咖啡酸苯乙酯对实验性肝损伤大鼠的保护作用

目的 探讨不同给药途径及不同剂量咖啡酸苯乙酯(CAPE)对四氯化碳(CCl4)等复合因素诱导大鼠肝损伤的保护作用.方法 选取95只雄性SD大鼠,随机分为9组,A:正常对照组;B:溶剂对照组,皮下注射橄榄油,腹腔注射10％乙醇;C:单纯模型组,腹腔注射10％乙醇;D:维生素E组,腹腔注射维生素E 10 mg/kg,1次/...     

咖啡酸苯乙酯对激活的大鼠血管平滑肌细胞增殖的抑制作用

目的探讨脂多糖（lipopolysaccharide,LPS）激活血管平滑肌细胞（vascular smooth muscle cells,VSMC）后,咖啡酸苯乙酯（caffeic acid phenethyl ester,CAPE）对细胞过度增殖的影响。方法采用不同浓度的CAPE处理LPS激活的VSMC。MTT法用于检测细胞的生长情况;免疫细胞化学法检测增殖核抗原（PCNA）和存活素（Survivin）蛋白的阳性率;流式细胞仪检测细胞周期分布。结果5、10、20、40、80 mg/L CAPE处理LPS激活的VSMC后,MTT检测发现细胞的生长明显受到抑制并呈剂量和时间依赖;VSMC经CAPE处理72 h后,细胞中PCNA和Survivin蛋白阳性表达率随药物剂量增加而显著降低（P<0.05）;细胞周期分析表明5、10、20 mg/L CAPE处理VSMC 24 h后,对照组和实验组G0/G1期细胞百分率分别为（40±4.3）%和（52±6）%,（65±1）%,（76±4）%,S期细胞百分率由对照组的（32.4%±2.6）%逐渐下降20 mg/L组的（11.2%±1.4）%,呈剂量依赖性（P<0.05）。结论CAPE可能通过调控细胞周期,减少PCNA和Survivin蛋白的表达而发挥细胞增殖抑制作用,其效能与作用时间和药物剂量存在一定的相关性。     

咖啡酸苯乙酯对ox-LDL诱导的THP-1源性巨噬细胞炎症因子分泌的抑制作用

目的研究咖啡酸苯乙酯(CAPE)对ox-LDL诱导的THP-1源性巨噬细胞炎症因子分泌的影响及其可能机制。方法用不同浓度CAPE预处理THP-1源性巨噬细胞2 h,再用40 mg/L ox-LDL处理24 h,ELISA检测炎症因子TNF-α,IL-6以及MCP-1的分泌情况;用40 mg/L ox-LDL分别处理THP-1源性巨噬细胞不同时间,Westernblot检测细胞COX-2蛋白表达的情况;最后,采用Western blot分析CAPE对ox-LDL诱导的THP-1源性巨噬细胞COX-2蛋白表达、IκB-α降解及其NF-κB核转位的影响。结果 40 mg/L ox-LDL可诱导THP-1源性巨噬细胞TNF-α,IL-6以及MCP-1分泌增多,而CAPE明显抑制了ox-LDL诱导的细胞炎症因子分泌,呈浓度依赖性(P<0.05);随着ox-LDL处理时间的延长,THP-1源性巨噬细胞COX-2蛋白表达逐渐增高,以24 h最为明显(P<0.05),而CAPE能抑制ox-LDL诱导的THP-1源性巨噬细胞COX-2蛋白表达上调,并可抑制ox-LDL诱导的THP-1源性巨噬细胞IκB-α降解及其NF-κB核转位。结论 CAPE对ox-LDL诱导的巨噬细胞炎症因子分泌应具有抑制作用,作用机制可能与其抑制NF-κB激活,下调COX-2蛋白表达有关。     

血管平滑肌细胞中Survivin的表达和咖啡酸苯乙脂的干预效应

目的 观察脂多糖(LPS)激活血管平滑肌细胞(VSMC)后,咖啡酸苯乙酯(CAPE)对细胞表达存活素(Survivin)的影响.方法 MTT法检测LPS及CAPE对VSMC增殖能力的影响.分别用免疫细胞化学法和实时荧光定量RT-PCR法检测细胞Survivin蛋白和mRNA的表达.结果 0.1～10.0 mg/L的LPS刺激VSMC生长并诱导细胞表达Survivin,而5、10、20、40、80 mg/L CAPE呈剂量依赖性地抑制激活的VSMC生长,同时降低细胞中Survivin的表达水平.结论 CAPE可能通过降低激活的VSMC中表达的Survivin,增加细胞的凋亡而抑制其增殖.     

