二十二碳六烯酸对氧糖剥夺环境下大鼠脑星形胶质细胞凋亡及血管生成因子分泌的影响

摘要 目的：评价二十二碳六烯酸(Docosahexaenoic Acid,DHA)预处理对氧糖剥夺环境下(Oxygen and glucose deprivation,OGD)大鼠脑星形胶质细胞凋亡及血管生成因子分泌的影响。方法：大鼠脑星形胶质细胞传代培养,第3~4代用于实验。采用随机数字法将培养的细胞分为6组：正常对照组、OGD组、OGD+10 μMDHA组、OGD+40 μMDHA组、OGD+10 μMDHA+GW9662组、OGD+40 μMDHA+GW9662组。在所有缺氧模型组(除正常对照组外)先用无糖、无血清的DMEM液置换原培液;其次在OGD+10 μMDHA、OGD+40 μMDHA、OGD+10 μMDHA+GW9662、OGD+40 μMDHA+GW9662组加入相应浓度的DHA,同时在OGD+10 μMDHA+GW9662组和OGD+40μMDHA+GW9662组加入5 μM GW9662(过氧化物酶体增殖物激活受体PPARγ的抑制剂)。预处理完成后,正常对照组和其余各组分别在5% CO₂:95%空气和94％N₂:5％ CO₂:1％O₂条件下培养24 h。采用流式细胞技术检测细胞凋亡率,酶联免疫吸附剂测定（Enzyme linked immunosorbent assay,ELISA）检测培养上清液中的促血管生成素1（Angiopoietin-1,Ang1）、促血管生成素2（Angiopoietin2,Ang2）、血管内皮生长因子（vascular endothelial growth factor,VEGF）的分泌量,Western blotting法检测Bax、Bcl-2、caspase-3的表达。结果：与正常对照组比较,其余组细胞凋亡率、Bax、Caspase-3表达水平明显增加(P＜0.01),而Bcl-2、Bcl-2/Bax表达明显降低(P＜0.01)。与OGD组比较,OGD+10 μMDHA组和OGD+40 μMDHA组细胞凋亡率、Bax、Caspase-3表达水平明显降低(P＜0.01),而Bcl-2、Bcl-2/Bax表达明显增加(P＜0.01);Ang1分泌量明显增加(P＜0.01),而Ang2和VEGF分泌量明显降低(P＜0.01);上述各指标的差异在OGD+40 μMDHA组里更加显著(P＜0.01)。与OGD组比较,OGD+10 μMDHA+GW9662和OGD+40 μMDHA+GW9662组各个观察指标均无明显差异(P＞0.05)。相关性统计分析结果显示细胞凋亡率与Ang1水平呈显著负相关(P<0.01),与Ang2和VEGF的水平呈显著正相关(P<0.01)。结论：二十二碳六烯酸(DHA)预处理能够减少大鼠脑星形胶质细胞在氧糖剥夺(OGD)环境下的凋亡,其机制与增加Ang1分泌,减少Ang2和VEGF分泌,进而调控Ang/Tie2信号通路相关。     

小檗碱对缺血性脑梗死大鼠氧化应激/炎症反应、血管生成的作用研究

摘要 目的：探讨小檗碱对缺血性脑梗死大鼠氧化应激/炎症反应、血管生成的作用。方法：选取60只SPF级SD大鼠,随机分为对照组、模型组和小檗碱组各20只。建立大鼠脑缺血再灌注损伤模型。术后及给药后7d采用Longa标准评分评估大鼠神经功能。检测各组大鼠脑组织的抗氧化活性和炎症因子水平。采用免疫组化检测脑缺血再灌注皮质微血管密度（MVD）。采用实时定量聚合酶链反应（qRT-PCR）检测低氧诱导生长因子- 1 （HIF-1 ）和血管内皮生长因子（VEGF） mRNA表达水平。采用蛋白免疫印迹试验检测VEGF和HIF-1 蛋白表达水平。结果：模型组和小檗碱组大鼠术后具有神经功能缺损症状表现,Longa评分均高于对照组。给药7 d后,模型组和小檗碱组大鼠Longa评分均高于对照组（P＜0.05）,且小檗碱组大鼠Longa评分低于模型组（P＜0.05）。与对照组比较,模型组丙二醛（MDA）水平显著升高,而谷胱甘肽过氧化物酶（GSH-Px）和超氧化物岐化酶（SOD）活性显著降低（P＜0.05）。与模型组比较,小檗碱组MDA水平显著降低,而GSH-Px和SOD活性显著升高（P＜0.05）。与对照组比较,模型组白细胞介素-1β（IL-1β）、肿瘤坏死因子-α（TNF-α）水平显著升高（P＜0.05）。与模型组比较,小檗碱组IL-1β、TNF-α水平显著降低,差异有统计学意义（P＜0.05）。给药7 d后,模型组和小檗碱组MVD、VEGF mRNA和HIF-1 mRNA表达水平均高于对照组（P＜0.05）,而小檗碱组MVD、VEGF mRNA和HIF-1 mRNA表达水平高于模型组（P＜0.05）。给药7 d后,小檗碱组和模型组VEGF和HIF-1 蛋白表达水平均高于对照组（P＜0.05）,而小檗碱组VEGF和HIF-1 蛋白表达水平高于模型组（P＜0.05）。结论：小檗碱通过抑制氧化应激/炎症反应、促进血管生成从而达到脑保护作用,其机制可能与激活HIF-1 /VEGF信号通路有关。     

