复方黄柏液对糖基化内皮细胞增殖迁移成管的影响

目的：通过研究复方黄柏液对糖基化内皮细胞增殖迁移成管的影响,寻找复方黄柏液治疗糖尿病足溃疡的依据。方法：为说明晚期糖基化终产物受体（RAGE）,用晚期糖基化终产物（AGEs）诱发内皮细胞功能障碍的重要受体,本研究设置了RT-PCR实验沉默了RAGE,结果发现沉默RAGE后,经AGEs处理内皮细胞,检测其内皮细胞增殖迁移成管作用出现上升,因此检测RAGE表达水平的变化能作为后续评价复方黄柏液改善内皮细胞功能的重要指标。如果RAGE水平出现下降,就证明内皮细胞功能障碍得到了修复,复方黄柏液治疗糖尿病足溃疡是有效的。为了评估内皮细胞的增殖能力,设置CCK-8实验;用划痕实验检查内皮细胞的迁移水平变化;通过成管实验来测试内皮细胞的成管作用;利用蛋白质印迹法检测RAGE蛋白表达水平。结果：RAGE-3 siRNA沉默效率最好,可用于后续实验。与空白组比较,AGEs处理后内皮细胞增殖活性、迁移水平、成管作用都下降,RAGE蛋白表达水平上升。与AGEs-BSA处理组比较,沉默RAGE或加入复方黄柏液后均较AGEs处理后内皮细胞的增殖迁移成管作用有了提高,RAGE蛋白的表达下降,差异有统计学意义（P<0.05）。结论：复方黄柏液可能通过改善AGEs诱发的内皮细胞功能障碍即促进内皮细胞增殖迁移成管来实现对糖尿病足溃疡的愈合。     

血府逐瘀汤促血管新生中VEGF通路的作用研究

目的:探讨VEGF通路在血府逐瘀汤促血管新生中的作用.方法:运用血清药理学方法,以1.25％,2.5％,5％的血府逐瘀汤含药血清和空白血清诱导处理血管内皮细胞ECV304,分别采用MTT比色法,Boyden小室迁移法和体外成血管实验检测内皮细胞增殖、迁移和成血管能力,并通过酶联免疫法、免疫组化和RT-PCR检测血管内皮生长因子(vascular endothelial growth factor,VEGF)和血管内皮生长因子受体-2(vascular endothelial growth factor receptor-2,VEGFR2)分泌和表达的情况.结果:1.25％含药血清除了能提高体外成血管能力外,对其余指标均无影响;2.5％含药血清明显促进内皮细胞增殖、迁移及成血管,并能显著提高VEGF及VEGFR2的表达;5％含药血清仅上调胞内VEGF的表达,进而促进内皮细胞的迁移和黏附.结论:血府逐瘀汤通过上调VEGF-VEGFR2途径,诱导内皮细胞增殖、迁移和血管形成,部分说明了药物促血管新生的作用机制.     

复方斑蝥胶囊抑制血管新生的体内体外研究

目的:通过体内和体外实验研究复方斑蝥胶囊对肿瘤血管新生相关因子的作用及可能机制。方法:通过免疫印迹试验检测复方斑蝥胶囊对Hep G2细胞中血管内皮生长因子(VEGF)蛋白表达的影响;选择裸鼠移植性肝癌(H-22)荷瘤模型,观察药物对荷瘤小鼠的抑癌作用,并通过免疫印迹试验检测肿瘤组织中VEGF蛋白的表达情况。结果:复方斑蝥胶囊对Hep G2细胞中VEGF的表达呈浓度和时间依赖性;复方斑蝥胶囊能够抑制H-22荷瘤小鼠肿瘤的生长,并且肿瘤组织中VEGF的表达水平随着药物浓度升高而降低。结论:复方斑蝥胶囊可能通过抑制肿瘤细胞VEGF的表达起到抑制肿瘤血管新生的作用。     

