Antiaggregation potential of berry fractions against pairs of Streptococcus mutans with Fusobacterium nucleatum or Actinomyces naeslundii

Coaggregation is an interspecies adhesion process, which is essential to the development of dental plaque. This is an in vitro study of the composition of the soluble solids in the berry juice molecular size fractions (<10 kDa, FI; 10–100 kDa, FII; >100 kDa, FIII) derived from apple, bilberry, blackcurrant, cloudberry, crowberry and lingonberry and their ability to inhibit and reverse coaggregation of the pairs of common species in dental plaque: Streptococcus mutans with Fusobacterium nucleatum or Actinomyces naeslundii. Inhibitory and reversal activity was found in the molecular size fractions FII and FIII of bilberry, blackcurrant, crowberry and lingonberry. The active fractions contained higher amounts of polyphenols (5–12% of soluble solids) than those without activity (<2% of soluble solids). Proanthocyanidins dominated in the active lingonberry juice fractions FII and FIII and also small amounts of anthocyanins were detected. Anthocyanins, proanthocyanidins and flavonol glycosides were prevalent in FII and FIII fractions of bilberry, blackcurrant and crowberry juices. Comparable amounts of sugars and titratable acids were present in the latter three berry juice fractions of different size. The results indicate that the high molecular size fractions of lingonberry, bilberry, blackcurrant and crowberry juices have antiaggregation potential on common oral bacteria, the potential being associated with their polyphenolic content. Copyright © 2010 John Wiley & Sons, Ltd.     

Tart cherry (Prunus cerasus L.) fractions inhibit biofilm formation and adherence properties of oral pathogens and enhance oral epithelial barrier function

Dental caries, candidiasis, and periodontal disease are the most common oral infections affecting a wide range of the population worldwide. The present study investigated the effects of two tart cherry (Prunus cerasus L.) fractions on important oral pathogens, including Candida albicans, Streptococcus mutans, and Fusobacterium nucleatum, as well as on the barrier function of oral epithelial cells. Procyanidins and quercetin and its derivatives were the most important constituents found in the tart cherry fractions. Although the fractions showed poor antimicrobial activity, they inhibited biofilm formation by the three oral pathogens in a dose‐dependent manner. The tart cherry fractions also attenuated the adherence of C. albicans and S. mutans to a hydroxylapatite surface as well as the adherence of F. nucleatum to oral epithelial cells. Treating oral epithelial cells with the tart cherry fractions significantly enhanced the barrier function as determined by monitoring the transepithelial electrical resistance. In conclusion, this study showed that the tart cherry fractions and their bioactive constituents could be promising antiplaque compounds by targeting biofilm formation and adherence properties of oral pathogens. Furthermore, its property of increasing the epithelial barrier function may protect against microbial invasion of the underlying connective tissue.     

Inhibition of Helicobacter pylori-induced inflammation in human gastric epithelial AGS cells by Phyllanthus urinaria extracts

AIM OF THE STUDY: Helicobacter pylori is linked to a majority of peptic ulcers and to some types of gastric cancer, and its resistance to antibiotic treatment is now found worldwide. This study is aimed at evaluating the antimicrobial activity of Phyllanthus urinaria Linnea (Euphorbiaceae), chloroform (PUC) and methanol (PUM) extracts, and its eight isolates on H. pylori-infected human gastric epithelial AGS cells. MATERIALS AND METHODS: The in vitro anti-bacterial activity of P. urinaria chloroform (PUC) and methanol (PUM) extracts, and its eight isolates were determined. Additional experiments were also performed to know the PUC and PUM ability to inhibit the H. pylori adhesion to and invasion of AGS cells, in addition to the effect of PUC on NF-kappaB activity as well as IL-8 synthesis during H. pylori infection of AGS cells. RESULTS: The results revealed that crude extracts PUC and PUM showed potent antimicrobial activity against H. pylori than pure isolates. On the other hand, in vitroH. pylori-infection model revealed that the inhibition of bacterial adhesion and invasion to AGS cells has dramatically reduced by treatment of extract PUC, while PUM has the same moderate effect. Furthermore, H. pylori-induced nuclear factor (NF)-kappaB activation, and the subsequent release of interleukin (IL)-8 in AGS cells were also inhibited by the extract PUC. CONCLUSIONS: These results open the possibility of considering P. urinaria a chemopreventive agent for peptic ulcer or gastric cancer, but this bioactivity should be confirmed in vivo in the future.     