黄芪多糖基于PI3K/Akt通路对食管癌大鼠抑瘤作用及免疫功能的影响

摘要 目的：探究黄芪多糖（APS）对食管癌（EPC）模型大鼠的抑瘤作用、免疫功能及磷脂酰肌醇-3-激酶（PI3K）/蛋白激酶B（Akt）信号通路的影响。方法：选取6周龄SPF级健康SD大鼠50只,随机分为模型组(M组)、顺铂组（S组）、黄芪多糖低剂量组（APSL）、黄芪多糖中剂量组（APSM）、黄芪多糖高剂量组（APSH）,每组10只。通过移植人食管癌Eca109细胞建立EPC模型,分别给予3 mg/kg顺铂和100、200、400 mg/kg的APS干预。测量各组大鼠肿瘤体积和肿瘤质量,计算肿瘤生长抑制率、胸腺指数和脾指数,HE染色观察肿瘤组织形态学,采用流式细胞仪检测外周血CD3⁺、CD4⁺、CD8⁺T淋巴细胞群,Western blot测定各组肿瘤组织中PI3K和Akt蛋白的相对表达。结果：经干预后,S组和各APS组大鼠肿瘤体积和肿瘤质量均显著小于M组（P＜0.05）;S组和APSH组肿瘤体积和肿瘤质量相显著小于APSM和APSL组（P＜0.05）;APSM组大鼠肿瘤体积和肿瘤质量显著小于APSL组（P＜0.05）;APSH、APSM和APSL组的抑瘤率分别为45.59%、32.35%和17.65%。S组大鼠的胸腺指数和脾指数均显著低于M组（P＜0.05）;各APS组大鼠的胸腺指数和脾指数均显著高于S组（P＜0.05）;APSH和APSM组大鼠胸腺指数和脾指数均显著高于M组（P＜0.05）。各APS组大鼠CD3⁺、CD4⁺和CD4⁺/CD8⁺均显著高于S组和M组,而CD8+均显著低于S组和M组（P＜0.05）;APSH、APSM和APSL组之间两两相比亦均有统计学差异（P＜0.05）。S组和各APS组PI3K/Akt信号通路蛋白的相对表达均显著低于M组（P＜0.05）;APSH、APSM和APSL组之间两两相比亦均有统计学差异（P＜0.05）。结论：APS可通过调控PI3K/Akt 信号通路和改善免疫功能,发挥对EPC的抑制作用,为临床治疗EPC提供了新的思路和理论依据。     

扶正方对Lewis肺癌小鼠免疫功能、PI3K/AKT信号通路和外周血IL-2、IL-6、INF-γ的影响

摘要 目的：观察扶正方对Lewis肺癌小鼠免疫功能、磷脂酰肌醇3激酶（PI3K）/蛋白激酶B（AKT）信号通路和外周血白细胞介素（IL）-2、IL-6、γ干扰素（INF-γ）的影响。方法：将40只Lewis肺癌小鼠随机分为模型组（M组）、扶正方低剂量组（A组）、扶正方高剂量组（B组）、顺铂组（S组）,每组10只,A组、B组分别给予扶正方0.4 mL/20 g、0.8 mL/20 g灌胃,M组给予生理盐水0.4 mL/20 g灌胃,S组给予顺铂1 mg/mL,0.4 mL灌胃,连续14d,比较各组小鼠一般情况、肿瘤重量,胸腺指数、脾脏指数、脾脏CD3⁺细胞、CD4⁺细胞、CD8⁺细胞比例细胞百分比,鼠肿瘤组织PI3K/AKT信号通路蛋白表达水平及外周血IL-2、IL-6、INF-γ水平。结果：A组、B组、S组小鼠肿瘤重量低于M组,S组小鼠肿瘤重量低于A组、B组（P<0.05）,治疗前各组小鼠体重比较无统计学差异（P>0.05）,治疗后A组、B组小鼠体重高于S组、M组（P<0.05）。A组、B组小鼠胸腺指数显著高于M组、S组（P<0.05）。A组、B组CD3⁺、CD4⁺、CD4⁺/CD8⁺显著高于M组、S组,CD8⁺显著低于M组、S组（P<0.05）,B组CD3⁺、CD4⁺、CD4⁺/CD8⁺显著高于A组,CD8⁺低于A组（P<0.05）。A组、B组、S组小鼠肿瘤组织PI3K蛋白、AKT蛋白表达水平显著低于M组（P<0.05）。A组、B组、S组小鼠外周血IL-2、INF-γ水平显著高于M组,IL-6水平显著低于M组（P<0.05）。结论：扶正方可以提升Lewis肺癌小鼠免疫功能,调节IL-2、IL-6、INF-γ细胞因子水平,抑制PI3K/AKT信号通路起到抗肺癌的作用。     

