阿魏酸对人脐静脉血管内皮细胞ECV304的增殖作用

目的：观察阿魏酸对于培养人脐静脉内皮细胞ECV304增殖的影响,为治疗性血管新生的靶基因选择提供依据。方法：取对数生长期的内皮细胞,分为阿魏酸低、中、高浓度组,同步化于G0期,ELISA法检测BrdU的掺入;RT-PCR法检测血管内皮细胞生长因子（VEGF）mRNA的表达情况。结果：与对照组相比,不同浓度阿魏酸均可显著提高BrdU的掺入量,上调VEGFmRNA的表达,且在10^2ng/ml～10^4ng/ml之间呈一定的量效关系。结论：阿魏酸能显著促进体外培养的ECV304细胞的增殖,可能具有血管新生的作用。     

Cyclin D1启动子在养精胶囊促进小鼠精原干细胞增殖中的作用※

目的 研究养精胶囊促进小鼠精原干细胞（SSCs）增殖的分子机制。方法 将不同浓度的养精胶囊提取物加入SSCs中培养48 h,CCK-8检测细胞的增殖活性,流式细胞仪检测细胞周期,荧光素酶报告基因检测Cyclin D1启动子的活性,qRT-PCR以及免疫荧光检测Cyclin D1的表达。之后进行阻断实验,在加入养精胶囊前预先加入siRNA-Cyclin D1,同样的方法检测细胞的增殖活性、细胞周期、Cyclin D1启动子的活性以及Cyclin D1的表达情况。结果 低、中、高浓度的养精胶囊可以促进SSCs的增殖,提高S期细胞的比例,增强Cyclin D1启动子的活性,促进Cyclin D1的表达。阻断Cyclin D1后,SSCs的增殖活性降低,S期细胞比例减少,Cyclin D1启动子的活性降低,Cyclin D1的表达减少。结论 养精胶囊通过增强Cyclin D1启动子的活性提高Cyclin D1的转录和翻译水平,进而促进SSCs增殖。     

HPLC同时测定当归建中汤中阿魏酸和桂皮醛的含量

目的:采用HPLC同时测定当归建中汤中阿魏酸、桂皮醛的含量。方法:采用Dikma C18(250mm×4.6mm,5μm)色谱柱为固定相,以乙腈-0.5%醋酸水溶液(体积分数)为流动相进行梯度洗脱,流速为1.0mL·min-1,检测波长为325nm,进样量20μL。结果:阿魏酸在3.684~36.84mg·L-1线性关系良好,r=0.9996,平均回收率为100.47%(n=6),RSD为1.54%;桂皮醛在38.56~385.6mg·L-1线性关系良好,r=0.9997,平均回收率为100.52%(n=6),RSD为1.75%。仪器与方法精密度均小于2.0%。结论:该方法快速、灵敏、准确、重复性好,可用于当归建中汤中指标性成分的含量测定,更加全面控制该方的质量。     

Ferulic acid enhances insulin secretion by potentiating L-type Ca2+ channel activation

Objective: To investigate the effect of ferulic acid, a natural compound, on pancreatic beta cell viability,Ca²⁺ channels, and insulin secretion.Methods: We studied the effects of ferulic acid on rat insulinoma cell line viability using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide viability assay. The whole-cell patch-clamp technique and enzyme-linked immunosorbent assay were also used to examine the action of ferulic acid on Ca²⁺ channels and insulin se...     

阿魏酸对bFGF诱导PC12细胞分化的影响

目的:探讨阿魏酸在低浓度碱性成纤维生长因子(bFGF)诱导大鼠肾上腺髓质嗜铬瘤细胞(PC12细胞)分化的作用及其初步机制。方法:在0 ng/mL或1 ng/mL bFGF条件下培养PC12细胞,以PC12细胞的突起长度及诱导率确定药效。结果:阿魏酸在无bFGF的情况下不能诱导PC12分化,但在低浓度bFGF(1 ng/mL)情况下,与bFGF(1 ng/mL)对照组比较可明显诱导PC12细胞分化(P<0.01),这种作用能被有丝分裂原激活的蛋白激酶(MAPK)特异性抑制剂PD98059所抑制。结论:阿魏酸可增强bFGF诱导PC12细胞分化,其机制可能与MAPK信号通路有关。     

