加减青娥方体外对RAW264.7细胞向破骨细胞分化的影响

目的 探讨加减青娥方抑制骨质疏松的作用机制。方法 在体外培养的小鼠破骨前体RAW264.7细胞中加入不同浓度的加减青娥方提取物,利用抗酒石酸酸性磷酸酶(TRAP)染色法检测核因子κ B受体活化因子配体(RANKL)诱导的RAW264.7细胞分化为破骨细胞的数量及其活性的影响;利用荧光定量PCR(RT-PCR)法检测RANKL诱导的RAW264.7细胞分化为破骨细胞雌激素受体(ER)mRNA表达。结果 加减青娥方可抑制RANKL诱导的RAW264.7细胞分化为破骨细胞,该作用可能是通过调控ERα mRNA的表达实现的。结论 加减青娥方可通过调控ERα基因的表达,抑制破骨细胞的分化、增殖,从而实现增加骨密度、防治骨质疏松的作用。     

CRISPR/Cas 9介导的基质Gla蛋白基因敲除对破骨细胞分化的影响

目的利用 CRISPR/Cas 9基因编辑技术,构建基质 Gla蛋白(MGP)基因敲除的 RAW264.7巨噬细胞系,明确 MGP基因在破骨细胞分化过程中的作用。方法利用在线软件分析并筛选出 3个针对 MGP基因外显子区的单链向导 RNA(gRNA)人工合成 gRNA寡核苷酸序列,并将其插入线性化的 LentiCRISPRv2质粒中,构建成 LentiCRISPRv2?MGP?gRNA重组质粒。将重组,质粒与 psPAX2、VSVG包装质粒一同转染 293?T细胞,包装并收集重组慢病毒,将其感染 RAW264.7细胞。经嘌呤霉素筛选得到稳定 RAW264.7细胞系,实时荧光定量多聚核苷酸链式反应(qPCR)、蛋白质印迹法(Western Blot)验证 MGP mRNA及蛋白表达水平。 qPCR检测破骨细胞分化标志分子的表达情况。结果成功构建了 LentiCRISPRv2?MGP?gRNA重组质粒,成功包装了含有 MGP?gRNA的重组慢病毒,筛选得到了稳定低表达 MGP的 RAW264.7细胞系。 qPCR及蛋白质印迹法检测显示,MGP mRNA、蛋白表达显著性下调(P＜0.05)。 qPCR结果显示,最具有代表性的 LentiCRISPRv2?MGP?gRNA1细胞破骨标志分其子 Itgb3(2.29±0.17)、 Acp5(2.86±0.15)、 Ctsk(2.07±0.13)的 mRNA水平均显著上调(P＜0.05)。结论利用 CRISPR/Cas 9基因编辑技术成功构建了 MGP基因敲除的 RAW264.7细胞系,敲除 MGP后 RAW264.7细胞破骨分化能力增强。     

木豆叶提取物对人的类成骨细胞TE85成骨功能和体外破骨细胞分化的影响

木豆素是从天然植物木豆叶中提取的单体化合物，结构类似乙烯雌酚。本文观察木豆素及4种木豆叶提取物总成分(32-1，35-1，35-2和35-3)对人的类成骨细胞HOS TE85成骨功能、间质矿化及体外破骨细胞分化的影响。以木豆叶提取物作用于细胞48 h，观察细胞增殖、³H-脯氨酸掺入量及碱性磷酸酶活性；用抗酒石酸酸性磷酸酶(TRAP)染色法观察体外破骨细胞形成；作用于细胞18 d，用茜素红染色法观察细胞间质矿化。结果表明，作用于细胞48 h后，木豆素可促进细胞增殖，在1×10^-8 g·mL^-1时细胞数增加57.7%，³H-脯氨酸掺入量增加98.5%。35-1和35-3还可明显增加碱性磷酸酶活性。长时程作用后，32-1和35-3可明显增加成骨细胞间质矿化程度。木豆素(1×10^-7 g·mL^-1)对破骨细胞形成有明显的抑制作用，抑制率为22.8%，32-1(1×10^-7 g·mL^-1)的抑制率为37.9%。木豆素及木豆叶提取物都具有刺激成骨细胞骨形成、促进细胞间质矿化及抑制破骨细胞形成的活性，提示木豆素在骨细胞上有类雌激素样作用，具有抗骨质疏松新药的研究前景。     