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。     

芪归解毒汤对硫酸镍诱导晶状体上皮细胞凋亡的抑制作用

目的观察芪归解毒汤对硫酸镍诱导正常人晶状体上皮细胞(Lens epithelium cell,LEC)凋亡的影响。方法体外培养正常人晶状体上皮细胞,用血清药理学方法制备含芪归解毒汤药物血清,用药物血清和硫酸镍处理培养的LEC,采用流式细胞术检测LEC凋亡率;透射电子显微镜观察LEC超微结构改变和凋亡小体形成。结果硫酸镍组LEC凋亡率(26.99±2.58)高于中药组LEC(12.21±2.42),硫酸镍组和中药组LEC凋亡率均高于对照组(0.8±0.16),三组间差异有统计学意义(x2=-20.489,P0.01),两两比较均有统计学意义(P0.01)。透射电镜观察到硫酸镍组LEC各阶段均有细胞凋亡,可见到LEC凋亡后的继发性坏死。中药组LEC呈凋亡早期改变,只有少数LEC呈凋亡改变,未见有凋亡后继发性坏死的LEC。结论芪归解毒汤药物血清可有效地抑制硫酸镍诱导的LEC凋亡。     

益糖康对高糖诱导人肾小管上皮HK-2细胞上皮间质转化的影响

目的:探讨益糖康是否通过抑制肾小管上皮细胞发生上皮-间质转化(EMT)进而减轻糖尿病对肾脏的早期损伤.方法:40只SD雄性大鼠随机分成生理盐水组(1mL/100g)、益糖康组(2.10g/100g)、二甲双胍组(9.11g/100g)、二甲双胍+益糖康组(4.55g/100g二甲双胍+1.05g/100g益糖康煎剂),...     

急性高眼压对兔晶状体上皮细胞的影响

目的观察急性高眼压状态下兔晶状体上皮细胞凋亡,Bcl-2、PCNA表达的动态变化,以证实凋亡是青光眼性白内障的早期表现。方法36只健康无眼疾成年灰兔随机分为A、B、C、D4组,每组18只眼。将每组再随机分入对照组、50 mm Hg组、80 mm Hg组,保证每只兔两只眼不在同一组。对照组做玻璃体穿刺,不灌注。50 mm Hg组、80 mm Hg组玻璃体穿刺后灌注乳酸钠林格注射液,调整灌注高度控制相应眼压。A、B、C、D组动物分别在灌注3、6、9、12I小时后处死并摘除眼球,取晶状体前囊膜制作晶状体上皮细胞铺片,进行TUNEL染色,Bcl-2、PCNA免疫组化染色。结果1.对照组极少见凋亡细胞,随眼压升高、灌注时间的延长,晶状体上皮细胞出现凋亡并且数量增加(P<0.01)。2.BCL-2蛋白在对照组中有少量表达,高眼压状态下表达随时间延长、眼压增高而增加、染色增强。与对照组相比有显著性差异(P<0.01)。3.PCNA阳性染色位于晶状体赤道部,中央区无阳性细胞。对照组与实验组无统计学差异(P>0.05)。结论在急性高眼压状态下,晶状体上皮细胞开始出现凋亡并随眼压程度、高眼压持续时间的延长而增加。认为凋亡也是青光眼性白内障起始的细胞学基础。     