Salvianolic acid A promotes the acceleration of neovascularization in the ischemic rat myocardium and the functions of endothelial progenitor cells

Ethnopharmacological relevance

Salvia miltiorrhiza (SM, also known as DanShen) is one of the well-known widely used Chinese herbal medicines in clinical, containing phenolic compounds and potent antioxidant properties. Salvianolic acid A (SAA) is the most potent component of SM. A modern experimental strategy for treating myocardial ischemia is to induce neovascularization of the heart by the use of "angiogens", mediators that induce the formation of blood vessels, or angiogenesis. Studies demonstrated that coronary collateral vessels protect ischemic myocardium after coronary obstruction; therefore, we sought to examine whether SAA could stimulate myocardial angiogenesis.

Materials and methods

Male Sprague–Dawley rats myocardial infarct (MI) induced by ligation of left anterior descending coronary artery (LAD) were randomly divided into five groups: sham-operated group; LAD occlusion + administration of physiological saline (vehicle treated group); LAD occlusion + administration of different concentrations of SAA (10, 5.0 and 2.5 mg/kg/d). Infarct size and capillary density in the infarct region were measured with a previous experimental method. Immunohistological analysis was performed to measure vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor-2 (VEGFR-2) expressions. The secretion of matrix metalloproteinase type X (MMP-9) was evaluated in serum of post-ischemic rats. We also performed the experiments of SAA on rat endothelial progenitor cells (EPCs) numbers and the capacity of migration and vasculargenesis.

Results

SAA potentiated the ischemia-induced neovascularization after 1 week post-operation when compared to vehicle treated group. This effect could be attributed to an increased formation of VEGF, VEGFR-2, and MMP-9 as well as the promotion of numbers and functions of EPCs.

Conclusion

These findings show that SAA has potent proangiogenic properties by promoting the expression of proangiogenic factors, and the functions of EPCs, indicating that SAA might contribute to the protective effect against coronary disease. Chemical compound studied in this paper is salvianolic acid A (PubChem CID: 5281793)     

丹参酸A通过调控血管内皮细胞谷胱甘肽过氧化物酶活性及丙二醛含量抑制内毒素诱导的凋亡

目的：探寻丹参素（丹参酸A,SAA）对脂多糖（LPS）处理的血管内皮细胞（VEC）的保护作用及可能机制。方法：以LPS处理培养的人脐静脉血管内皮细胞,造成细胞凋亡,培养液中同时加入系列浓度的SAA;倒置相差显微镜观察细胞形态变化;吖啶橙染色＋激光共聚焦显微镜观测凋亡小体：PI染色＋流式细胞仪测定细胞周期分布;MTT法检测细胞存活率;生化试剂盒检测细胞内谷胱甘肽脱氢酶（GSH—Px）活性及丙二醛（MDA）水平。结果：SAA可明显抑制细胞凋亡,提高细胞存活率。且该作用具浓度依赖性。对于LPS导致的细胞周期异常、GSH—Px活性下降、MDA水平升高,SAA均可明显逆转。结论：丹参酸A通过抗氧化作用抑制内毒素诱导的血管内皮细胞凋亡。     

Crocetin inhibits cell cycle G1/S transition through suppressing cyclin D1 and elevating p27kip1 in vascular smooth muscle cells