柚皮苷对破骨细胞分化和极化影响的初步探究

目的　探究柚皮苷对破骨细胞分化和极化功能的影响。方法　体外培养RAW264.7细胞,设置对照组（培养基不含柚皮苷）和实验组（培养基中柚皮苷剂量分别为2、20、200 μg/L）连续培养7 d,通过抗酒石酸酸性磷酸酶（TRAP）染色对比组间诱导分化结果,通过实时荧光定量逆转录聚合酶链反应（qPCR）对比组间组织蛋白酶K（CK）、基质金属蛋白酶-9（MMP-9）、整合素β3（integrin β3）和非受体酪氨酸激酶（c-src）mRNA表达水平,通过蛋白免疫印迹法对比组间CK、MMP-9、integrin β3、磷酸化产物p-src蛋白表达水平。结果　与对照组相比,各实验组TRAP染色阳性细胞数显著降低（P＜0.05）;与对照组相比,20 μg/L与200 μg/L组CK、MMP-9、integrin β3及c-src mRNA表达水平显著降低（P＜0.05）;与对照组相比,20 μg/L与200 μg/L组CK、MMP-9、integrin β3及p-src蛋白表达水平显著降低（P＜0.05）。结论　柚皮苷对破骨细胞的分化和极化有一定的抑制作用,其作用机制可能与下调integrin β3、c-src及p-src表达有关。     

葡萄糖对大鼠骨髓破骨细胞分化的影响

目的观察葡萄糖对大鼠骨髓破骨细胞(OC)分化的影响,探讨糖尿病骨质疏松的发病机制。方法用巨噬细胞克隆刺激因子(M-CSF)、RANKL诱导大鼠骨髓单个核细胞分化为OC,同时给予不同浓度的葡萄糖(0、5.5、15、25mmol/L)干预,通过观察抗酒石酸酸性磷酸酶(TRAP)染色阳性OC数、TRAP活性测定、OC膜表面RANKmRNA表达量来分析葡萄糖对OC分化的影响。结果高糖(25mmol/L)组培养7d时TRAP染色阳性OC数及TRAP活性测定均高于对照组(0mmol/L)、葡萄糖5.5mmol/L组(P<0.01);与对照组相比高糖组培养3d时TRAP染色阳性OC数明显增多(P<0.01);葡萄糖呈浓度依赖性上调OC膜表面RANKmRNA的表达,其中高糖组培养的各时间点与其他三组相比差异有显著性(P<0.01,P<0.05)。结论高糖可促进OC的分化,其促进作用始于诱导分化的早期;骨髓微环境中高糖可引起OC分化增多,可能是糖尿病骨质疏松的发病机制之一。     

