体(PPAR)γ、核因子-E2相关因子(Nrf)2表达的影响。结果MTT比色法显示,水蛭浓度≥6.88μg/ml,虻虫浓度≥4.25μg/ml对BV-2具有明显的抑制作用(P<0.05);免疫荧光法观察到BV-2吞噬了RBC,显示脑出血体外模型成立。流式细胞检测显示,与模型组相比,水蛭-虻虫组小胶质细胞未吞噬RBC数量明显下降(P<0.05)。Western印迹显示,与空白组比较,模型组CD36、TLR4、PPARγ、Nrf2蛋白表达明显上调(P<0.05);与模型组相比,水蛭-虻虫组TLR4蛋白表达明显下调(P<0.05),CD36、TLR4、PPARγ、Nrf2蛋白表达明显上调(P<0.05)。结论水蛭-虻虫配伍可以有效促进RBC诱导的BV-2 ICH体外模型血肿的内源性清除。其作用机制与抑制TLR4的表达,上调CD36、PPARγ、Nrf2蛋白表达相关。Objective To study the effect of the combination of leech and tabanus on endogenous removal of hematoma in model of intracerebral hematoma(ICH)in vitro,to explore the mechanism of action which promoted the absorption of hematoma,and reveal the compatibility mechanism of Chinese herbs.Methods Single-cell suspensions of well-cultured BV-2 microglia were prepared,and were divided into blank group,model group,simvastatin group,different concentration of leech water extract groups(0.1075,0.2150,0.4300,0.8600,1.7200,3.4400,6.8800,13.7600μg/ml)and different concentration of tabanus water extract groups(0.134,0.269,0.538,1.075,2.150,4.250,8.500,17.000μg/ml),corresponding drugs were added according to different groups.The effects of different concentrations of leech and tabanus extracts on the survival rate of BV-2 were determined by thiazolyl blue(MTT)assay;the ICH model of BV-2 cells induced by red blood cell(RBC)in vitro was observed through immunofluorescence method;the effects of leeches,tabanus and leeches-tabanus on the removal of RBC by BV-2 cells were detected by flow cytometry;the effects of leeches,tabanus and leeches-tabanus on CD36,Toll like receptor(TLR)4,peroxide-proliferating receptor(PPAR)γand nuclear factor-E2 related factor(Nrf)2 expressions in BV-2 cells were detected by Western blot.Results MTT assay showed that leech concentration≥6.88μg/ml and tabanus concentration≥4.25μg/ml which had a significant inhibititory effect on BV-2 cells(P<0.05).Immunofluorescence method showed that BV-2 cells swallowed the RBC which showed the intracerebral hemorrhage model in vitro was establish.Flow cytometry showed that compared with the model group,the amounts of RBC which not swallowed by microglia in leeches-tabanus group were significantly decressed(P<0.05).Western blot showed that compared with the blank group,the expressions of CD36,TLR4,PPARγand Nrf2 protein in model group were significantly incressed(P<0.05);compared with the model group,the expression of TLR4 protein in leeches-tabanus group was sig

关 键 词：水蛭 虻虫 脑出血 BV-2小胶质细胞 血肿清除 

分 类 号：R743.34[医药卫生—神经病学与精神病学]