Crocetin is a natural carotenoid compound isolated from Gardenia jasminoids Ellis. Our previous study shows that crocetin inhibits angiotensin II (Ang II)‐induced vascular smooth muscle cells (VSMCs) proliferation. To further explore the mechanism by which crocetin inhibits VSMCs proliferation, in the present study we examined the effect of crocetin on cell cycle progression and cell cycle regulatory proteins. Flow cytometry analysis showed that Ang II elicited significant increase in the percentage of VSMCs in the S phase, with a concomitant decline in the percentage of VSMCs in the G₀/G₁ phase. However, on pretreatment of VSMCs with crocetin, the percentage of VSMCs in the S phase decreased, while that in the G₀/G₁ phase increased significantly. In addition, Ang II‐induced increase of cell proliferation index was also decreased by crocetin. Western blotting analysis indicated that crocetin markedly inhibited the protein expression of cyclin D1 but not cyclin E. Crocetin also increased the level of cyclin‐dependent kinase inhibitor (CDKI) p27^kip1 but not CDKI p21^waf1/cip1. In conclusion, our present results suggest that the inhibition of cell cycle G₁/S transition in VSMCs by crocetin can be attributed, at least in part, to its suppression of cyclin D1 and elevation of CDKI p27^kip1. Copyright © 2009 John Wiley & Sons, Ltd.     

23-羟基白桦酸对血管内皮细胞增殖和迁移的影响

目的:探讨23-羟基白桦酸(23-HBA)对血管内皮细胞(ECV-304)形态变化,增殖和迁移的影响。方法:SRB方法测定23-HBA对ECV-304细胞的增殖抑制率,体外琼脂法检测内皮细胞的迁移能力,并用光镜观察内皮细胞形态变化。结果:23-HBA对ECV-304细胞的IC50为39.74μg/ml。当23-HBA浓度提高到20μg/ml以上,ECV-304细胞的凋亡现象非常明显。23-HBA浓度在1μg/ml时即能抑制细胞的迁移。结论:23-HBA对ECV-304增殖和迁移均有显著抑制作用。     

阿魏酸和异阿魏酸对HepG2细胞增殖及其细胞色素P450酶的影响

目的观察阿魏酸和异阿魏酸对HepG2细胞中细胞色素P450同工酶1A1、3A4(CYP1A1、CYP3A4)的作用特点。方法采用MTT比色法测定不同质量浓度阿魏酸和异阿魏酸对体外培养人肝癌HepG2细胞增殖的影响;采用流式细胞术测定阿魏酸和异阿魏酸对HepG2细胞周期的影响;实时定量PCR技术检测阿魏酸和异阿魏酸处理后CYP1A1、CYP3A4 mRNA的表达;Western blotting检测CYP3A4蛋白表达。结果作用48 h后,阿魏酸和异阿魏酸对HepG2细胞均有抑制作用,且有明显的剂量依赖关系;阿魏酸和异阿魏酸(50μg/mL)均阻滞HepG2细胞周期于G2/M期;阿魏酸和异阿魏酸在不同作用质量浓度下均是CYP1A1、CYP3A4 mRNA的抑制剂;50μg/mL阿魏酸和异阿魏酸处理细胞48 h后,CYP3A4蛋白表达均明显低于对照组,相比于对照组,阿魏酸和异阿魏酸的表达量分别为0.57、0.39。结论阿魏酸和异阿魏酸均能抑制HepG2细胞的增殖,其机制之一是影响细胞周期使其阻滞于G2/M期,能够抑制药物代谢酶CYP1A1、CYP3A4 mRNA的表达,同时抑制CYP3A4的蛋白表达,但作用程度差别较大,可能与其羟基和甲氧基异构有关。     

丹参多酚酸盐对大鼠骨髓间充质干细胞增殖及血管内皮生长因子表达的影响

目的观察中药丹参多酚酸盐对SD大鼠骨髓间充质干细胞的增殖及血管内皮生长因子(VEGF)表达的影响,以探讨其对促骨折愈合的可能机制。方法将0.5 mg/ml浓度丹参多酚酸盐与大鼠骨髓间充质干细胞共培养,对照组为无药物单位培养的大鼠骨髓间充质干细胞。培养48小时镜下观察细胞情况,1、3、5、7天行MTT检测细胞增殖情况。行实时荧光定量PCR检测方法,对培养7天的两组细胞的VEGF mRNA水平进行检测分析。结果共培养48小时后骨髓间充质干细胞生长密集程度明显优于对照组;MTT检测也显示实验组细胞增殖情况优于对照组;实验组VEGF mRNA表达高于对照组。结论丹参多酚酸盐能促大鼠骨髓间充质干细胞增殖,并提高其VEGF的表达。可能是其促骨折愈合的作用机制之一。     