女贞子提取物对破骨细胞分化及成骨细胞增殖的影响

目的:探讨女贞子提取物对破骨细胞分化及成骨细胞增殖的影响。方法:以1α,25-二羟基维生素D3诱导兔原代骨髓细胞获取破骨细胞,经女贞子醇提物和水煎物低、中、高剂量(均为2、20、200 mg/L)处理后,采用抗酒石酸酸性磷酸酶(TRACP)活性测定法检测各组细胞裂解液中TRACP活性及细胞中TRACP阳性细胞数,采用甲苯胺蓝染色法检测各组细胞骨吸收陷窝数量及骨陷窝面积百分率。以L-抗坏血酸、β-甘油磷酸钠、地塞米松诱导小鼠颅顶前成骨细胞亚克隆14细胞获取成骨细胞,采用MTT法检测各组细胞的相对增殖率,采用碱性磷酸酶(APK)活性测定法检测各组细胞中APK活性。结果:经不同剂量女贞子提取物处理后,TRACP阳性细胞数、骨吸收陷窝数量及其面积均有不同程度的改变,其中女贞子醇提物各剂量组和水煎物高剂量组TRACP活性及阳性细胞数、骨吸收陷窝数量及面积百分率,以及女贞子水煎物中剂量组TRACP活性及阳性细胞数、骨陷窝面积百分率均显著降低或减少,而水煎物低剂量组细胞的骨吸收陷窝数量显著增多(P<0.05或P<0.01);女贞子提取物低、中剂量组细胞的相对增殖率以及提取物各剂量组细胞的APK活性均显著升高,而女贞子提取物高剂量组细胞的相对增殖率均显著降低(P<0.01)。结论:女贞子提取物可抑制破骨细胞的TRACP活性,改变破骨细胞的骨吸收功能和成骨细胞的增殖行为,并增加成骨细胞的APK活性。     

维生素K对成骨细胞骨形成和破骨细胞骨吸收的影响

目的 比较维生素K₁(VK₁)、维生素K₂(MK₄)、维生素K₂(MK₇)和维生素K₃(VK₃)促进骨形成和抑制骨吸收的作用。方法 采用新生大鼠颅盖骨分离成骨细胞和以核因子κB受体活化因子配体(RANKL)诱导骨髓单核细胞的破骨细胞为模型,用磷酸苯二钠法检测成骨碱性磷酸酶(ALP)和抗酒石酸酸性磷酸酶(TRAP)活性;用CellTilter试剂盒检测破骨细胞代谢活力;用Z-FR-MCA荧光底物和胶原底物降解检测组织蛋白酶K(CTSK)抑制作用。结果 MK₄和MK₇在0.1~1 μmol/L时显著促进成骨细胞增殖(P<0.05),1 μmol/L时显著提高ALP活性和骨结节形成面积,VK₃抑制了骨结节形成(P<0.05)。VK₁、VK₃、MK₄和MK₇在1 μmol/L时对破骨细胞代谢活力均无影响,MK₄和MK₇在0.1~1 μmol/L时显著抑制TRAP活性(P<0.05),而VK₁和VK₃无抑制作用。MK₄在25 μmol/L可对CTSK与Z-FR-MCA底物结合的抑制率达58.9%,在100 μmol/L对CTSK胶原降解的抑制率达73.2%。结论 相较于VK₁和VK₃,MK₇和MK₄有显著促进成骨细胞活性和抑制破骨细胞骨吸收的作用,MK₄能显著抑制破骨细胞CTSK酶活性。     

补肾方骨青颗粒对破骨细胞分化及骨吸收功能的影响

目的 探讨补肾方骨青颗粒对破骨细胞分化及骨吸收功能的影响.方法 体外诱导破骨前体细胞系RAW264.7向破骨细胞分化,通过抗酒石酸酸性磷酸酶(TRAP)染色观察破骨细胞数目,图像分析计算骨吸收陷窝面积,荧光定量RT-PCR检测骨青颗粒对破骨细胞骨吸收功能标志性基因水平.结果 与对照组相比,骨青颗粒可显著抑制TRAP染色阳性细胞的产生,减少破骨细胞骨吸收陷窝的面积,明显降低破骨细胞骨吸收功能标志性基因[组织蛋白酶K(Ctsk)、基质金属蛋白酶9(MMP-9)]mRNA的表达.结论 骨青颗粒可显著抑制破骨细胞的分化及骨吸收功能.     