独一味胶囊对复发性口腔溃疡大鼠免疫功能和溃疡组织NF-κB炎症通路蛋白表达的影响

摘要 目的：探讨独一味胶囊对复发性口腔溃疡（ROU）大鼠免疫功能和溃疡组织核因子-κB（NF-κB）炎症通路蛋白表达的影响。方法：将60只SD大鼠随机分为正常组、模型组和独一味胶囊低、中、高剂量组,每组12只。除正常组外其余各组均采用免疫法建立ROU模型,建模8周后独一味胶囊低、中、高剂量组分别灌胃给予0.01 mg/mL、0.02 mg/mL、0.03 mg/mL独一味胶囊溶液治疗,正常组和模型组灌胃给予等量生理盐水,均连续治疗20 d。记录各组大鼠口腔溃疡数目、持续时间和口腔溃疡面积,采用酶联免疫吸附法检测血清白介素（IL）-1β、IL-6、肿瘤坏死因子-α（TNF-α）水平,流式细胞术检测外周血CD3⁺、CD4⁺、CD8⁺百分比并计算CD4⁺/CD8⁺比值,免疫印迹法检测口腔黏膜组织NF-κB p65、磷酸化NF-κB抑制蛋白α（p-IκBα）、IkappaB激酶α（IKKα）蛋白水平。结果：正常组未出现口腔溃疡,模型组、独一味胶囊低剂量组、独一味胶囊中剂量组、独一味胶囊高剂量组溃疡数目依次降低,持续时间依次缩短,溃疡面积依次缩小（P＜0.05）。模型组、独一味胶囊低剂量组、独一味胶囊中剂量组、独一味胶囊高剂量组、正常组IL-1β、IL-6、TNF-α、CD8⁺、NF-κB p65、p-IκBα、IKKα水平依次降低,CD3⁺、CD4⁺、CD4⁺/CD8⁺依次升高（P＜0.05）。结论：独一味胶囊能显著减少ROU大鼠口腔溃疡数目和面积,缩短愈合时间,且效果呈剂量依赖性,其机制可能与改善免疫功能和抑制NF-κB炎症通路有关。     

基于PI3K/Akt信号通路探讨桃红四物汤促进老年股骨粗隆间骨折患者PFNA术后骨折愈合的疗效及其机制

摘要 目的：基于磷酯酰肌醇3激酶（PI3K）/丝氨酸蛋白激酶（Akt）信号通路探讨桃红四物汤促进老年股骨粗隆间骨折患者股骨近端抗旋髓内钉（PFNA）术后骨折愈合的疗效及其机制。方法：选取2021年7月-2022年12月期间遂宁市中医院收治的90例老年股骨粗隆间骨折行PFNA术患者,按照随机数字表法将患者分为对照组和研究组,各为45例。对照组术后接受常规干预,研究组在对照组基础上接受桃红四物汤干预。对比两组中医证候积分、骨折愈合时间、Harris髋关节功能评分、血液流变学、PI3K/Akt信号通路相关指标。结果：治疗后研究组髋部疼痛、痛有定处、神疲乏力、患肢软而无力、头晕眼花、失眠健忘、自汗畏风寒、胸闷气短、肌肤甲错、面色晄白无华评分和总分低于对照组（P<0.05）。研究组的骨折愈合时间短于对照组,Harris髋关节功能评分高于对照组（P<0.05）。研究组的红细胞压积、血浆比黏度、全血比黏度、红细胞电泳时间低于对照组（P<0.05）。研究组治疗后PI3K mRNA、AktmRNA高于对照组（P<0.05）。结论：老年股骨粗隆间骨折患者PFNA术后使用桃红四物汤,可促进患者临床预后转归,降低中医证候积分,促进髋关节功能恢复,缩短骨折愈合时间,改善机体血液流变学,调节PI3K/Akt信号通路表达。     

基于PI3K/AKT/mTOR信号通路探讨半枝莲总黄酮对脑缺血再灌注损伤大鼠神经功能和氧化应激损伤的影响

摘要 目的：基于磷脂酰肌醇3激酶（PI3K）/丝氨酸苏氨酸蛋白激酶（Akt）/哺乳动物的雷帕霉素（mTOR）信号通路探究半枝莲总黄酮对脑缺血再灌注损伤（CIRI）大鼠神经功能和氧化应激损伤的影响。方法：选取70只SPF级雄性SD大鼠,采用线栓法制备大脑中动脉闭塞（MCAO）模型,将造模成功的60只大鼠随机分为模型组、半枝莲总黄酮低剂量组（半枝莲L组）、半枝莲总黄酮中剂量组（半枝莲M组）、半枝莲总黄酮高剂量组（半枝莲H组）、control组、LY294002组,每组10只,剩余10只大鼠作为sham组。半枝莲L、M、H组分别给予半枝莲总黄酮0.1、0.2、0.4 g/kg溶于2 mL生理盐水灌胃,control组给予尼莫地平0.3 g/kg溶于2 mL生理盐水灌胃,LY294002组给予半枝莲总黄酮0.4 g/kg溶于2 mL生理盐水灌胃,同时侧脑室注射PI3K抑制剂LY294002 20 μmol/L,模型组和sham组给予等量的生理盐水灌胃。分别比较各组大鼠神经功能评分、脑组织积水量、脑梗死体积、脑组织病理学变化、脑组织氧化应激水平、脑组织中p-PI3K/PI3K、p-Akt/Akt、p-mTOR/mTOR蛋白表达。结果：各组大鼠神经功能评分、脑组织含水量、脑梗死体积、丙二醛（MDA）水平比较,模型组明显高于sham组,半枝莲L、M、H组、control组均低于模型组,且半枝莲H组优于半枝莲L、M组,但LY294002组高于半枝莲H组（P<0.05）;各组大鼠超氧化物岐化酶（SOD）水平和p-PI3K/PI3K、p-Akt/Akt、p-mTOR/mTOR蛋白表达比较,模型组明显低于sham组,半枝莲L、M、H组、control组均高于模型组,且半枝莲H组优于半枝莲L、M组,但LY294002组低于半枝莲H组（P<0.05）。结论：半枝莲总黄酮可能通过激活PI3K/Akt/mTOR信号通路发挥对CIRI大鼠神经功能和氧化应激损伤的改善作用,且高剂量半枝莲总黄酮的改善作用最显著。     