基于斑马鱼模型和分子对接技术的心可舒片促血管生成活性成分研究

目的 基于斑马鱼模型和分子对接技术研究心可舒片的促血管生成活性成分。方法 以人脐静脉内皮细胞HUVECs为模型,采用CCK-8与Ed U-488细胞增殖检测试剂盒检测细胞活性与增殖能力,采用划痕实验与Transwell实验评价细胞迁移能力,采用成管实验检测各组细胞成管腔能力,评价心可舒片对HUVECs的促血管生成活性。以斑马鱼为模型,采用血管内皮细胞生长因子受体抑制剂PTK787建立斑马鱼节间血管损伤模型,根据各组节间血管长度,评价心可舒片对斑马鱼的促血管生成活性。心可舒片29个成分与表皮生长因子受体（epidermal growth factor receptor,EGFR）、血管内皮生长因子受体1(vascular endothelial growth factor receptor 1,VEGFR1)、蛋白激酶B(protein kinase B,AKT)和VEGFR2的7个晶体结构进行分子对接,通过打分值与对接能筛选潜在活性成分,采用斑马鱼模型评价潜在活性成分的促血管生成活性。结果 心可舒片可显著提高细胞增殖能力（P<0.001）,提高细胞迁移能力及成管能力（P<0...     

当归补血汤对内皮祖细胞氧化应激损伤的保护作用

目的:研究当归补血汤(DBT)对过氧化氢(H2O2)诱导的小鼠骨髓来源内皮祖细胞(EPCs)氧化应激损伤的保护作用。方法:采用密度梯度离心法获取小鼠骨髓来源的单核细胞,通过特定培养基培养并鉴定获得EPCs,实验分为空白组,模型组,DBT组(100,200,400 mg·L-1)。采用噻唑蓝(MTT)比色法测定EPCs细胞存活率,构建H2O2诱导的细胞损伤模型;通过MTT,迁移小室(transwell),Matrigel基质胶,超氧化物荧光阴离子探针(DHE),分别检测EPCs增殖、迁移与体外血管形成能力,以及活性氧(ROS)水平,蛋白免疫印迹法(Western blot)检测细胞自噬相关蛋白泛素结合蛋白SQSTM1(p62),微管相关蛋白1轻链3Ⅱ型蛋白(LC3-Ⅱ)的蛋白表达。结果:与空白组比较,模型组EPCs细胞增殖能力、迁移能力、成管腔数及小管分支长度均显著降低(P0.01),胞内ROS水平显著升高(P0.01),自噬标志性蛋白p62显著升高(P0.01),LC3-Ⅱ蛋白表达降低(P0.01);与模型组比较,DBT组能呈浓度依赖性地升高EPCs增殖能力与迁移能力(P0.01),增加细胞成管腔数和小管分支长度(P0.01),降低胞内ROS水平(P0.01);此外,DBT呈浓度依赖性上调了LC3-Ⅱ蛋白的表达(P0.01),降低p62蛋白的表达(P0.01)。结论:当归补血汤能提高氧化应激状态下的EPCs自噬水平,促进损伤的EPCs迁移、增殖能力与血管形成能力,保护氧化应激条件下内皮祖细胞的生物学功能。     

当归补血汤含药血清对大鼠心肌微血管内皮细胞缺氧/复氧损伤的影响

目的:观察当归补血汤含药血清对缺氧/复氧模型大鼠心肌微血管内皮细胞增殖及促血管内皮生长因子的影响。方法:将缺氧/复氧心肌微血管内皮细胞模型大鼠随机分为模型组及当归补血汤含药血清低、中、高剂量(5%、10%、20%)组,另设正常对照组,干预8小时后,MTT法检测各组大鼠细胞的细胞增殖活力,同时检测乳酸脱氢酶(LDH)漏出量;Transwell法检测细胞迁移能力;酶联免疫吸附法检测细胞上清液中血管内皮生长因子、受体蛋白内源性配体(Apelin)含量。结果:当归补血汤含药血清干预后心肌微血管内皮细胞损伤明显减轻,细胞增殖活力及迁移能力明显改善,细胞上清液中血管内皮生长因子、Apelin含量明显增加,呈剂量依赖。结论:当归补血汤含药血清能明显促进大鼠心肌微血管内皮细胞血管新生能力,其机制可能与促进血管生长相关细胞因子分泌有关。     