11种中药提取物对脂多糖诱导的RAW264.7细胞生成一氧化氮的影响

目的研究11味中药提取物对脂多糖(LPS)诱导巨噬细胞RAW264.7产生一氧化氮(NO)的抑制作用。方法使用MTT法测试样品对RAW264.7的细胞毒作用,Griess法测定样品对LPS刺激RAW264.7细胞产生NO的含量,N-单甲基-L-精氨酸单乙酸酯(L-NMMA)为阳性对照。结果 11味中药甲醇提取物均具有一定的抑制RAW264.7细胞生成NO的作用,其中秦艽、五加皮、独活、雷公藤、仙鹤草提取物的抑制作用最强(IC50在4.3±0.6~14.8±2.6μg·m L-1),且选择指数较高(SI>27)。结论本研究系统评价了11味常用中药提取物对LPS诱导RAW 264.7细胞产生NO的影响,研究结果进一步证实这些中药的抗炎活性。     

Effects of marjoram volatile oil and grape seed extract on ethanol toxicity in male rats

Natural dietary antioxidants are extensively studied for their ability to protect cells from miscellaneous damages. Marjoram volatile oil (Origanum majorana L., Lamiaceae) and grape seed extract (Vitis vinifera L., Vitaceae) are potent antioxidants. Effects of administration of marjoram volatile oil or grape seed extract on oral administration of ethanol, simultaneously, daily for 10 weeks were studied through determining epididymal spermatozoal analysis, serum testosterone level, weight and histopathological examination of testis, liver and brain. Glutathione level and lipid peroxidation content as malondialdehyde in the testis, liver and brain were measured. The repeated intake of a great amount of ethanol (10 ml/kg body weight, 25% v/v) was followed by fertility disturbances with low sperm count, impaired sperm motility and decrease in serum testosterone level. Moreover, ethanol toxicity induced significant alterations in the histological structures of the testis, liver and brain. The results revealed a significant increase in lipid peroxidation and decrease in the level of glutathione in the testis, liver and brain in the ethanol-treated group. However, co-administration of the extracts of protective plants resulted in minimizing the hazard effects of ethanol toxicity on male fertility, liver and brain tissues. It may be concluded that marjoram volatile oil and grape seed extract are useful herbal remedies, especially for controlling oxidative damages.     

Antioxidant properties of grape seed extract on human lymphocyte oxidative defence

The distribution of polyphenolic compounds in a grape (Vitis vinifera) seed extract (GSE) was determined using LC/ESI-TOF MS, HPLC/DAD, and (13)C-NMR. The 17 identified compounds comprised gallic and protocatechuic acid, catechin and epicatechin monomers, procyanidin oligomers, and procyanidin gallates. This study addresses the in vitro effects of grape seed extract (GSE) on the frequency of micronuclei with reference to the antioxidant status in human lymphocytes. To establish the most effective protective support, we used four different concentrations of GSE, in the range 1-6 microg/mL. Treatment of lymphocytes with GSE at a concentration of 2.5 microg/mL induced a significant decrease in the frequency of micronuclei by 40%, reduction of malonyldialdehyde production by 30%, while a concentration of 5 microg/mL increased catalase and glutathione S-transferase activity by 10% and 15%, respectively. These results demonstrate that GSE may be effective in the prevention of oxidative lymphocyte damage by ROS.     

葡萄籽原花青素对兔动脉粥样硬化血管病理形态的影响

目的：通过建立新西兰兔的高脂血症模型,观察葡萄籽原花青素（GSPE）对兔动脉粥样硬化（AS）血管病理形态的影响。方法选用新西兰大白兔,随机分为空白对照组、高脂模型组、GSPE 小剂量组、中剂量组、大剂量组、洛伐他汀阳性对照组,复制高脂食饵性兔 AS 模型,每组7只。空白对照组喂饲基础饲料;其余各组给予高脂饲料（80％普通饲料＋14％蛋黄粉＋1％胆固醇＋5％猪油）喂饲,实验共8周。给予高脂饲料第1天起开始给药,GSPE 组每兔每天每公斤体重给予0.1 g、0.2 g、0.4 g,洛伐他汀阳性对照组每公斤体重给予洛伐他汀1 mg,以上药物均胶囊给药,模型组、空白对照组给以空胶囊。观察原花青素预防性用药后对兔主动脉内皮损伤的病理形态影响。结果GSPE 大、中剂量组与高脂模型组比较,从大体形态、组织学、扫描电镜三方面显示内皮细胞损伤均有一定的改善,斑块/内膜面积比分别减少29.5％,27.2％,差异有统计学意义（ P ＜0.05）,内/中膜厚度比分别减少72.5％,60.2％,差异有统计学意义（ P ＜0.05）。对主动脉内膜超微结构也有良好的修复和保护作用。但小剂量 GSPE 与高脂模型组比较,差异无统计学意义（ P ＞0.05）。结论大、中剂量 GSPE 对于减轻 AS 兔主动脉内膜损伤病变有一定作用。     