龟板提取物上调维生素D受体表达促骨髓间充质干细胞向成骨分化

目的 探讨龟板提取物诱导大鼠骨髓间充质干细胞(mesenchymal stem cells,MSCs)向成骨分化和对维生素D受体(VDR)表达的影响.方法 从大鼠骨髓中分离MSCs.体外培养,利用流式细胞术检测MSCs表面抗原CD44细胞标志,体外培养MSCs分成不同的组观察其向成骨分化,在培养基中不加任何处理的作为对照组,培养基中加成骨诱导液诱导MSCs向成骨分化作为阳性对照组,龟板提取物组在培养基中加龟板提取物,诱导7 d后,通过免疫化学染色、Western blotting、原位杂交、RT-PCR等方法观察龟板提取物对原代培养的MSCs向成骨分化的成骨分化标记分子碱性磷酸酶(ALP)、骨桥蛋白(OPN)及VDR阳性表达及VDR mRNA的表达情况.结果 免疫组化染色显示,龟板提取物组成骨分化标记分子ALP、OPN和VDR的阳性百分比明显高于对照组,Western blotting结果也显示龟板提取物组的ALP、OPN和VDR的蛋白表达水平高于对照组;同时原位杂交、RT-PCR结果表明.龟板提取物诱导MSCs向成骨分化过程可明显促进VDR mRNA的表达.结论 龟板提取物可促MSCs向成骨分化,其机制可能与VDR的上调有关.     

Toona sinensis (leaf extracts) inhibit vascular endothelial growth factor (VEGF)-induced angiogenesis in vascular endothelial cells

Aim of the study

Toona sinensis is well known as a traditional Chinese medicine; also, it has been shown to exhibit anticancer and anti-inflammatory effects. This study was aimed at evaluating the anti-angiogenesis effect of the aqueous extracts of Toona sinensis (TS extracts) or gallic acid, a major component of TS extracts, against both VEGF-induced EA.hy 926 and human umbilical vein endothelial cells (HUVECs).

Materials and methods

Anti-proliferative activity of TS extracts or gallic acid, was determined against EA.hy 926 and HUVECs by trypan blue exclusion method. Invasion, tube formation and chick chorioallantoic membrane assay were carried out to determine the in vitro and in vivo anti-angiogenic effects.

Results

Non-cytotoxic concentration of TS extracts (50-100 μg/mL) and gallic acid (5 μg/mL) inhibited the proliferation of VEGF-stimulated EA.hy 926 and HUVECs. Inhibitory effects of TS extracts and gallic acid on angiogenesis were assessed by VEGF-induced migration/invasion and capillary-like tube formation by EA.hy 926 and HUVECs. Additionally, gelatin zymography assays showed that TS extracts and gallic acid suppressed the activity of metalloproteinase (MMP)-9 and MMP-2 activated by VEGF. In vivo, TS extracts and gallic acid strongly suppressed neovessel formation in the chorioallantoic membrane of chick embryos. Flow cytometry analyses and Western blot demonstrated that treatment with TS extracts and gallic acid induced G₀/G₁ arrest in VEGF-stimulated EA.hy 926 cells via a reduction in the amounts of cyclin D1, cyclin E, CDK4, hyperphosphorylated retinoblastoma protein (pRb), VEGFR-2, and eNOS.

Conclusions

These results support an anti-angiogenic activity of Toona sinensis that may contribute critically to its cancer and inflammation chemopreventive potentials.     