双歧杆菌对母婴分离大鼠成年后肠道敏感性及结肠脑源性神经营养因子表达的影响

摘要 目的：探讨双歧杆菌对母婴分离大鼠成年后肠道敏感性及结肠脑源性神经营养因子表达的影响。方法：将60只新生期SD大鼠随机分为NS组（正常组）、MS组（模型组）、MS+Bif组（双歧杆菌干预）,每组20只大鼠,应用避水应激模型(chronic water avoidance stress,WAS)避水实验构建大鼠应激波形,通过兴盛时期母婴分离建立大鼠的肠道高敏感模型,新生鼠在断奶之后对溶剂组与双歧杆菌组分别进行灌胃干预,取大鼠的新鲜粪便进行选择性培养基平皿技术方法检测大鼠粪便的菌群代表性菌种数量。到大鼠8 w成年之后应用阶梯体积直肠求精扩张对三组大鼠肠道敏感性进行评价。结果：与对照组相比,模型组大鼠的大肠杆菌和类杆菌数量明显增多,且通过双歧杆菌干预之后大肠杆菌和类杆菌数量下降,三组大鼠大肠杆菌和类杆菌数量差异显著（P＜0.05）,三组大鼠的双歧杆菌和乳酸菌对比无明显差异（P＞0.05）;对比大鼠成年时利用阶梯体积为0 mL、0.4 mL、0.8 mL和1.2 mL直肠球囊扩张评价三组大鼠的肠道敏感性发现,NS组与MS组在0 mL和0.4 mL体积球囊出现扩张的时候血管运动反应性(vascular motor reactivity,VMR)对比无明显差异（P＞0.05）,从0.4 mL到1.2 mL两组大鼠显著提高,且对比出现显著差异（P＜0.05）,MS组与MS+Bif组,在0 mL到0.4 mL体积球囊出现扩张的时候VMR对比无明显差异（P＞0.05）,在0.4 mL到1.2 mL之后VMR差异显著（P＜0.05）;对比大鼠血清中的促肾上腺皮质激素(adrenocorticotropic hormone,ACTH)和促肾上腺皮质激素释放因子(corticotropin releasing factor,CRF)发现,三组ACTH和CRF表达差异显著（P＜0.05）;对比大鼠结肠中脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)发现,三组大鼠的BDNF表达与SP（P物质）表达对比出现显著差异（P＜0.05）。结论：母婴分离大鼠成年之后会出现肠道高敏感性现象,应用双歧杆菌干预之后能够调节肠道菌群,稳定结肠脑源性神经因子表达,改善肠道敏感性现象。     

象皮生肌膏对肛瘘术后创面修复及PI3K/Akt/mTOR信号通路的影响

摘要 目的：探究象皮生肌膏对肛瘘切除术后大鼠创面的治疗作用及其与PI3K/Akt/mTOR信号通路的相关性。方法：将SD雄性大鼠随机分为假手术组、模型组、象皮生肌膏组、湿润烧伤膏组,共4组,每组10只。4组均采用0号钢丝制造肛瘘模型,造模成功后,除假手术组外,均在麻醉下行肛瘘切除术,创面保持开放,使之形成"开放、渗血、渗液、有脓性分泌物"的感染性肛瘘术后创面,假手术组保留瘘管,不予换药,模型组予以生理盐水冲洗、络合碘消毒后不予换药,治疗组分别予以相应药物进行创面换药,共10天。10天后采用常规面积检测法比较模型组、象皮生肌膏组、湿润烧伤膏组大鼠肛瘘术后创面的创面愈合率及创面肉芽组织覆盖率;HE染色观察各组大鼠肛周组织病理情况;ELISA检测各组大鼠血清中bFGF、EGF、VEGF的表达水平,WB检测比较各组大鼠肛周肉芽组织中PI3K、Akt、mTOR、p70 S6K、p-PI3K（S473）、p-AKT（S473）、p-mTOR（Ser2448）、p-p70 S6K的蛋白表达水平。结果：与模型组比较,治疗第3、7、10天象皮生肌膏组和湿润烧伤膏组的创面愈合率及创面肉芽组织填充率显著升高（P＜0.01）。病理切片显示,假手术组炎性细胞浸润较少,其余各组均可见不同程度的炎性细胞浸润,且创面区可见不同程度炎性修复型肉芽组织增生、胶原纤维新生及血管扩张;其中模型组病理切片显示大量炎性细胞浸润,血管扩张明显,并有明显的血管出血;象皮生肌膏组病理切片显示炎性细胞较少,成纤维细胞成熟且分布整齐,真皮层内胶原纤维丰富且排列整齐,可见血管新生,无明显血管扩张及出血;湿润烧伤膏组病理切片显示少量炎性细胞浸润及血管扩张,真皮层内可见成纤维细胞生成。ELISA检测结果显示,与假手术组比较,模型组血清中bFGF、EGF、VEGF的含量显著降低（P＜0.01）;与模型组比较,象皮生肌膏组血清中bFGF、EGF、VEGF的含量显著升高（P＜0.01）,湿润烧伤膏组大鼠血清中EGF、VEGF的含量显著升高（P＜0.01）。WB检测结果显示,与假手术组比较,模型组肛周组织中p-PI3K、p-Akt 、p-mTOR、p-p70 S6K蛋白表达显著降低（P<0.01）;与模型组比较,象皮生肌膏组肛周组织中p-PI3K、p-Akt 、p-mTOR、p-p70 S6K蛋白表达显著升高（P<0.05）。结论：象皮生肌膏能有效促进肛瘘术后创面修复,减轻肛瘘术后创面炎症反应,促进创面肉芽生长,其促愈机制可能与PI3K/Akt/mTOR信号通路相关蛋白表达有关,其通过上调PI3K/Akt/mTOR信号通路相关蛋白激活PI3K/Akt/mTOR信号通路,进而加快创面修复进程。     