Ameliorative effect of grape seed proanthocyanidin extract on thioacetamide-induced mouse hepatic fibrosis

The present study was designed to examine the effect of the grape seed proanthocyanidin extract (GSPE) on developing hepatic fibrosis that was induced by thioacetamide (TAA) in mice. Administration of TAA for 9 weeks led to a serious necrosis and apoptosis of the parenchymal cells, which resulted in an accumulation of excessive collagen in the liver and an increase of transformed hepatic stellate cells (HSCs). In addition, the mRNA expression of transforming growth factor β1 (TGF-β1), α-smooth muscle actin (α-SMA), as the marker of the activated HSCs, and α1-(I)-collagen were all up-regulated significantly when compared with the control. However, combined oral administration of GSPE at 100mg/kg suppressed the mRNA expression of TGF-β1 and α-SMA, with decreased collagen accumulation as demonstrated by histomorphological evaluation and quantitative RT-PCR. The mRNA expression of the pro-inflammatory factors, including inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), was remarkably enhanced by TAA treatment. However, their levels displayed a down-regulated trend beyond simultaneous GSPE treatment. Moreover, GSPE administration markedly suppressed lipid peroxidation. In conclusion, as a plant antioxidant, GSPE manifested effective hepatocellular protective action to ameliorate the developing liver fibrosis induced by chronic TAA administration in mice.     

葡萄籽原花青素对小鼠深低温脑缺血-再灌注损伤的影响

目的探讨葡萄籽原花青素(GSPE)对小鼠脑缺血-再灌注(I-R)损伤的保护作用。方法 60只C57/BL6小鼠随机均分为GSPE(A)组、模型(B)组和假手术(C)组,建立深低温I-R模型,再灌注24 h后处死小鼠取脑,RT-PCR技术检测小鼠脑组织半胱天冬氨酸蛋白酶(Caspase)3mRNA,TUNEL法检测小鼠大脑皮层细胞凋亡。结果与C组比较,A组和B组Caspase-3 mRNA表达和细胞凋亡明显增加(P<0.05);而A组各观察指标低于B组(P<0.05)。结论 GSPE对深低温I-R损伤鼠脑有保护作用。     

葡萄籽原花青素提取物对大鼠乙酸性结肠炎的保护作用

目的:研究葡萄籽原花青素提取物(GSPE)对大鼠乙酸性结肠炎的治疗作用。方法:制备大鼠乙酸性结肠炎模型,分别设正常对照组、模型对照组、阳性对照组和GSPE低、中、高剂量组,共6组。每天给药1次,共7d。于给药d8时进行大鼠病变结肠大体及组织学评分;用邻联茴香胺法检测结肠组织中MPO活性以考察中性白细胞浸润程度。结果:实验1周时,模型对照组大鼠体重明显下降,结肠湿重明显增加,和模型对照组比较,GSPE低、中剂量组体重下降程度减轻(P<0.05),GSPE高剂量组体重呈增加趋势(P<0.01)。GSPE各剂量组结肠湿重增加幅度较模型对照组明显降低(P<0.05)。大体观察,模型对照组结肠粘膜大面积溃疡、坏死,和模型对照组比较,GSPE各剂量组结肠粘膜溃疡面积缩小,粘膜修复明显。光镜下观察,模型对照组结肠粘膜组织内可见大量炎性渗出物和组织坏死;GSPE各剂量组溃疡面可见多量再生上皮和新生腺体。模型对照组结肠粘膜组织内MPO含量较正常对照组明显升高(P<0.01),GSPE各剂量组MPO活性较模型对照组明显降低(P<0.05)。结论:GSPE对大鼠乙酸性结肠炎有明显的治疗作用,且呈剂量依赖性。     

