不同连翘制剂对甲型流感病毒NP基因转染后表达的影响

目的：观察连翘苷、连翘醇提液、连翘水煎剂等3种连翘制剂对甲型流感病毒甲1型流感病毒核蛋白（ nucleoprotein,NP）基因转染后表达的影响。方法：将甲型流感病毒NP基因转染Hela细胞,用甲型流感病毒胶体金检测3种连翘制剂对转染后细胞内和上清核蛋白表达情况,用RT_PCR检测转染后Hela细胞内NP基因的拷贝数。结果：NP重组质粒组上清胶体金检测为阳性,连翘苷组上清为阴性、细胞内为弱阳性;连翘水煎剂及连翘醇提物组上清均为弱阳性。连翘苷组NP基因表达量较NP重组质粒组减少（ P<0.05）。结论：连翘苷抑制甲型流感病毒NP基因转染后表达作用最强。     

连翘对重症急性胰腺炎大鼠肝组织中NF-κB和Foxp3表达的影响

目的探讨核因子NF-κB和Foxp3在重症急性胰腺炎（SAP）肝损伤中的作用及连翘对其表达活性的影响。方法雄性Wistar大鼠80只,随机分成假手术组（SO组）、SAP组和干预组,其中干预组分连翘高、中、低剂量组和阳性对照组（PDTC）。牛磺胆酸钠溶液在胰胆管远端注射造模,SO和SAP组于术后3、6、12 h,干预组于术后12 h处死大鼠,分别留取标本。测各组血淀粉酶（AMY）、ALT及TNFα水平,鲎试剂法测血浆内毒素水平,流式细胞术测外周血Treg百分数,对胰腺及肝脏进行病理学检查及评分,RT-PCR法检测肝脏组织中NF-κBmRNA和Foxp3mRNA表达量。组间比较采用单因素方差分析,进一步进行多重比较,采用LSD法进行统计学处理,各指标间相关性分析采用直线相关分析。结果与SO组比较,SAP组中各项指标均随时间升高,于12 h达高峰。与SAP12 h组相比,干预组（大鼠死亡率为0）肝脏组织中的NF-κBmRNA和Foxp3mRNA表达明显降低（P〈0.01）,与Treg呈正相关（r=0.738,P〈0.01）。随连翘剂量增加,AMY、ALT及TNFα水平均明显降低,肝脏和胰腺组织炎症明显减轻,高剂量组和阳性对照组相比较无明显差异（P〉0.05）。结论 NF-κB的激活参与SAP肝损伤的发生,连翘能显著降低NF-κB的活性及肝脏组织中NF-κBmRNA和Foxp3mRNA的表达,减轻SAP肝损伤的严重程度。     

连翘中连翘苷含量测定方法的试验研究

目的改进高效液相色谱法测定连翘中连翘苷的含量测定方法。方法采用甲醇索氏提取,中性氧化铝柱层析分离纯化、高效液相色谱法测定连翘中连翘苷含量。结果改进了高效液相色谱法测定连翘中连翘苷的含量测定方法。结论该连翘苷含量测定方法准确、重现性好、回收率为97.09%、含量限度制定合理、可行,可较好地控制连翘质量。     

金银花、连翘配伍提取物体外抗甲型流感病毒FM1株的实验研究

目的:观察金银花、连翘配伍提取物(JLT)体外抑制甲型流感病毒FM1株的作用。方法:采用血凝试验方法,测定不同质量浓度JLT体外和鸡胚内抑制流感病毒效价,分析JLT对病毒增殖的影响。结果:与病毒对照组比较,JLT组血凝效价降低,其抑制作用时间可从1 h持续到24 h,其对病毒的抑制作用随着药物质量浓度的降低而逐渐减弱;JLT在400 mg/mL、200 mg/mL及100 mg/mL时,对感染流感病毒鸡胚具有预防和保护治疗作用(P<0.001),而在400 mg/mL时的预防作用明显优于阳性对照组(P<0.05)。结论:JLT可有效抑制甲型流感病毒FM1株。     

金翘感冒口服液的质量标准研究

目的研究金翘感冒口服液的质量标准。方法对金翘感冒口服液中主要药物金银花、连翘、甘草进行薄层色谱鉴别;采用RP-HPLC法测定绿原酸的含量。结果薄层色谱检出绿原酸、连翘药材、甘草次酸特征斑点,绿原酸进样量在0.224~1.120μg线性关系良好,平均加样回收率99.81%,RSD为0.89%。结论方法简便易行,灵敏,结果准确,重现性好,可有效控制金翘感冒口服液的质量。     