黄连解毒汤对人胃癌SGC-803细胞株作用的实验研究

目的:观察黄连解毒汤对人胃癌细胞株SGC-803的作用及其机制。方法:体外培养胃癌细胞株SGC-803,应用黄连解毒汤干预胃癌SGC-803细胞。运用CCK-8实验检测SGC-803细胞增殖;划痕实验检测细胞迁移能力;流式细胞仪检测细胞凋亡水平; qRT-PCR法检测细胞相关基因mRNA的表达水平。结果:CCK-8实验结果表明,黄连解毒汤可有效抑制胃癌MGC-803细胞株的增殖,且呈现出药物浓度依赖性及时间依赖性。划痕实验结果表明,黄连解毒汤干预的实验组细胞迁移率的增长明显慢于对照组,具有统计学差异(P 0. 05)。流式细胞术检测细胞凋亡结果显示,黄连解毒汤可以有效促进胃癌细胞的凋亡,实验组MGC-803细胞的细胞凋亡率与对照组相比均有显著性差异(P 0. 05)。qRT-PCR结果显示,经黄连解毒汤干预,Bax、Caspase3基因mRNA表达上调,Bcl-2基因mRNA表达下调。结论:黄连解毒汤能够通过诱导SGC-803抑制胃癌细胞MGC-803增殖,抑制胃癌细胞迁移能力,推测其机制可能是通过影响凋亡相关蛋白Bcl-2、Bax的表达,发挥抗肿瘤作用。     

薄荷醇对肝癌HepG2细胞增殖、迁移及IL-8，CXCL-12，VEGF表达的影响

目的:体外实验观察不同剂量薄荷醇对肝癌HepG2细胞增殖、迁移及白细胞介素-8(IL-8),趋化因子-12(CXCL-12)和血管内皮生长因子(VEGF)表达的影响,阐释薄荷醇抗肝癌的作用及其相关机制。方法:设立薄荷醇(25,50,100,200μmol·L-1)组以及空白组作用于肝癌HepG2细胞,利用细胞增殖与毒性检测(CCK-8)法和5-乙炔基-2'脱氧尿嘧啶核苷(EDU)检测方法检测薄荷醇对HepG2细胞增殖的影响,利用迁移实验(Transwell)检测薄荷醇对HepG2细胞迁移能力的影响,利用实时荧光定量聚合酶链式反应检测薄荷醇处理HepG2细胞炎症和趋化因子IL-8,CXCL-12 mRNA表达水平,通过蛋白免疫印迹法(Western blot)检测薄荷醇处理对HepG2细胞中VEGF蛋白的表达水平。结果:与空白组比较,薄荷醇(25,50,100,200μmol·L-1)在体外对HepG2细胞增殖和迁移均有抑制作用。薄荷醇100μmol·L-1时抑制作用明显(P 0. 05);薄荷醇(50,100,200μmol·L-1)对HepG2细胞迁移能力有明显抑制作用(P 0. 05),呈剂量依赖性。与空白组比较,薄荷醇(100,200μmol·L-1)均能显著抑制HepG2细胞中的IL-8,CXCL-12 mRNA和VEGF蛋白的表达水平(P 0. 05),进一步证实薄荷醇通过抑制炎症趋化因子起到抗癌作用。结论:薄荷醇在体外对肝癌HepG2细胞增殖、迁移具有明显抑制作用,潜在机制可能与其抑制细胞IL-8,CXCL-12,VEGF的表达有关。此结论为薄荷醇抗肝癌作用的阐释提供了实验依据。     