人参皂苷Rh2对大鼠C6胶质瘤细胞Siah-1、Synaptophysin、MMP9及VEGF表达的影响

摘要 目的：探讨人参皂苷Rh2对大鼠C6胶质瘤细胞Siah-1、突触素（Synaptophysin）、基质金属蛋白酶-9（MMP-9）血管内皮生长因子（VEGF）表达的影响。方法：将大鼠C6胶质瘤细胞分为对照组、人参皂苷Rh2低剂量组（16 μg/mL）、人参皂苷Rh2中剂量组（32 μg/mL）、人参皂苷Rh2高剂量组（48 μg/mL），CCK-8法和平板克隆实验检测细胞增殖；流式细胞术检测细胞凋亡；Transwell检测细胞侵袭；实时荧光定量-聚合酶链式反应（qRT-PCR）检测C6胶质瘤细胞中VEGF、Siah-1、Synaptophysin、MMP-9 mRNA表达；蛋白质印迹法（Western blot）检测C6胶质瘤细胞中VEGF、Siah-1、Synaptophysin、MMP-9蛋白表达。结果：与对照组比较，人参皂苷Rh2低剂量组、人参皂苷Rh2中剂量组、人参皂苷Rh2高剂量组大鼠C6胶质瘤细胞OD₄₅₀值（24 h、48 h）、克隆形成率、细胞侵袭数、VEGF、Synaptophysin、MMP-9 mRNA及蛋白表达降低，细胞凋亡率、Siah-1 mRNA及蛋白表达升高，且呈剂量依赖性（P<0.05）。结论：人参皂苷Rh2可能通过上调Siah-1，下调VEGF、Synaptophysin、MMP-9表达来抑制大鼠C6胶质瘤细胞增殖与侵袭，促进细胞凋亡。     

梅花针叩刺联合桃红四物汤加味治疗气滞血瘀型白癜风的临床疗效及对血清免疫球蛋白和自身抗体的影响

摘要 目的：观察气滞血瘀型白癜风经桃红四物汤加味、梅花针叩刺联合治疗后的临床疗效及对血清免疫球蛋白（Ig）和自身抗体的影响。方法：根据随机数字表法将中国中医科学院广安门医院2019年3月~2022年4月期间收治的80例气滞血瘀型白癜风患者分为对照组（常规治疗的基础上结合梅花针叩刺治疗）和观察组（对照组的治疗基础上结合桃红四物汤加味治疗），各为40例。对比两组疗效、中医证候评分、白斑面积、出现黑素细胞时间、白斑的色素积分、血清自身抗体、Ig水平以及不良反应发生率。结果：观察组的临床总有效率为95.00%（38/40），高于对照组的75.00%（30/40）（P<0.05）。观察组治疗3个月后抗甲状腺球蛋白抗体（A-TG）、抗甲状腺过氧化物酶抗体（A-TPO）阳性率低于对照组（P<0.05）。观察组治疗3个月后面色晦暗、肌肤甲错、心烦不安、气郁不舒、失眠多梦评分低于对照组（P<0.05）。观察组治疗3个月后出现黑素细胞时间短于对照组，白斑的色素积分高于对照组，白斑面积小于对照组（P<0.05）。观察组治疗3个月后血清IgA、IgG、IgM高于对照组（P<0.05）。两组不良反应发生率比较无差异（P>0.05）。结论：气滞血瘀型白癜风经桃红四物汤加味联合梅花针叩刺治疗，临床症状得到显著改善，自身抗体阳性率降低，机体免疫力提高，且治疗安全性较高。     

VEGFR-2-mediated increased proliferation and survival in response to oxygen and glucose deprivation in PlGF knockout astrocytes