蜕皮甾酮对氧化损伤的HLEC线粒体超微结构的影响

目的探讨植物雌激素蜕皮甾酮（ecdysterone,ECR）保护氧化损伤的人晶状体上皮细胞（human lens epithelial cells,HLEC）亚细胞水平的形态结构的变化。方法实验分4组：正常对照组、过氧化氢（hy—drogen peroxide,H2O2）组、雌二醇（B—Estradiol,E2）和ECR组。密度梯度离心法分离出HLEC线粒体,透射电镜观察HLEC线粒体超微结构。结果正常对照组线粒体双层膜结构完整,嵴结构完整,线粒体几乎未受损伤;H2O2组线粒体双层膜结构不完整,嵴结构断裂消失,线粒体受损伤;ECR组和E2组线粒体双层膜结构稍完整,嵴结构断裂,线粒体受损伤,但较H2O2组情况好。结论植物雌激素ECR对H2O2诱导氧化损伤的HLEC线粒体超微结构有保护作用。     

人参皂甙Rbl对人肾小管上皮细胞表达蛋白质组的影响

目的:研究人参皂甙Rbl对人肾小管上皮细胞表达蛋白质组的影响。方法:人肾小管上皮细胞株(HK-2)常规培养,随机分为两组,实验组加入5μg/ml的Rbl,对照组加入等量的培养基,药物作用20min,裂解细胞,提取全细胞蛋白。双向电泳(2-DE)分离,ImageMaster 2D Platinum v5.0软件进行差异表达蛋白质组分析,基质辅助激光解吸附离子化飞行时间质谱(MALDI-TOF-MS)鉴定蛋白质。结果:通过对2-DE图谱蛋白斑点的匹配及对比分析,实验组的2-DE图谱共检出蛋白斑点为429个,其中236个为表达增强的蛋白斑点;经质谱鉴定的与Rbl作用相关的4个差异表达的蛋白斑点包括:普通转录因子ⅡH亚基1变体、双链断裂修复蛋白rad-21同源物、富含亮氨酸重复序列蛋白-45、DNA依赖性蛋白激酶,它们均属于磷酸化蛋白质。结论:Rbl作用于人肾小管上皮细胞的表达蛋白质组与对照组相比较存在明显的差异;Rbl对人肾小管上皮细胞的作用极有可能是通过相应的细胞信号转导网络系统来实现的。     

益肾软坚散含药血清拮抗马兜铃酸对人近端肾小管上皮细胞的作用

目的:研究益肾软坚散含药血清能否拮抗马兜铃酸(aristolochicacid,AA)诱发的人近端肾小管上皮细胞株(HKC)促纤维化效应。方法:用马兜铃酸钠盐(AA Na,40mg·L^-1)加或不加10%大鼠含药血清与HKC细胞孵育,而后检测信使RNA(mRNA)和蛋白质表达。结果:大鼠含药血清能显著下调AA Na刺激后HKC细胞对转化生长因子β1(TGFβ1)、结缔组织生长因子(CTGF)和金属蛋白酶组织抑制物1(TIMP1)mRNA及蛋白质的高表达(P<0.05),但对纤溶酶原激活物抑制物1(PAI1)mRNA及蛋白质高表达无显著作用(P>0.05)。结论:益肾软坚散含药血清可下调AA刺激的HKC细胞促细胞外基质(ECM)合成因子及抗ECM降解因子的表达。     