复方苦参注射液对膀胱癌细胞增殖、侵袭的影响及相关机制分析

目的:探讨复方苦参注射液对膀胱癌细胞血管内皮生长因子(VEGF)、细胞凋亡蛋白Caspase-3、缺氧诱导因子-1α(HIF-1α)、干细胞标志物SOX-2及癌细胞增殖、侵袭的影响。方法:选用人膀胱癌细胞株S637,分为对照组(等体积DMSO)及复方苦参注射液0.25、0.5、1.0 mg/m L组,CCK-8法检测各组细胞增殖情况,FCM检测细胞凋亡情况,Transwell法检测细胞侵袭情况,RT-PCR检测VEGF、Caspase-3、HIF-1α及SOX-2 mRNA表达。结果:与对照组比较,复方苦参注射液各浓度组增殖率、侵袭能力及VEGF、HIF-1α、SOX-2 mRNA表达显著降低,凋亡率及Caspase-3 mRNA表达显著升高,且其作用呈浓度依赖性(P0.05)。结论:复方苦参注射液对膀胱肿瘤细胞的侵袭、增殖有抑制作用,其机制可能与降低VEGF、HIF-1α、SOX-2 mRNA表达,上调Caspase-3 mRNA表达有关。     

复方银杏叶颗粒改善人脐静脉内皮细胞氧化应激损伤的作用及其机制研究

该实验于体外使用人脐静脉内皮细胞(HUVEC)与复方银杏叶颗粒(80,160,320,640 mg·L-1)预孵育,合并使用H2O2(1 200μmol·L-1)建立氧化应激损伤模型。采用MTT法研究药物对HUVEC细胞增殖情况的影响,检测细胞培养上清中的LDH,MDA,NO含量及SOD活性,判断药物对内皮细胞的保护作用。通过Casepase-3活性检测以及Annexin V-FITC/PI流式细胞术,检测药物对细胞凋亡的保护作用。使用Western blot法测定凋亡相关蛋白Bcl-2和Bax的表达。研究发现1 200μmol·L-1H2O2能够诱导内皮细胞产生氧化应激损伤,使细胞存活率降低,且细胞增殖抑制程度与H2O2的作用时间呈正相关。而80,160,320,640 mg·L-1的复方银杏叶颗粒能够明显减轻该模型下内皮细胞的氧化应激损伤,恢复细胞正常增殖水平,改善细胞状态,抑制细胞凋亡,同时能够上调Bcl-2的表达以及下调Bax的蛋白表达。该实验证明了复方银杏叶颗粒对H2O2诱导的内皮细胞氧化应激损伤及细胞凋亡具有一定的保护作用,其机制可能与复方银杏叶颗粒抑制了内皮细胞凋亡的线粒体途径有关。     