葡萄籽

植物名 Vitis vinifera：V．coignetiae, 活性成分与药理作用葡萄籽是葡萄酒生产的副产物,低聚原花青素类（OPCs）或原花青素类是葡萄籽的活性成分。葡萄籽中的OPCs具有抗氧化和潜在的抗脂质过氧化活性．可防治心血管和循环系统疾病。OPCs还可抑制蛋白水解酶胶原酶、弹性蛋白酶、透明质酸酶和β-葡萄糖苷酸酶,这些酶会导致脉管系统和皮肤结构成分的破坏。葡萄籽还可通过抑制变异链球菌的生长,阻止蔗糖转化为葡聚糖而预防虫牙。     

The effect of grape seed extract on the pharmacokinetics of dextromethorphan in healthy volunteers

Purpose

Grape seed extract (GSE) has been shown to inhibit the cytochrome P450 (CYP) 2D6 isoenzyme in vitro. To determine the clinical effect of GSE on CYP2D6, the pharmacokinetic interaction between GSE and the sensitive CYP2D6 probe dextromethorphan in healthy adult volunteers was examined.

Methods

In this open label, randomized, cross-over study, 30 subjects were assigned to cohort A or B. Both cohorts ingested 30 mg dextromethorphan hydrobromide on day 1 and day 10. Cohort A received 100 mg GSE capsules three times daily on days 8, 9 and 10, while cohort B started with GSE on day ?1 until day 1. After urine collection (0–8 h) on day 1 and day 10, the urinary dextromethorphan to dextrorphan metabolic ratio was determined.

Results

Among 28 evaluable subjects, an increase of the urinary metabolic ratio was observed in 16 subjects (57 %). The mean metabolic ratio (± standard deviation) before and after GSE supplementation was 0.41 (± 0.56) and 0.48 (± 0.59), respectively. This result was neither statistically (P?=?0.342) nor clinically [geometric mean ratio 1.10, 90 % CI (0.93–1.30)] significant. Further, the majority (73 %) of the included subjects did not experience any adverse events after intake of dextromethorphan or GSE.

Conclusions

Supplementation of GSE did not significantly affect the urinary dextromethorphan to dextrorphan metabolic ratio in healthy volunteers. The results of this clinical study indicate that GSE appears to be safe to combine with drugs extensively metabolized by CYP2D6, such as dextromethorphan and tamoxifen.     

葡萄籽原花青素对大鼠肾缺血再灌注损伤的保护作用

目的探讨葡萄籽原花青素(Grape seed proanthocyanidin extract,GSPE)对大鼠急性肾缺血再灌注损伤的保护作用及其机制。方法32只SD大鼠随机分为4组,每组8只,分别为假实验组(SG),缺血再灌注组(I/RG),急性GSPE(450mg/kg灌胃1次)治疗+I/R组(ATG),慢性GSPE(150mg/kg灌胃喂养1周)治疗+I/R组(CTG)。采用一侧肾蒂结扎、另一侧肾动脉夹闭的肾缺血再灌注损伤模型,测定肾组织丙二醛(MDA)水平和超氧化物歧化酶(SOD)的活性,同时测定血清尿素氮(BUN)和肌酐(Cr)水平。结果I/RG与SG相比,MDA、BUN和Cr含量明显升高(P<0.05),SOD活性下降(P<0.05)。与I/RG相比,ATG的MDA和Cr含量下降(P<0.01,P<0.05),CTG的MDA、BUN和Cr含量明显下降(P<0.05,P<0.01),SOD活力显著升高(P<0.05)。结论葡萄籽原花青素对肾缺血再灌注损伤具有保护作用,其机制可能与抗自由基损伤有关。