异硫氰酸苯乙酯联合阿霉素诱导骨肉瘤细胞凋亡的作用研究

目的探讨异硫氰酸苯乙酯(PEITC)联合阿霉素(ADM)诱导骨肉瘤细胞凋亡的作用。方法选用U2-OS细胞株,根据处理方法的不同将其分为PEITC组、ADM组、PEITC+ADM组和空白对照组。采用MTT法检测不同处理组药物对U2-OS细胞增殖能力的影响。采用TUNEL法测定不同处理组U2-OS细胞的凋亡情况。采用Western blot法检测不同处理组U2-OS细胞Caspase-3、Fas、FasL蛋白的表达情况。结果PEITC和ADM浓度对U2-OS细胞的半抑制浓度(IC50)分别为4.37μM/ml和6.61μg/ml。与单独使用PEITC或ADM处理相比,联合使用两种药物对U2-OS细胞的增殖抑制率更高(P<0.05)。低剂量的PEITC联合ADM产生协同效应,而高剂量的两种药物联合产生相加效应。与单药物处理相比,PEITC联合ADM能够显著提高U2-OS细胞凋亡率(P<0.05),增加Caspase-3蛋白的活性及表达(P<0.05)。结论PEITC增强了ADM诱导骨肉瘤细胞凋亡的作用,可能与Caspase-3蛋白活性升高和表达量上调有关,这为PEITC联合ADM的临床应用提供了参考依据。     

异硫氰酸苯乙酯对胃癌细胞体外抑制作用相关研究

[目的]研究异硫氰酸苯乙酯（PEITC）体外实验中对人胃癌BGC-823细胞的增殖抑制作用及诱导其凋亡相关分子机制.[方法]应用MTT法、Annexin V/PI双染法检测PEITC人胃癌BGC-823细胞增值以及凋亡的影响;应用RT-PCR以及Western Bolt检测PEITC诱导胃癌细胞凋亡相关基因蛋白的表达情况.[结果]PEITC作用人胃癌细胞BGC-823给药24 h后,与空白组比较,能显著抑制胃癌细胞的增殖,抑制作用与浓度和时间成依赖关系,诱导胃癌细胞的凋亡,能上调Bax表达,下调Bcl-2、Bcl-xl、Survivin等基因表达,激活Caspase-3、Caspase-9、PARP活性.[结论]PEITC能有效抑制人胃癌BGC-823细胞的增殖,其机制可能是通过激活Caspase家族蛋白酶活性而诱导胃癌细胞凋亡.     

咖啡酸苯乙酯对实验性肝损伤大鼠的抗氧化作用

目的探讨蜂胶提取物咖啡酸苯乙酯（CAPE）对四氯化碳（CCl4）等复合因素诱导的肝损伤大鼠的抗氧化作用。方法取95只健康雄性SD大鼠,随机分为9组。A：正常对照组;B：溶剂对照组,皮下注射橄榄油、腹腔注射10％乙醇,纯水灌胃;C：单纯模型组,腹腔注射10％乙醇;D：维生素E组,10mg／kg,腹腔注射,1次/d;E～I：CAPE（10％乙醇溶液）干预组：腹腔注射,3mg／kg、6mg／kg和12rag／kg;灌胃,12mg／kg和24mg／kg,1次／d。C—I组均予以40％CCl4橄榄油溶液皮下注射、30％乙醇灌胃,高脂饲料作为单一饲料。同时每组给予对应药物处理10周。实验第10周处死大鼠。测定肝组织匀浆中氧化应激指标：丙二醛（MDA）、谷胱甘肽（GSH）水平以及过氧化氢酶（CAT）、超氧化物歧化酶（SOD）活力。肝组织标本行常规HE、Van Gieson染色。结果与单纯模型组的各项氧化应激指标相比较,腹腔注射CAPE6、12mg／kg两剂量组的肝组织中MDA含量明显降低、GSH水平升高、CAT和SOD活力增加（P〈0．05）。腹腔注射CAPE12mg／kg组对肝脏氧化应激指标的改善程度比灌胃给药两个剂量组好（P〈0．05）。腹腔注射CAPE12mg／kg组经HE和VG染色观察可见小部分区域肝细胞炎症坏死,少量纤维增生,较单纯模型组肝损伤程度轻。结论CAPE可以抑制脂质过氧化,提高肝脏抗氧化能力。在本课题观察的剂量范围内,CAPE腹腔给药的抗氧化作用好于灌胃给药途径。     