葛根素对人肾近曲小管上皮细胞ABCG2表达影响

  目的：探讨葛根素(Pur)对人肾近曲小管上皮细胞(HK2)内三磷酸腺苷结合盒转运体G2(ABCG2) 基因与蛋白表达的影响,阐明葛根素治疗痛风的可能机制。  方法：体外培养HK2细胞,分为对照组与25、50、100、200 mg/L 葛根素给药组。CCK-8法检测葛根素对HK2细胞的影响;检测ALP活性,观察HK2细胞内酶促反应状态;荧光定量PCR法、Western blotting法检测对照组与给药组HK2细胞内ABCG2基因蛋白表达。  结果：葛根素对HK2细胞有促进增殖作用(P<0.05);100 mg/L葛根素组ALP活性最佳(P<0.01)。荧光定量 PCR和Western blotting法检测结果显示,葛根素组HK2细胞内ABCG2基因及蛋白表达明显高于对照组,其中100 mg/L葛根素组HK2细胞内ABCG2最为明显,差异有统计学意义(P<0.01)。  结论：葛根素能够增加HK2细胞内ABCG2基因蛋白的表达,这可能是葛根素调节痛风患者血尿酸水平的重要分子机制之一。     

Protective effects of Puerariae radix extract and its single compounds on methylglyoxal-induced apoptosis in human retinal pigment epithelial cells

Ethnopharmacological relevance

In Korea, Puerariae radix is a medicinal plant traditionally used to treat various diseases including diabetes mellitus. To provide pharmacological basis for Puerariae radix in the treatment of diabetes, we investigated the protective effects of the ethanolic extract and its single compounds on apoptosis associated with glycation in human retinal pigment epithelial (RPE) cells.

Materials and methods

In the present work, a quantified ethanolic extract or single compounds of Puerariae radix were selected to determine its anti-apoptotic effect in human RPE cells cultured with methylglyoxal (MG), which is a stimulator of glycation. To assess the protective effect of the extract or single compounds, the cytotoxicity assessment was performed using an MTT assay in the human RPE cells. Selected active compounds or extracts were tested by FACS analysis with annexin V staining for apoptosis.

Results

Daidzein (1), daidzin (2), puerarin (3), 3'-hydroxy-daidzein 8-C-apiosyl (1→6) glucoside (4), and daidzein 8-C-apiosyl (1→6) glucoside (5), and pueroside B (6) were isolated from an ethanolic extract of Puerariae radix. MG-induced apoptosis was completely inhibited by Puerariae radix, ethanolic extract, and single compounds. Of the six major compounds, daidzin (2) and 3'-hydroxy-daidzein 8-C-apiosyl (1→6) glucoside (4) significantly inhibited MG-induced apoptosis.

Conclusion

Our results provide the first evidence that, due to its anti-glycation effect, Puerariae radix extract could inhibit MG-induced apoptosis in the cultured human RPE cells. These data suggest that Puerariae radix extract, especially its single compounds daidzin and 3'-hydroxy-daidzein 8-C-apiosyl (1→6) glucoside, has potential utility as a preventive agent for glycation-related diabetic retinopathy.     

Inhibition of enteropathogenic Escherichia coli adhesion on host epithelial cells by Holarrhena antidysenterica (L.) WALL.

Bacterial adhesion is the first step in the sequence of events leading to infection. Previous data are available on the effect of Holarrhena antidysenterica on antidiarrhoeal and antibacterial action, but there is little information on the mechanism of action of the various aspects of EPEC‐induced diarrhoea, namely adherence and translocation of the effector molecule to intestinal epithelial cells. The aim of the present study was to investigate the effects of alkaloids of Holarrhena antidysenterica (AHA) on interference in the mechanism of enteropathogenic Escherichia coli (EPEC) adhesion on host epithelial cells (INT 407 and HEp2). To determine the impact of AHA on epithelial cells, cytotoxicity (LDH), adherence, apoptotic and ultrastructural studies were performed. To analyse the effect of AHA on EPEC secreted proteins, especially EspD, INT 407 monolayers were infected with EPEC and AHA‐treated EPEC, followed by immunoblotting, probed with anti EspD antisera. The maximum percentage of LDH leakage was reduced in AHA‐treated EPEC (400 µg/mL) in both cell lines. Reduced bacterial adherence was observed under light microscopy and altered apoptotic changes were visualized using propidium iodide staining in conjunction with fluorescence microscopy, in both cell lines infected with AHA‐treated EPEC and these results were confirmed with transmission electron microscope images. The suppression of type III secretory proteins (TTSPs), EspD (～40 kDa), was detected in INT 407 cells infected with AHA‐treated EPEC. In conclusion, AHA reduces initial bacterial adhesion to intact epithelial cells and it may exert an antiadherence effect against the pathogenesis of EPEC in host epithelial cells. Thus, the investigations provide a rational basis for the treatment of EPEC‐mediated diarrhoea with AHA. Copyright © 2009 John Wiley & Sons, Ltd.     