抗血管内皮生长因子受体1和2双靶向寡肽融合蛋白A7/G6-力达霉素辅基蛋白的构建及其抗血管生成活性

目的 构建抗血管内皮生长因子受体1和血管内皮生长因子受体2双靶向寡肽融合蛋白A7/G6-力达霉素辅基蛋白并研究其抗血管生成活性及作用机制。方法 构建抗血管生成寡肽A7、G6与力达霉素辅基蛋白的融合蛋白A7/G6-力达霉素辅基蛋白表达载体;利用亲和层析进行蛋白纯化,产物进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳和高效液相色谱法分析;酶联免疫吸附法和免疫细胞化学分析A7/G6-力达霉素辅基蛋白与内皮细胞的结合活性;CCK-8法检测A7/G6-力达霉素辅基蛋白对人微血管内皮细胞-1增殖的影响;蛋白质印迹法分析A7/G6-力达霉素辅基蛋白对血管内皮生长因子信号通路影响;划痕法分析A7/G6-力达霉素辅基蛋白对人微血管内皮细胞-1迁移的影响;小管形成分析A7/G6-力达霉素辅基蛋白对人微血管内皮细胞-1血管生成的影响。结果 成功获得A7/G6-力达霉素辅基蛋白融合蛋白,产量20 mg·L^-1培养基,纯度较高;A7/G6-力达霉素辅基蛋白具有与人微血管内皮细胞-1和人脐静脉内皮细胞结合的活性;较高浓度能够抑制人微血管内皮细胞-1的增殖;能抑制内皮细胞的迁移和体外血管生成;其作用机制是通过G6和A7抑制血管内皮生长因子信号通路中AKT蛋白的磷酸化引起的。结论 针对血管内皮生长因子受体构建的双靶向寡肽融合蛋白A7/G6-力达霉素辅基蛋白具有抗血管生成的活性,为A7/G6-力达霉素辅基蛋白进一步体内活性研究和作为药物输送载体研究奠定了基础。     

灵芝孢子油对MCF-7细胞凋亡及迁移的影响

目的：观察灵芝孢子油对人乳腺癌MCF-7细胞增殖、迁移及凋亡的影响,并探讨其可能的作用机制。方法：CCK-8法检测灵芝孢子油对MCF-7增殖的影响;伤口愈合实验观察灵芝孢子油对MCF-7细胞迁移的作用;流式细胞术检测细胞凋亡率;RT—PCR法检测抗凋亡基因Bcl-2、促凋亡基因Bax及血管内皮生长因子（VEGF）mRNA表达水平。结果：不同浓度的灵芝孢子油对MCF-7的增殖和迁移均有一定程度的抑制作用,且呈时间、剂量依赖性;灵芝孢子油能显著诱导MCF-7细胞凋亡;RT—PCR结果显示,灵芝孢子油能上调Bax同时下调Bcl-2和VEGF的mRNA表达水平。结论：灵芝孢子油能抑制人乳腺癌MCF-7细胞增殖和迁移,并促进细胞凋亡,其作用机制可能与下调VEGF和Bcl-2／Bax的比值有关。     

苦参碱对NCI-N87胃癌细胞增殖凋亡及bcl-2蛋白表达的影响

目的:探讨不同浓度苦参碱对体外培养的人胃癌细胞NCI-N87的增殖、凋亡及bcl-2蛋白表达的影响。方法:NCI-N87细胞经不同浓度苦参碱处理后,采用CCK-8法检测药物对肿瘤细胞的抑制作用,显微镜观察细胞形态学改变;应用流式细胞仪分析苦参碱对细胞凋亡和bcl-2蛋白的影响。结果:苦参碱浓度>3mg/mL,细胞毒作用较大;浓度在0.5～2.0mg/mL时,对肿瘤细胞的生长有抑制作用,且呈时间剂量依赖性;浓度在0.1mg/mL,对肿瘤细胞生长几乎无抑制作用。流式细胞仪分析结果显示,细胞凋亡百分率随着药物浓度的增加和作用时间的延长而增加,而bcl-2蛋白则随之下降。结论:苦参碱对人胃癌细胞NCI-N87增殖有抑制作用,并能诱导细胞凋亡,下调bcl-2蛋白水平。     