异硫氰酸苯乙酯对结肠癌SW480细胞PI3K/Akt/mTOR信号通路的影响

目的探讨异硫氰酸苯乙酯(PEITC)对结肠癌SW480细胞PI3K/Akt/mTOR信号通路的影响。方法选择6~8周龄的SPF级BALB/c(nu/nu)雄性裸鼠24只,将其随机分为对照组、低剂量组、中剂量组和高剂量组,各6只。体外培养结肠癌细胞系SW480,对照组加入等体积的二甲基亚砜(DMSO),低剂量组、中剂量组和高剂量组分别给予10、30和50μmol/L PEITC作用24 h,Western blot检测PI3K和PTEN的表达和Akt、mTOR磷酸化情况。最后,建立裸鼠异种移植瘤动物模型,观察PEITC对异种移植瘤生长的抑制作用。结果 PEITC能抑制PI3K的表达以及Akt和mTOR磷酸化。肿瘤质量:高剂量组中剂量组低剂量组对照组,抑瘤率:高剂量组中剂量组低剂量组(P0.05)。结论 PEITC可能通过影响PI3K/Akt/mTOR通路发挥抗肿瘤作用。     

生脉散对急性肝衰竭大鼠内毒素诱导细胞因子的影响

[目的]探讨生脉散对急性肝衰竭大鼠内毒素（LPS）诱导细胞因子水平的影响。[方法]采用胁氨基半乳糖腹腔注射法制作急性肝衰竭大鼠模型。予生脉散灌胃2h及8h后,检测血清LPS与细胞因子水平。[结果]D-氨基半乳糖能明显增加大鼠血清白细胞介素6（IL-6）、细胞间黏附分子1（ICAM-1）和IL-1β水平（P〈0．01）,随着D-氨基半乳糖作用时间延长的趋势明显减弱,但对血清LPS、肿瘤坏死因子α（TNF-α）水平无明显影响（P〉0．05）;生脉散能显著降低D氨基半乳糖肝衰竭大鼠血清IL-6、ICAM-1和IL-1β水平（P〈0．01,〈O．05）。LPS攻击2h和8h后,能明显诱导D-氨基半乳糖大鼠血清LPS、TNF-α、IL-6、ICAM-1和IL-1β水平增加（P〈0．01）;除在8h对TNF-α无影响外,生脉散能明显减轻LPS攻击D-氨基半乳糖大鼠后对血清LPS、TNF-α、IL-6、ICAM-1和IL-1β水平的影响（P〈0．01,〈O．05）。[结论]SD大鼠在急性肝衰竭状态下,存在严重LPS血症,并引起细胞因子水平变化的炎症级联反应,生脉散可通过调节细胞及炎症因子水平起到治疗作用。     