黄芪注射液对高糖诱导的肾小管上皮细胞凋亡的影响

[目的]研究黄芪注射液对高糖环境下肾小管上皮细胞(HK-2)凋亡的影响。[方法]传代培养人近曲肾小管上皮细胞,细胞用无血清培养基同步化24 h后,将细胞分为5组:低糖对照组、高糖组、高糖+不同浓度黄芪注射液组(2、20、200μg/m L),每组设3个复孔。将细胞置于37℃恒温培养箱中培养至24、48、72 h,收集细胞及细胞上清液。用流式细胞仪测定细胞凋亡率。[结果]24、48、72 h,低糖对照组细胞凋亡率明显低于高糖组(P0.05)。细胞培养24 h,黄芪干预组200和20μg/m L细胞凋亡率明显低于高糖组(P0.05)。细胞培养48和72 h,黄芪注射液干预组细胞凋亡率明显低于高糖组(P0.05)。黄芪注射液干预组各组之间凋亡率比较差异也都有显著性(P0.05),其干预作用呈剂量依赖性。[结论]黄芪注射液能抑制高糖诱导的肾小管上皮细胞的凋亡,有助于糖尿病肾病的治疗。     

扶正解毒汤对染镍体外培养人支气管上皮细胞抗氧化酶活性及丙二醛含量的影响

目的:研究灌服中药扶正解毒汤(FJD)后兔血清拮抗硫酸镍(NiSO4)的细胞毒作用及其机制。方法:以NiSO4和不同剂量FJD含药血清共同处理体外培养人支气管上皮(16HBE)细胞,测定其存活率及活力,同时观察细胞内谷胱甘肽过氧化物酶(GSH-Px)、超氧化物歧化酶(SOD)活性以及丙二醛(MDA)含量的变化。结果:在镍暴露细胞培养体系中加入FJD含药血清,可提高该细胞的存活率和活力,增强细胞内GSH-Px及SOD活性,减少MDA的生成,其中以高剂量FJD含药血清的作用最为明显。结论:NiSO4可诱发细胞氧化应激的产生,FJD具有拮抗硫酸镍细胞毒性的作用,可能与其抗氧化应激功能有关。     

Ocimum sanctum extracts attenuate hydrogen peroxide induced cytotoxic ultrastructural changes in human lens epithelial cells

Hydrogen peroxide (H₂O₂) is the major oxidant involved in cataract formation. The present study investigated the effect of an aqueous leaf extract of Tulsi (Ocimum sanctum) against H₂O₂ induced cytotoxic changes in human lens epithelial cells (HLEC). Donor eyes of the age range 20–40 years were procured within 5–8 h of death. After several washings with gentamicin (50 mL/L) and betadine (10 mL/L), clear transparent lenses (n = 6 in each group) were incubated in Dulbecco's modified Eagle's medium (DMEM) alone (normal) or in DMEM containing 100 µm of H₂O₂ (control) or in DMEM containing both H₂O₂ (100 µm ) and 150 µg/mL of Ocimum sanctum extract (treated) for 30 min at 37 °C with 5% CO₂ and 95% air. Following incubation, the semi‐hardened epithelium of each lens was carefully removed, fixed and processed for electron microscopic studies. Thin sections (60–70 mm) were contrasted with uranyl acetate and lead citrate and viewed under a transmission electron microscope. Normal epithelial cells showed intact, euchromatic nucleus with few small vacuoles (diameter 0.58 ± 0.6 µm) in well‐demarcated cytoplasm. After treatment with H₂O₂, they showed pyknotic nuclei with clumping of chromatin and ill‐defined edges. The cytoplasm was full of vacuoles (diameter 1.61 ± 0.7 µm). The overall cellular morphology was typical of dying cells. Treatment of cells with Ocimum sanctum extract protected the epithelial cells from H₂O₂ insult and maintained their normal architecture. The mean diameter of the vacuoles was 0.66 ± 0.2 µm. The results indicate that extracts of O. sanctum have an important protective role against H₂O₂ injury in HLEC by maintaining the normal cellular architecture. The protection could be due to its ability to reduce H₂O₂ through its antioxidant property and thus reinforcing the concept that the extracts can penetrate the HLEC membrane. Copyright © 2009 John Wiley & Sons, Ltd.     