In hypoxic/ischemic conditions, astrocytes are involved in neuroprotection and angiogenesis. Vascular endothelial growth factor (VEGF) induces angiogenesis and exhibits neuroprotective and neurotrophic properties. However, the role of placental growth factor (PlGF), a VEGF homolog, in these processes is unclear. Therefore, proliferation and survival studies were performed on PlGF knockout (PlGF-/-) and wild-type (PlGF+/+) mouse astrocytes. A significant increase in cell proliferation and survival to oxygen and glucose deprivation (OGD) was observed in PlGF-/- compared to PlGF+/+ astrocytes. Interestingly, no PlGF protein expression was detected in PlGF+/+ astrocytes and no changes in VEGF protein levels were observed between the two genotypes. Real-time PCR and immunocytochemistry showed over-expression of VEGF receptor-2 (VEGFR-2) in PlGF-/- compared with PlGF+/+ astrocytes. Confocal microscopy revealed nuclear, membrane, and cytoplasmic localization of VEGFR-2. In vivo over-expression of VEGFR-2 mRNA was also detected in PlGF-/- compared with PlGF+/+ astrocytes. Stimulation with VEGF165 resulted in increased proliferation in PlGF-/- compared with PlGF+/+ astrocytes. This effect was blocked by the VEGFR-2 antagonist, VEGF165b. The enhanced proliferation of PlGF-/- astrocytes correlated with increased phospho-extracellular-signal-regulated kinase-1/2 levels, while the resistance to OGD was independent of the phosphatidylinositol 3'-kinase/Akt pathway. These results suggest that VEGFR-2 mediates the enhanced proliferative/OGD resistant phenotype observed in PlGF-/- astrocytes.     

Vascular endothelial growth factor acts through novel, pregnancy-enhanced receptor signalling pathways to stimulate endothelial nitric oxide synthase activity in uterine artery endothelial cells

During pregnancy, VEGF (vascular endothelial growth factor) regulates in part endothelial angiogenesis and vasodilation. In the present study we examine the relative roles of VEGFRs (VEGF receptors) and associated signalling pathways mediating the effects of VEGF(165) on eNOS (endothelial nitric oxide synthase) activation. Despite equal expression levels of VEGFR-1 and VEGFR-2 in UAECs (uterine artery endothelial cells) from NP (non-pregnant) and P (pregnant) sheep, VEGF(165) activates eNOS at a greater level in P- compared with NP-UAEC, independently of Akt activation. The selective VEGFR-1 agonist PlGF (placental growth factor)-1 elicits only a modest activation of eNOS in P-UAECs compared with VEGF(165), whereas the VEGFR-2 kinase inhibitor blocks VEGF(165)-stimulated eNOS activation, suggesting VEGF(165) predominantly activates eNOS via VEGFR-2. Although VEGF(165) also activates ERK (extracellular-signal-regulated kinase)-1/2, this is not necessary for eNOS activation since U0126 blocks ERK-1/2 phosphorylation, but not eNOS activation, and the VEGFR-2 kinase inhibitor inhibits eNOS activation, but not ERK-1/2 phosphorylation. Furthermore, the inability of PlGF to activate ERK-1/2 and the ability of the VEGFR-2 selective agonist VEGF-E to activate ERK-1/2 and eNOS suggests again that both eNOS and ERK-1/2 activation occur predominantly via VEGFR-2. The lack of VEGF(165)-stimulated Akt phosphorylation is consistent with a lack of robust phosphorylation of Ser(1179)-eNOS. Although VEGF(165)-stimulated eNOS phosphorylation is observed at Ser(617) and Ser(635), pregnancy does not significantly alter this response. Our finding that VEGF(165) activation of eNOS is completely inhibited by wortmannin but not LY294002 implies a downstream kinase, possibly a wortmannin-selective PI3K (phosphoinositide 3-kinase), is acting between the VEGFR-2 and eNOS independently of Akt.     

Anti-tumor effects of vascular endothelial growth factor/vascular endothelial growth factor receptor binding domain-modified chimeric antigen receptor T cells

Background aimsThe vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor (VEGFR) signaling pathway plays an important role in angiogenesis and lymphangiogenesis, which are closely related to tumor cell growth, survival, tissue infiltration and metastasis. Blocking/interfering with the interaction between VEGF and VEGFR to inhibit angiogenesis/lymphangiogenesis has become an important means of tumor therapy.MethodsHere the authors designed a novel chimeric antigen receptor (CAR) lentiviral vector expressing the VEGF-C domain targeting both VEGFR-2 and VEGFR-3 (VEGFR-2/3 CAR) and then transduced CD3-positive T cells with VEGFR-2/3 CAR lentivirus.ResultsAfter co-culturing with target cells, VEGFR-2/3 CAR T cells showed potent cytotoxicity against both VEGFR-2- and VEGFR-3-positive breast cancer cells, with increased simultaneous secretion of interferon gamma, tumor necrosis factor alpha and interleukin-2 cytokines. Moreover, CAR T cells were able to destroy the tubular structures formed by human umbilical vein endothelial cells and significantly inhibit the growth, infiltration and metastasis of orthotopic mammary xenograft tumors in a female BALB/c nude mice model.ConclusionsThe authors’ results indicate that VEGFR-2/3 CAR T cells targeting both VEGFR-2 and VEGFR-3 have significant anti-tumor activity, which expands the application of conventional CAR T-cell therapy.     