益生菌对结肠炎大鼠黏膜细胞因子表达的影响

目的探讨益生菌对实验性结肠炎大鼠肠道炎症、菌群及黏膜肿瘤坏死因子(TNF)-α表达的影响。方法建立2,4,6-三硝基苯磺酸(TNBS)实验性结肠炎大鼠模型,30只Wistar大鼠均分为正常对照组(NC组)、模型对照组(UC组)和益生菌治疗组(PC组)。PC组大鼠采用双歧三联活菌悬液(2．2×10~9 CFU/只)灌胃治疗,1次/d,共4周,光镜下观察肠黏膜炎症并评分,检测各组大鼠部分肠道菌群、肠黏膜中TNF-α、白细胞介素(IL)-6表达及血浆内毒素水平,分析其变化。结果PC组大鼠结肠炎症评分较UC组明显改善(7．94±0．85比10．25±1．36,P＜0．05),但未恢复至NC组水平(7．94±0．85比4．35±0．88,P＜0．01)。PC组肠道内乳酸杆菌和双歧杆菌较UC组明显增加,大肠杆菌和真菌则减少(P＜0．05)。PC组血浆内毒素、TNF-α水平较UC组降低[内毒素：(93．33±21．22)pg/ml比(1 21．25±39．07)pg/ml；TNF-α：67．51±14．63比85．99±18．17,P值均＜0．05],但高于NC组[内毒索：(93．33±21．22)pg/ml比(35．20±15．12)pg/ml；TNF-α：67．51±14．63比43．28±19．98,P值均＜0．01]。与UC组比较,PC组IL-6水平有所降低,但差异无统计学意义(155．22±34．01比184．09±29．11,P＞0．05),且高于NC组(155．22±34．01比108．73±37．35,P＜0．01)。结论双歧三联活菌可调整肠道菌群紊乱,减轻大鼠结肠炎症,改善菌群紊乱、减少内毒素吸收及局部促炎性细胞因子的分泌。     

Effect of caffeic acid phenethyl ester on cartilage in experimental osteoarthritis

Systemic lupus erythematosus (SLE) is characterized by the presence of various autoantibodies and the deposition of immune complex, which is cleared by Fcgamma receptors. Genotype analysis was done to investigate whether the FcgammaRIIIA-176F/V polymorphism is a risk factor for SLE in Koreans. We genotyped 145 Korean SLE patients and 75 control subjects for FcgammaRIIIA-176F/ V. After amplifying a 1.7-kb fragment containing the Fcgamma/RIIIA-176F/V polymorphic site using two FcgammaRIIIA gene-specific primers, we performed a nested polymerase chain reaction (PCR) for allele-specific genotyping at position 559 in FcgammaRIIIA. FcgammaRIIIA genotype or allele distribution was not significantly different between lupus patients and controls, and also between lupus nephritis patients and healthy controls. Neither creatinine clearance, 24 h urine proteinuria, number of American College of Rheumatology (ACR) criteria, nor the Systemic Lupus International Collaborating Clinics (SLICC)/ACR damage index was different according to the genotype. In conclusion, FcgammaRIIIA-176F/V polymorphism is not associated with SLE in Koreans.     

Effect of caffeic acid phenethyl ester on proliferation and apoptosis of hepatic stellate cells in vitro

AIM: To investigate the role of nuclear factor-κB (NF-κB)inhibitor caffeic acid phenethy1 ester (CAPE) in the proliferation, collagen synthesis and apoptosis of hepatic stellate cells (HSCs) of rats. METHODS: The HSCs from rats were isolated and cultured in Dulbecco's Modified Eagle's Medium (DMEM) and treated with CAPE. The proliferation and collagen synthesis of HSCs were determined by 3H-TdR and 3H-proline incorporation respectively, and the expression of type Ⅰ, Ⅲ procollagen genes was further explored byin situ hybridization. Apoptosis cell indices (AIs) were examined using terminal deoxynucleotidyl transferase- mediated DIG-dUTP nick end labeling (TUNEL). RESULTS: Tn activated HSC in culture, CAPE significantly inhibited 3H-TdR and 3H-proline incorporation by HSCs at concentrations of 5 μmol/L and 10 μmol/L respectively. CAPE also reduced the type I procollagen gene expression (P＜0.05)at higher concentration. Apoptosis of HSC was induced by CAPE and the AIs were time-and dose-dependently increased from 2.82+0.73 % to 7.66±1.25 % at 12 h (P＜0.01) and from 3.15±0.88 % to 10.6L±2.88 % at 24 h (P＜0.01). CONCLUSION: CAPE inhibits proliferation and collagen synthesis of HSC at lower concentration and induces HSC apoptosis at higher concentration.     