阿魏酸钠防护人晶状体上皮细胞紫外线损伤的研究

目的探讨阿魏酸钠(sodium ferulate,SF)对紫外线(UV)照射诱导的人晶状体上皮细胞(humanlens epithelia cell,HLEC)损伤的防护作用。方法采用SF与HLEC共同孵育,以吡喏克辛滴眼液(PS)作为阳性对照,再用UV照射HLEC后,观察SF和PS对紫外线照射的HLEC损伤的影响。结果 UV照射后细胞活性降低,组织结构改变,流式细胞仪检查出现凋亡二倍体峰,线粒体膜电位水平下降;SF可改善UV照射后细胞损伤。结论 UV照射可引起HLEC凋亡,SF可减轻HLEC凋亡,其机制是通过提高HLEC核内DNA含量、降低细胞凋亡率、升高线粒体跨膜电位、提高HLEC活性来实现的。     

水蛭提取液对体外培养牛晶状体上皮细胞生长抑制的实验研究

目的观察水蛭提取液对体外培养的牛晶状体上皮细胞（LEC）生长的影响，探讨水蛭用于防治后发性白内障的可能性。方法采用植块培养法，对牛晶状体前囊膜进行培养。利用水蛭水提醇沉提取液，观察传代培养48小时的LEC加入不同浓度提取液培养24和72小时后的生长情况，以MTT法测定OD值，并求出半效抑制量（ID50）。传代的LEC加入提取液高剂量（ID50）及低剂量（1／10ID50）培养24小时，观察贴壁抑制情况。结果水蛭水提醇沉提取液抑制LEC生长的24和72小时ID50分别为31．85mg／ml、30．69mg／ml；与空白组比较，水蛭水提醇沉液对LEC贴壁有明显抑制作用（P〈0．01）。结论水蛭水提醇沉液能同时抑制体外培养的LEC生长及贴壁，为水蛭提取液用于防治后发性白内障提供了实验依据。     

热休克蛋白70在老年性白内障晶状体上皮细胞的表达及意义

目的探讨热休克蛋白70（heat shock protein70，HSP70）在老年性白内障晶状体上皮细胞中的表达及意义，进一步阐明老年性白内障的发病机制。方法以人类晶状体前囊膜为研究对象，正常对照组20例（20眼），老年性白内障组104例，104例白内障分为3组：皮质性组、核性组、后囊下组。按同组中性别相同、年龄相近原则将两个囊膜合二为一作为一个标本，用逆转录聚合酶链反应（RT-PCR）方法检测HSP70基因在20例皮质性白内障、17例核性白内障、15例后囊下性白内障及10例正常对照组中晶状体上皮细胞中的表达。以β-Aetin作为内参校正，并对获得的指标进行统计学分析。结果老年性白内障组HSP70的表达高于正常对照组，差异有显著性（P〈0．001）。老年性白内障各型间HSP70的表达不同，皮质性组〉核性组〉后囊下性组，差异有显著性（P〈0．05）。结论热休克蛋白70参与老年性白内障的发生发展，在皮质型的发生发展中起重要作用，各型老年性白内障发病机理不同。