香青兰有效部位对OGD/R损伤诱导的人脑微血管内皮细胞保护作用研究

目的 采用泛半胱氨酸天冬氨酸蛋白酶(cysteine aspartic acid protease,Caspase)抑制剂Z-VAD-FMK联合氧糖剥夺/复氧(oxygen-glucose deprivation/re-oxygenation, OGD/R)建立体外脑缺血再灌注损伤模型,探讨了香青兰有效部位(effective parts of Dracocephalum moldavica, EPDM)对人脑微血管内皮细胞(human brain microvascular endothelial cells,HBMECs)的保护作用。方法 CCK-8法检测细胞活力,倒置显微镜下观察细胞形态,NO测定试剂盒检测NO含量,transwell法检测细胞迁移情况,免疫荧光检测血管内皮-钙黏蛋白(VE-Cadherin)的表达情况,免疫印迹检测酪氨酸激酶(SRC)、内皮素-1(ET-1)、紧密连接蛋白Claudin 5、内皮性一氧化氮合酶(eNOS)、及磷酸化eNOS的蛋白表达。结果 与对照组比较,Z-VAD-FMK联合OGD/R可使HBMECs活力下降,细胞形态变差,细胞迁移减少,NO释放量降低,VE-cadherin表达减少,Src、Claudin 5、磷酸化eNOS表达减少,ET-1表达升高;而EPDM预处理可部分逆转Z-VAD-FMK联合OGD/R引起的上述因素的变化。结论 EPDM对体外脑缺血再灌注模型下的HBMECs具有保护作用,可促进细胞增殖、迁移,增强血管舒缩调节能力,并通过增强紧密连接和黏附连接维持屏障功能。     

Angiogenic efficacy of simplified 2-herb formula (NF3) in zebrafish embryos in vivo and rat aortic ring in vitro

Ethnopharmacological relevance

Diabetic foot ulceration results in high risk of lower extremity amputation, and represents a significant health care expenditure worldwide. Radix Astragali (RA) and Radix Rehmanniae (RR) are widely used Chinese medicinal herbs in treating diabetes, and have shown positive effects in enhancing wound healing in diabetic foot ulcer animal model.

Materials and methods

The angiogenic efficacy of NF3, a simplified 2-herb formula consisting of RA and RR in 2:1 ratio, was investigated. Median lethal concentration (LC50) and median effective concentration (EC50) were determined by treating zebrafish embryos with different concentrations of NF3 from 20 hpf to 72 hpf. The angiogenic activity of NF3 was examined in zebrafish embryos in vivo and by rat aortic ring assay in vitro. Cell cycle analysis of endothelial cells induced by NF3 was analyzed by flow cytometry using transgenic zebrafish Tg(fli1:EGFP). Real-time PCR was used to analyze mRNA expression profiles of selected genes involved in VEGF, FGF and MAPK pathways.

Results

NF3 enhanced blood vessel formation as indicated by extra growth of intersegmental vessels in zebrafish embryos, and increased microvessels formation in rat aortic ring. NF3 also enhanced endothelial cells proliferation as shown by increased percentage of cells accumulating in S phase and G2/M phase of the cell cycle. NF3 exposure significantly induced up-regulation of VEGF-A, Flk-1, fgf1 and bRaf expression in zebrafish embryos.

Conclusions

Our results demonstrated that NF3 was effective in promoting angiogenesis in zebrafish embryos and by rat aortic ring assay, which provided scientific basis to support the use of NF3 as potential therapeutics in treating diabetic foot ulceration.     

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??OBJECTIVE To investigate the inhibitory effects of bispecific antibody-drug conjugate Fv-LDM-NGR targeting EGFR and CD13 on human tumor cells and endothelial cells,and possible mechanisms. METHODS Human breast cancer cells MCF-7, human lung adenocarcinoma cells A549 and human microvascular endothelial cells HMEC-1, were studied. MTT assay was applied to measure proliferative activity of tumor cells. The influence of Fv-LDM-NGR on tube formation of HMEC-1 was observed. Transwell assay was applied to measure migration and invasion capacity in tumor cells. Western blot was applied for analyzing intracellular signaling transduction pathways. Flow cytometry, Hochest stain and Annexin ??-FITC/PI were used to detect cell cycle and apoptosis. RESULTS Fv-LDM-NGR could inhibit the proliferation of tumor cells and microvascular endothelial cells with IC₅₀ values of 10^-14-10^-12mol??L^-1. Fv-LDM-NGR prevented tube formation in microvascular endothelial cells, and suppressed migration and invasion in tumor cells. Fv-LDM-NGR interfered with the intracellular signaling transduction pathways, then caused G₂/M and S phase arrest and induced apoptosis. CONCLUSION Bispecific antibody-drug conjugate Fv-LDM-NGR could prevent cell invasion in tumor cells and tube formation in microvascular endothelial cells through blocking activity of CD13. And it could down-regulate the expression and the phosphorylation of EGFR, interfere with cellular signal pathways, induce cell cycle arrest and cell apoptosis, and inhibit cell proliferation and migration.     