Effect of caffeic acid phenethyl ester on proliferation and apoptosis of colorectal cancer cells in vitro

ABM: To study the effect of caffeic add phenethyl ester (CAPE) on proliferation, cell cycle, apoptosis and expression of β-catenin in cultured human colorectal cancer (CRC) cell line HCT116. METHODS: HCT116 cells were treated with CAPE at serial concentrations of 80,40,20,10,5,2.5 mg/L. The proliferative status of HCT116 cells was measured by using methaben-zthiazuron (MTT) assay. Cell cycle was analyzed by using flow cytometry (FCM) with propidium iodide (PI) labeling method. The rate of apoptosis was detected by using FCM with annexin V-FITC and PI double labeling method, β-catenin levels were determined by Western blotting, β-catenin localization in HCT116 was determined by indirect immunofluorescence. RESULTS: After HCT116 cells were exposed to CAPE (80, 40, 20, 10, 5, and 2.5 mg/L) for 24, 48, 72, 96 h, CAPE displayed a strong growth inhibitory effect in a dose- and time-dependent manner against HCT116 cells. FCM analysis showed that the ratio of G0/G1 phase cells increased, S phase ratio decreased and apoptosis rate increased after HCT116 cells were exposed to CAPE (10, 5, and 2.5 mg/L) for 24 h. CAPE treatment was associated with decreased cytoplasmic β-catenin, nuclear p-catenin and a concurrent increase in β-catenin protein expression at cell-cell junctions. CONCLUSION: CAPE could inhibit HCT116 cell proliferation and induce cell cycle arrest and apoptosis. Decreased β-catenin protein expression may mediate the anti-proliferative effects of CAPE.     

Therapeutic effect of caffeic acid phenethyl ester on cerulein-induced acute pancreatitis

AIM: To evaluate the therapeutic role of caffeic acid phenethyl ester (CAPE) in a rat model of ceruleaninduced acute pancreatitis (AP). METHODS: Seventy male Wistar albino rats were divided into seven groups. Acute edematous pancreatitis was induced by subcutaneous cerulein injection (20 μg/kg) four times at 1-h intervals. CAPE (30 mg/kg) was given by subcutaneous injection at the beginning (CAPE 1 group) and 12 h after the last cerulein injection (CAPE 2 group). Serum amylase, lipase, white blood cell count, and tumor necrosis factor (TNF)-α levels were measured, and pancreatic histopathology was assessed. RESULTS: In the AP group, amylase and lipase levels were found to be elevated and the histopathological evaluation showed massive edema and inflammation of the pancreas, with less fatty necrosis when compared with sham and control groups. Amylase and lipase levels and edema formation decreased significantly in the CAPE therapy groups (P < 0001); especially in the CAPE 2 group, edema was improved nearly completely (P = 0001). Inflammation and fatty necrosis were partially recovered by CAPE treatment. The pathological results and amylase level in the placebo groups were similar to those in the AP group. White blood cell count and TNF-α concentration was nearly the same in the CAPE and placebo groups. CONCLUSION: CAPE may be useful agent in treatment of AP but more experimental and clinical studies are needed to support our observation of beneficial effects of CAPE before clinical usage of this agent.     

咖啡酸苯乙基酯缓解L02细胞脂毒性机制研究*

目的 探讨咖啡酸苯乙基酯(CAPE)对棕榈酸酯诱导的L02细胞脂毒性的保护机制。方法 取正常肝脏L02细胞和过氧化物酶体增殖激活受体共激活因子1α(PGC1α)敲除细胞,分别分为对照组、棕榈酸酯处理组和CAPE联合棕榈酸酯处理组,后两组分别给予终浓度300mM棕榈酸酯处理7 d或是联合终浓度10μM CAPE处理7 d。分别检测细胞甘油三酯(TG)、上清肿瘤坏死因子α(TNF-α)和白介素6(IL-6)水平,使用流式细胞术检测线粒体活性氧(ROS)水平,采用qPCR定量法检测PGC1α和过氧化物歧化酶2(SOD2)mRNA水平,采用Western-blot法检测PGC1α蛋白表达。结果 棕榈酸酯处理组细胞TG含量为(16.92±1.43)mg/g protein,显著高于CAPE联合棕榈酸酯处理组【(10.53±0.81)mg/g protein,P<0.05】;棕榈酸酯处理组细胞上清TNF-α和IL-6水平分别为(117.6±3.72)pg/ml和(67.9±2.7)pg/ml,均显著高于CAPE联合棕榈酸酯处理组【分别为(74.88±3.37)pg/ml和(53.94±2.39)pg/ml,P<0.05】;棕榈酸酯处理组线粒体ROS水平为(1.7±0.06),显著高于CAPE联合棕榈酸酯处理组【(1.36±0.04),P<0.05】;CAPE联合棕榈酸酯处理组SOD2和PGC1α mRNA水平分别为(0.76±0.03)和(0.73±0.04),显著高于棕榈酸酯处理组【(0.55±0.05)和(0.57±0.03),P<0.05】;CAPE联合棕榈酸酯处理组PGC1α 蛋白表达水平显著高于棕榈酸酯处理组;在敲除PGC1α后,CAPE联合棕榈酸酯处理组细胞TG含量为(23.73±1.95)mg/g protein,与棕榈酸酯处理组的(25.86±1.02)mg/g protein比,无显著性差异(P>0.05),上清TNF-α和IL-6水平分别为(128.33±4.41)pg/ml和(80.33±3.76)pg/ml,与棕榈酸酯处理组的(145.78±5.79)pg/ml和(87.23±4.85)pg/ml比,无显著性差异(P>0.05);线粒体ROS水平为(1.83±0.25),与棕榈酸酯处理组的(2.05±0.14)比,无显著性差异(P>0.05)。结论 通过上调PGC1α信号通路,CAPE对棕榈酸酯诱导的肝脏L02细胞脂毒性具有有效的保护作用。     