川芎嗪对花生四烯酸诱导血管内皮细胞凋亡的保护作用

目的　研究川芎嗪 (TMP)对花生四烯酸 (AA,16 0μm ol/ L )诱导的血管内皮细胞株 ECV 30 4损伤和凋亡的保护作用及其机制。方法　 MTT法检测细胞存活率 ,比色法测乳酸脱氢酶 (L DH)释放率和细胞丙二醛(MDA)含量 ,Hoechst 332 5 8荧光染色法观察细胞核形态变化并测定细胞凋亡率 ,琼脂糖凝胶电泳检测 DNA降解。结果　 TMP(0 .2 5～ 1.0 m mol/ L)能浓度依赖性地抑制 AA引起的细胞存活率下降 (P<0 .0 5 ) ,并降低细胞L DH释放率和 MDA含量 (P<0 .0 5 )。 AA处理 2 4 h后的细胞呈典型的凋亡核固缩表现 ,凝胶电泳显示 DNA凋亡梯带 ,TMP(0 .2 5～ 1.0 m mol/ L)能降低其凋亡率 (P<0 .0 5 )。结论　 TMP对 AA引起的 ECV30 4细胞损伤和凋亡具有保护作用 ,其机制可能与其抗脂质过氧化作用有关     

Ginsenoside Rg1 promotes glucose uptake through activated AMPK pathway in insulin-resistant muscle cells

Ginsenoside Rg1, a protopanaxatriols saponin, is one of the major active constituents from Panax ginseng and possesses various biological activities. A recent study reported that insulin resistance in skeletal muscle is a major contributor to the development of type 2 diabetes mellitus (T2DM). We examined the effects of ginsenoside Rg1 on glucose uptake and the associated molecular mechanisms of the glucose transport system in insulin-resistant muscle cells. The insulin resistance of the muscle cell was induced by treatment of differentiated C2C12 cells with chronic insulin. The results showed that chronic treatment of insulin resulted in reduced glucose uptake in the muscle cells. The treatment of ginsenoside Rg1 significantly enhanced glucose uptake in the differentiated muscle cells and the relative abundance of GLUT4 through the adenosine-monophosphate-activated protein kinase pathway. These results suggest that ginsenoside Rg1 improved the insulin resistance in C2C12 muscle cells, which might be useful for prevention of T2DM and metabolic syndromes.     

Effects of Panax notoginseng saponins on proliferation and apoptosis of vascular smooth muscle cells

Aim of the study

Atherosclerosis is a common cardiovascular disease, and linked with the development of many cardiovascular complications, such as myocardial ischemia and stroke. Although pathogenesis of atherosclerosis is not completely elucidated, increasing evidence has demonstrated that abnormal proliferation of vascular smooth muscle cells (VSMCs) plays an important role in formation of atherosclerosis. Previous studies showed that saponins from Panax notoginseng (PNS) possess anti-atherosclerotic properties. However, the mechanism of PNS against atherosclerosis is not well understood. Therefore, the present study observed the effects of PNS on proliferation and apoptosis of VSMCs.

Materials and methods

Rat VSMCs were cultured, and platelet-derived growth factor (PDGF) was used to stimulate cell proliferation. The viability of VSMCs was assessed with the MTT method. VSMCs apoptosis was detected by flow cytometry. Expressions of apoptosis related protein p53, Bax, caspase-3 and Bcl-2 were determined using Western blot.

Results

Pretreatment of the cells with PNS (200, 400, 800 μg/mL) significantly inhibited proliferation of PDGF-stimulated VSMCs, and induced apoptosis of the proliferated VSMCs in a concentration-dependent way. Western blot analysis showed that PNS upregulated expressions of pro-apoptotic protein p53, Bax and caspase-3, downregulated expression of anti-apoptotic protein Bcl-2, and enlarged Bax/Bcl-2 ratio in the proliferated VSMCs induced by PDGF.

Conclusions

This study demonstrates that PNS both inhibits VSMCs proliferation and induces VSMCs apoptosis through upregulating p53, Bax, caspase-3 expressions and downregulating Bcl-2 expression, which constitute the pharmacological basis of its anti-atherosclerotic action.     