Circulating vascular endothelial growth factor (VEGF) and its soluble receptors in patients with chronic lymphocytic leukemia

The vascular endothelial growth factor (VEGF) transduction pathway may be very active in B-cell chronic lymphocytic leukemia (B-CLL) cells and contributes to their enhanced survival. Vascular endothelial growth factor receptor-1 (VEGFR-1) and receptor-2 (VEGFR-2), are the high-affinity VEGF receptors, which play an important role in de novo blood vessel formation and hematopoietic cell development. The aim of our study was to compare the concentration of VEGF, VEGFR-1 and VEGFR-2 in the serum of 83, never-treated B-CLL patients in different stage of disease according to Rai classification, and 20 healthy volunteers. Of all the cytokines only the serum concentration of VEGF was found to be significantly higher in the CLL group when compared to the control group (median 468.2 pg/mL and 246.9 pg/mL, respectively) (p = 0.01). In the group of CLL patients, the serum concentrations of VEGF and VEGFR-2 were significantly higher in patients in Rai stage III and IV (median 890.0 pg/mL and 4680.4 pg/mL respectively) than in patients in Rai stage 0-II (347.8 pg/mL and 2411.6 pg/mL respectively) (p<0.0001). In the entire group of CLL patients, we have found a strong, positive correlation between the serum level of VEGF and VEGFR-2 (p = 0.00001, R = 0.46). We have also found a positive correlation between the number of lymphocytes in the peripheral blood of CLL patients and the level of VEGF (p = 0.05, R = 0.24) and VEGFR2 (p = 0.02, R = 0.29). In conclusion: VEGF and VEGF R2, but not VEGF R1, may have an important influence on the course of B-CLL.     

RUNX1对氧糖剥夺诱导PC12细胞凋亡的保护作用研究

目的:研究RUNX1在PC12细胞氧糖剥夺模型中的表达及其对PC12细胞的保护作用,并探讨其相关机制。方法:体外培养PC12细胞并构建氧糖剥夺模型,将细胞分为对照组、氧糖剥夺组、RUNX1 si RNA处理组、si RNA对照处理组(sicontrol)、pc DNA3.1-RUNX1处理组(pc RUNX1)和pc DNA3.1对照处理组(pc DNA 3.1)。q RT-PCR和western blot检测RUNX1、磷酸化Akt(p-Akt)和总Akt(t-Akt)表达水平;MTT法检测细胞存活率;Annexin V-FITC/PI双染法检测细胞凋亡。结果:与对照组比较,RUNX1在PC12细胞氧糖剥夺模型中表达水平显著升高;沉默RUNX1可下调PC12细胞的存活率,促进细胞的凋亡,有效抑制p-Akt蛋白表达,而过表达RUNX1显著提高细胞存活率,抑制细胞凋亡,并上调p-Akt蛋白表达;此外,PI3K/Akt通路抑制剂LY294002明显抑制RUNX1过表达对细胞存活率的促进作用和对细胞凋亡的抑制作用。结论:RUNX1可通过PI3K/Akt信号通路保护OGD对PC12细胞的损伤作用。     

Acetylbritannilactone Modulates Vascular Endothelial Growth Factor Signaling and Regulates Angiogenesis in Endothelial Cells

The present study was conducted to determine the effects of 1-O-acetylbritannilactone (ABL), a compound extracted from Inula britannica L., on vascular endothelial growth factor (VEGF) signaling and angiogenesis in endothelial cells (ECs). We showed that ABL promotes VEGF-induced cell proliferation, growth, migration, and tube formation in cultured human ECs. Furthermore, the modulatory effect of ABL on VEGF-induced Akt, MAPK p42/44, and p38 phosphorylation, as well as on upstream VEGFR-2 phosphorylation, were associated with VEGF-dependent Matrigel angiogenesis in vivo. In addition, animals treated with ABL (26 mg/kg/day) recovered blood flow significantly earlier than control animals, suggesting that ABL affects ischemia-mediated angiogenesis and arteriogenesis in vivo. Finally, we demonstrated that ABL strongly reduced the levels of VEGFR-2 on the cell surface, enhanced VEGFR-2 endocytosis, which consistent with inhibited VE-cadherin, a negative regulator of VEGF signaling associated with VEGFR-2 complex formation, but did not alter VE-cadherin or VEGFR-2 expression in ECs. Our results suggest that ABL may serve as a novel therapeutic intervention for various cardiovascular diseases, including chronic ischemia, by regulating VEGF signaling and modulating angiogenesis.     