雷公藤红素对胆管细胞的毒性作用及机制研究

目的研究雷公藤红素对人肝内胆管上皮细胞(human biliary epithelial cells,HIBEC)的毒性作用及机制。方法通过CCK-8实验及显微镜观察记录雷公藤红素对细胞形态和活力的影响;通过细胞划痕实验检测雷公藤红素对细胞迁移能力的影响;通过流式细胞术检测雷公藤红素对细胞周期的影响和对细胞凋亡的诱导作用;通过qRT-PCR和Western blotting检测雷公藤红素对凋亡相关基因Caspase-3、Bax、Bcl-2基因和蛋白的表达情况。结果雷公藤红素在400～2 000 nmol/L下,抑制细胞增殖,改变细胞形态;在200～800 nmol/L下,抑制细胞的迁移能力;在800～1 200 nmol/L下,出现G_0/G_1期阻滞作用;在400～1 200 nmol/L下,诱导细胞凋亡并增加相关基因的表达量。结论雷公藤红素可通过影响胆管细胞活力,改变其迁移能力,阻滞细胞周期并促进细胞凋亡的方式产生胆管毒性。     

虫草菌丝中3种活性成分对血管新生与内皮细胞功能的影响

目的探讨虫草菌丝中的3种活性成分(虫草素、腺苷、麦角甾醇)对血管新生和肝内皮细胞功能的影响。方法采用鸡胚绒毛尿囊膜实验和转基因斑马鱼实验分别观察虫草素、腺苷、麦角甾醇对新生血管面积、功能血管数量及碱性磷酸酶变化的影响;MTT法测定3个化合物对人肝癌SK-HEP-1细胞的细胞毒活性;采用血管内皮生长因子(VEGF)诱导SK-HEP-1细胞增殖为模型,以索拉非尼为阳性对照药,MTT法测定3个化合物对细胞增殖的影响,Transwell法观察细胞迁移,Matrigel管腔形成实验观察细胞管腔形成情况,荧光探针法检测胞内一氧化氮(NO)的量和一氧化氮合酶(NOS)活性。结果虫草菌丝中腺苷、麦角甾醇能够显著抑制鸡胚绒毛尿囊膜血管新生、减少转基因斑马鱼功能血管数量,而虫草素能够促进鸡胚绒毛尿囊膜血管新生;与对照组相比,VEGF可诱导SK-HEP-1细胞增殖,促进细胞迁移和管腔形成,并可使其胞内NO水平和NOS活性显著升高;与模型组相比,虫草素、腺苷、麦角甾醇呈剂量依赖性地抑制SK-HEP-1细胞增殖,腺苷、麦角甾醇能抑制SK-HEP-1细胞迁移,3者均可不同程度地抑制SK-HEP-1细胞管腔形成,且剂量依赖性地降低胞内NO水平和NOS活性。结论虫草菌丝中的虫草素、腺苷、麦角甾醇对SK-HEP-1细胞增殖、迁移、管腔形成有不同程度的抑制作用,其中腺苷的作用尤为明显;其机制与抑制细胞内NO水平和NOS活性有关。     

Synergistic interaction between Astragali Radix and Rehmanniae Radix in a Chinese herbal formula to promote diabetic wound healing

Ethnopharmacological relevance

Astragali Radix (AR) and Rehmanniae Radix (RR) are two traditional Chinese medicines widely used in China for treating diabetes mellitus and its complications, such as diabetic foot ulcer.