Effects of caffeic acid phenethyl ester on lipopolysaccharide-induced lung injury in rats

Extracts of propolis, a natural beehive product, have been known for centuries to have a variety of beneficial medical properties, among which their anti-inflammatory effect is a major one. Caffeic acid phenethyl ester (CAPE), an active propolis component, has antimicrobial, anti-inflammatory, antioxidant, carcinostatic and immunomodulatory properties. In this study, we aimed to investigate the efficacy of CAPE in endotoxin-induced lung injury in rats. Lung injury was induced by a footpad injection of lipopolysaccharide (LPS). In the treatment group, 10 micromol kg(-1) CAPE was injected intraperitoneally immediately after LPS injection. At 24 h after LPS and/or CAPE injection, blood and lung tissue specimens were collected. MDA levels and MPO activity in serum and lung tissue, serum total antioxidant levels, lung tissue Na(+)/K(+) ATP-ase activity and histopathological evaluation were determined to assess the efficacy of CAPE treatment. CAPE was found to be efficient in reducing inflammation and lung tissue damage induced by LPS in rats.     

Oleic acid-induced lung injury in rats and effects of caffeic acid phenethyl ester

Caffeic acid phenethyl ester (CAPE) is a phenolic antioxidant and is an active anti-inflammatory component of honeybee propolis. The authors evaluated the effects of CAPE on oxidative stress and lung damage in an oleic acid (OA)-induced lung-injury model. Rats were divided into 5 groups as sham, OA, CAPE, pre-OA-CAPE, and post-OA-CAPE. Acute lung injury was induced by intravenous administration of 100 mg/kg of OA. Pre-OA-CAPE group received CAPE (10 micromol/kg. intravenously) 15 minutes before OA infusion and post-OA-CAPE group received CAPE 2 hours after OA administration. Malondialdehyde (MDA) level of plasma, bronchoalveolar lavage fluid (BALF), and lung tissue; myeloperoxidase activity of BALF and lung tissue; Na(+)-K(+) ATPase activity of lung tissue; and total protein content of BALF were measured. Light microscopic analyses of lung specimens were performed. The increased MDA levels in lung homogenates (47.98+/-13.75 nmol/mL), BALF (31.12+/-3.07 nmol/mL), and plasma (61.84+/-15.34 nmol/mL) decreased significantly to 24.33+/-3.09 nmol/mL (P = 0.000), 23.19+/-4.97 nmol/mL (P = 0.002), and 27.36+/-5.37 nmol/mL (P = 0.000), respectively, following CAPE administration in pre-OA-CAPE group. Another important finding was the restoration of the enzymatic activity of Na(+)-K(+) ATPase from a value of 203.89+/-32.18 nmol Pi/mg Protein/h in OA group, to a value of 302.17+/-51.90 nmol Pi/mg Protein/h (P = 0.012) in pre-OA-CAPE group with CAPE treatment. CAPE has been shown to have a clear attenuating effect on oxidative damage in experimental animal studies. However, further investigations are necessary to suggest CAPE as a treatment agent in critically ill patients with lung injury.