川芎嗪对缺氧缺糖状态下培养的血管内皮细胞ECV-304的影响

目的 :观察缺氧缺糖条件下血管内皮细胞 (ECV-30 4 )释放乳酸脱氢酶 (LDH)、一氧化氮 (NO)、丙二醛(MDA)水平和细胞膜流动性的变化及川芎嗪对它们的影响。方法 :缺氧缺糖诱导血管内皮细胞损伤 ,自动生化分析仪测定培养液和细胞层中LDH活性 ;用比色法测定细胞培养液中NO水平 ;用荧光法检测细胞内脂质过氧化产物MDA以评价脂质过氧化程度 ;荧光偏振法测定内皮细胞膜流动性。结果 :缺氧缺糖引起的血管内皮细胞LDH释放增加、MDA生成增多和膜流动性增高 ,NO水平降低。而川芎嗪可抑制缺氧缺糖引起的血管内皮细胞释放LDH ,MDA生成和降低细胞膜流动性 ,提高NO水平。结论 :川芎嗪可保护缺氧缺糖诱导血管内皮细胞的损伤 ,其作用机制有待进一步研究。     

丹酚酸B及欧芹素乙对溶血磷脂酰胆碱刺激牛主动脉平滑肌细胞增殖的影响

目的　研究溶血磷脂酰胆碱 (L PC)对牛主动脉平滑肌细胞 (BASMC)增殖的影响及丹酚酸 B和欧芹素乙的抑制作用。方法　体外培养 BASMC,用 MTT法测定细胞增殖。结果　 L PC在 2 .5× 10 - 9～ 2 .5× 10 - 6 g/ L 剂量依赖性地促进 BASMC增殖 ;丹酚酸 B(1× 10 - 9～ 1× 10 - 6 mol/ L)和欧芹素乙 (1× 10 - 7～ 1× 10 - 3m ol/ L)浓度依赖性地抑制 L PC的作用。结论　 L PC可促进 BASMC增殖 ,丹酚酸 B和欧芹素乙可抑制 L PC的作用     

迷迭香酸对人结肠癌HCT-8细胞增殖和凋亡的影响及分子机制研究

目的研究迷迭香酸对人结肠癌HCT-8细胞增殖和凋亡的影响,并探究相关机制。方法采用CCK-8法检测不同浓度迷迭香酸作用不同时间后HCT-8细胞的增殖情况;根据CCK-8法结果确定迷迭香酸15、45、75μmol/L作用72 h,考察HCT-8细胞的凋亡和相关基因及蛋白的表达;通过流式细胞术检测HCT-8细胞的凋亡率;采用实时荧光定量PCR(RT-q PCR)法检测p53、Bax和Puma基因的m RNA表达;通过Western blotting法检测p53、Bax、Puma和active Caspase-9的蛋白表达水平。结果迷迭香酸能够抑制HCT-8细胞的增殖,并呈时间和浓度依赖性。15、45μmol/L的迷迭香酸主要诱导HCT-8细胞早期凋亡(P0.01),75μmol/L的迷迭香酸主要诱导HCT-8细胞晚期凋亡(P0.01)。迷迭香酸可以使Puma的m RNA表达上调,并呈浓度依赖性;45、75μmol/L的迷迭香酸可以使p53和Bax的m RNA表达升高(P0.05)。迷迭香酸可以上调Puma、Bax和active Caspase-9的蛋白表达水平,并呈浓度依赖性;75μmol/L的迷迭香酸可以使p53的蛋白表达明显升高(P0.05)。结论迷迭香酸对HCT-8细胞具有明显的增殖抑制作用,并且是通过诱导凋亡实现的,促凋亡蛋白p53、Puma、Bax和active Caspase-9诱导了凋亡的发生。     

泰山白花丹参提取物对人脐血内皮祖细胞增殖、黏附及分泌NO的影响

目的:观察单味中药泰山白花丹参提取物(SBE)对人脐血内皮祖细胞(EPCs)增殖、黏附及分泌一氧化氮(NO)的影响。方法:采用密度梯度离心法从脐血获得单个核细胞,将其接种在人纤维连接蛋白包被的培养板上,培养7d后收集贴壁细胞,加入不同浓度SBE继续培养24h;分别采用MTT比色法、黏附能力测定实验和硝酸还原酶法测定EPCs的增殖、黏附和分泌NO能力。结果:与对照组比较,SBE组EPCs增殖、黏附能力提高,培养上清中NO浓度增加(P<0.05),其作用在一定范围内随SBE浓度增加而增强。结论:白花丹参显著改善EPCs功能,这可能是其内皮保护的新机制。