Aim of study

In our previous study, a herbal formula NF3 comprising AR and RR in the ratio of 2:1 was found effective in enhancing diabetic wound healing in rats through the actions of tissue regeneration, angiogenesis promotion and inflammation inhibition. The aims of the present study were to investigate the herb–herb interaction (or the possible synergistic effect) between AR and RR in NF3 to promote diabetic wound healing and to identify the principal herb in the formula by evaluating the potencies of individual AR and RR in different mechanistic studies.

Materials and methods

A chemically induced diabetic foot ulcer rat model was used to examine the wound healing effect of NF3 and its individual herbs AR and RR. For mechanistic studies, murine macrophage cell (RAW 264.7) inflammation, human fibroblast (Hs27) proliferation and human endothelial cell (HMEC-1) migration assays were adopted to investigate the anti-inflammatory, granulation formation and angiogenesis-promoting activities of the herbal extracts, respectively.

Results

In the foot ulcer animal model, neither AR nor RR at clinical relevant dose (0.98 g/kg) promoted diabetic wound healing. However, when they were used in combination as NF3, synergistic interaction was demonstrated, of which NF3 could significantly reduce the wound area of rats when compared to water group (p < 0.01). For anti-inflammation and granulation formation, AR was more effective than RR in inhibiting lipopolysaccharide (LPS)-induced nitric oxide production from RAW 264.7 cells and promoting Hs27 fibroblast proliferation. In the aspect of angiogenesis promotion, only NF3 promoted cell migration of HMEC-1 cells.

Conclusions

AR plays a preeminent role in the anti-inflammatory and fibroblast-proliferating activities of NF3. The inclusion of RR, however, is crucial for NF3 to exert its overall wound-healing as well as the underlying angiogenesis-promoting effects. The results of present study justified the combined usage of AR and RR in the ratio of 2:1 as NF3 to treat diabetic foot ulcer and illustrated that AR is the principal herb in this herbal formula.     

The in vivo and in vitro diabetic wound healing effects of a 2-herb formula and its mechanisms of action

Ethnopharmacological relevance

The herbs Radix Astragali (RA) and Radix Rehmanniae (RR) have long been used in traditional Chinese Medicine and serve as the principal herbs in treating diabetic foot ulcer.

Aim of study

Diabetic complications, such as foot ulcer, impose major public health burdens worldwide. In our previous clinical studies, two Chinese medicine formulae F1 and F2 have achieved over 80% limb salvage. A simplified 2-herb formula (NF3) comprising of RA and RR in the ratio of 2:1 was used for further study. NF3 was examined for the ulcer healing effect in diabetic rats, and its potential mechanisms of action in fibroblast proliferation, angiogenesis and anti-inflammation in vitro.

Materials and methods

A chemically induced diabetic foot ulcer rat model was used for studying the wound healing effect. In the in vitro mechanistic studies, human fibroblast cells (Hs27), human umbilical vein endothelial cells (HUVEC) and mouse macrophage cells (RAW264.7) were assessed for tissue regeneration, angiogenesis and anti-inflammatory activities, respectively.

Results

Our in vivo results demonstrated a significant reduction of wound area at day 8 in NF3 (0.98 g/kg) group as compared to control (p < 0.01). NF3 could significantly stimulate Hs27 proliferation in a dose dependent manner (p < 0.05). Besides, NF3 could significantly increase the cell migration and tube formation (p < 0.05-0.001) of HUVEC in the angiogenesis study. Furthermore, significant inhibition of nitric oxide production (p < 0.01) was found in NF3-treated macrophage cells, suggesting its anti-inflammatory activity.

Conclusions

Our study presents for the first time scientific evidence towards the efficacy of the two-herb formula NF3 in enhancing diabetic wound healing through the actions of tissue regeneration, angiogenesis and anti-inflammation.