New molecular mechanisms of the unexpectedly complex role of VEGF in ulcerative colitis

The effects of VEGF on endothelial cells are mediated by different intracellular signaling cascades (e.g., Erk1/2, Akt, Src). VEGF plays a recently recognized role in ulcerative colitis (UC) pathogenesis, mostly by increasing vascular permeability and promoting the infiltration of inflammatory cells. We hypothesized that the excessive activation of signal transduction pathways, which is responsible for VEGF/VEGFR-2-mediated endothelial permeability (Src, Akt), is a new element in the pathogenesis of chronic UC. We demonstrated increased expression of pro-angiogenic growth factor VEGF and its receptor VEGFR-2 in colonic tissue during acute 6% iodoacetamide-induced UC in rats and chronic spontaneously developed UC in IL-10 knockout mice (IL-10 KO). Development of acute 6% iodoacetamide-induced UC in rats was accompanied by activation of Erk1/2 and Src kinase, while expression of total proteins Erk1/2 and Src was unchanged. During chronic colitis phosphorylation (i.e., activation) of Erk1/2 was significantly decreased in IL-10 KO mice vs. wild-type mice. Levels of total Erk1/2 proteins were unchanged, but the expression of total Src protein as well as its phosphorylated form was significantly increased in IL-10 KO vs. wild-type mice. There were no changes in total Akt proteins, while levels of activated Akt (pAkt) were slightly increased in IL-10 KO vs. wild-type mice. We conclude that VEGF/VEGFR-2-associated signal transduction pathways, that mediate increased vascular permeability (Src, Akt), might play a central role in perpetuation of chronic experimental UC.     

Blocking exosomal miRNA-153-3p derived from bone marrow mesenchymal stem cells ameliorates hypoxia-induced myocardial and microvascular damage by targeting the ANGPT1-mediated VEGF/PI3k/Akt/eNOS pathway

It has been widely reported that exosomes derived from mesenchymal stem cells (MSCs) have a protective effect on myocardial infarction (MI). However, the specific molecules which play a damaging role in MSCs shuttled miRNAs are much less explored. MiRNA-153-3p (miR-153-3p) is a vital miRNA which has been proved to modulate cell proliferation, apoptosis, angiogenesis, peritoneal fibrosis and aortic calcification. Here, we aim to study the effect and mechanism of miR-153-3p in MSC-derived exosomes on hypoxia-induced myocardial and microvascular damage. The exosomes of MSCs were isolated and identified, and the MSCs-exosomes with low expression of miR-153-3p (exo-miR-153-3p⁻) were constructed to interfere with the endothelial cells and cardiomyocytes in the oxygen-glucose deprivation (OGD) model. The viability, apoptosis, angiogenesis of endothelial cells and cardiomyocytes were determined. Additionally, ANGPT1/VEGF/VEGFR2/PI3K/Akt/eNOS pathway was detected by ELISA and/or western blot. The results illustrated that exo-miR-153-3p⁻ significantly reduced the apoptosis of endothelial cells and cardiomyocytes and promoted their viability. Meanwhile, exo-miR-153-3p⁻ can promote the angiogenesis of endothelial cells. Mechanistically, miR-153-3p regulates the VEGF/VEGFR2/PI3K/Akt/eNOS pathways by targeting ANGPT1. Intervention with VEGFR2 inhibitor (SU1498, 1 μM) remarkably reversed the protective effect of exo-miR-153-3p⁻ in vascular endothelial cells and cardiomyocytes treated by OGD. Collectively, MSCs-derived exosomes with low-expressed miR-153-3p notably promotes the activation of ANGPT1 and the VEGF/VEGFR2 /PI3K/Akt/eNOS pathways, thereby preventing the damages endothelial cells and cardiomyocytes against hypoxia.     

Selective inhibition of vascular endothelial growth factor receptor-2 (VEGFR-2) identifies a central role for VEGFR-2 in human aortic endothelial cell responses to VEGF

Vascular endothelial growth factor receptors (VEGFR) are considered essential for angiogenesis. The VEGFR-family proteins consist of VEGFR-1/Flt-1, VEGFR-2/KDR/Flk-1, and VEGFR-3/Flt-4. Among these, VEGFR-2 is thought to be principally responsible for angiogenesis. However, the precise role of VEGFRs1-3 in endothelial cell biology and angiogenesis remains unclear due in part to the lack of VEGFR-specific inhibitors. We used the newly described, highly selective anilinoquinazoline inhibitor of VEGFR-2 tyrosine kinase, ZM323881 (5-[[7-(benzyloxy) quinazolin-4-yl]amino]-4-fluoro-2-methylphenol), to explore the role of VEGFR-2 in endothelial cell function. Consistent with its reported effects on VEGFR-2 [IC(50) < 2 nM], ZM323881 inhibited activation of VEGFR-2, but not of VEGFR-1, epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), or hepatocyte growth factor (HGF) receptor. We studied the effects of VEGF on human aortic endothelial cells (HAECs), which express VEGFR-1 and VEGFR-2, but not VEGFR-3, in the absence or presence of ZM323881. Inhibition of VEGFR-2 blocked activation of extracellular regulated-kinase, p38, Akt, and endothelial nitric oxide synthetase (eNOS) by VEGF, but did not inhibit p38 activation by the VEGFR-1-specific ligand, placental growth factor (PIGF). Inhibition of VEGFR-2 also perturbed VEGF-induced membrane extension, cell migration, and tube formation by HAECs. Vascular endothelial growth factor receptor-2 inhibition also reversed VEGF-stimulated phosphorylation of CrkII and its Src homology 2 (SH2)-binding protein p130Cas, which are known to play a pivotal role in regulating endothelial cell migration. Inhibition of VEGFR-2 thus blocked all VEGF-induced endothelial cellular responses tested, supporting that the catalytic activity of VEGFR-2 is critical for VEGF signaling and/or that VEGFR-2 may function in a heterodimer with VEGFR-1 in human vascular endothelial cells.