反相高效液相色谱法测定连翘中乌索酸和齐墩果酸的含量

目的建立同时测定连翘中乌索酸和齐墩果酸含量的测定方法。方法采用高效液相色谱(HPLC)法测定其含量。色谱柱为Nova-Pak C18(3.9 mm×300 mm,4μm),流动相为甲醇-水(88∶12),流速0.8 m l/m in,检测波长210 nm,柱温25℃。结果乌索酸和齐墩果酸分别在0.912~4.560μg(r=0.999 8)和0.312~1.560μg(r=0.999 6)范围内峰面积与进样量呈良好的线性关系;乌索酸和齐墩果酸的平均回收率分别为98.2%(RSD=1.1%)和97.8%(RSD=1.4%)。结论此法简单、方便、快速、准确,可为中药连翘质量评价和控制提供科学依据。     

Relation of body mass index, periodontitis and Tannerella forsythia

Objective: To determine if there were differences in periodontal status and the composition of the subgingival microbiota in individuals who exhibited different body mass indices (BMI).
Material and Methods: One hundred and twenty-one periodontally healthy/gingivitis and 574 chronic periodontitis subjects had height and weight determined and were measured for probing pocket depth, clinical attachment level, bleeding on probing, gingival redness and presence of visible plaque. Subgingival plaque samples taken from each tooth were individually analysed for their content of 40 bacterial species using checkerboard DNA–DNA hybridization.
Results: Crude odds ratios (ORs) [95% confidence interval (CI)] of overweight and obese individuals exhibiting periodontitis were 3.1 (1.9–4.8) and 5.3 (2.8–9.5), respectively, when compared with subjects with normal BMI. Logistic regression analysis indicated an OR (95% CI) of 2.3 (1.2–4.5) for an obese subject to exhibit periodontitis after adjusting for age, gender and smoking status. Individuals <46.8 years (median age) were responsible for this association. Only Tannerella forsythia differed significantly in proportions among BMI groups and was significantly higher in obese periodontally healthy/gingivitis individuals.
Conclusion: The data suggest that an overgrowth of T. forsythia occurs in the subgingival biofilms of periodontally healthy, overweight and obese individuals that might put them at risk for initiation and progression of periodontitis.     

Virulence of major periodontal pathogens and lack of humoral immune protection in a rat model of periodontal disease

Oral Diseases (2010) 16 , 686–695 Objective: This study was designed to test the hypothesis that periodontal pathogens Tannerella forsythia and Porphyromonas gingivalis are synergistic in terms of virulence potential using a model of mixed‐microbial infection in rats. Materials and methods: Three groups of rats were infected orally with either T. forsythia or P. gingivalis in mono‐bacterial infections or as mixed‐microbial infections for 12 weeks and a sham‐infected group were used as a control. This study examined bacterial infection, inflammation, immunity, and alveolar bone loss changes with disease progression. Results: Tannerella forsythia and P. gingivalis genomic DNA was detected in microbial samples from infected rats by PCR indicating their colonization in the rat oral cavity. Primary infection induced significantly high IgG, IgG2b, IgG1, and IgG2a antibody levels indicating activation of mixed Th1 and Th2 immune responses. Rats infected with the mixed‐microbial consortium exhibited significantly increased palatal horizontal and interproximal alveolar bone loss. Histological examinations indicated significant hyperplasia of the gingival epithelium with moderate inflammatory infiltration and apical migration of junctional epithelium. The results observed differ compared to uninfected controls. Conclusion: Our results indicated that T. forsythia and P. gingivalis exhibit virulence, but not virulence synergy, resulting in the immuno‐inflammatory responses and lack of humoral immune protection during periodontitis in rats.     

Immune response profiling of primary monocytes and oral keratinocytes to different Tannerella forsythia strains and their cell surface mutants

The oral pathogen Tannerella forsythia possesses a unique surface (S‐) layer with a complex O‐glycan containing a bacterial sialic acid mimic in the form of either pseudaminic acid or legionaminic acid at its terminal position. We hypothesize that different T. forsythia strains employ these stereoisomeric sugar acids for interacting with the immune system and resident host tissues in the periodontium. Here, we show how T. forsythia strains ATCC 43037 and UB4 displaying pseudaminic acid and legionaminic acid, respectively, and selected cell surface mutants of these strains modulate the immune response in monocytes and human oral keratinocytes (HOK) using a multiplex immunoassay. When challenged with T. forsythia, monocytes secrete proinflammatory cytokines, chemokines and vascular endothelial growth factor (VEGF) with the release of interleukin‐1β (IL‐1β) and IL‐7 being differentially regulated by the two T. forsythia wild‐type strains. Truncation of the bacteria's O‐glycan leads to significant reduction of IL‐1β and regulates macrophage inflammatory protein‐1. HOK infected with T. forsythia produce IL‐1Ra, chemokines and VEGF. Although the two wild‐type strains elicit preferential immune responses for IL‐8, both truncation of the O‐glycan and deletion of the S‐layer result in significantly increased release of IL‐8, granulocyte‐macrophage colony‐stimulating factor and monocyte chemoattractant protein‐1. Through immunofluorescence and confocal laser scanning microscopy of infected HOK we additionally show that T. forsythia is highly invasive and tends to localize to the perinuclear region. This indicates, that the T. forsythia S‐layer and attached sugars, particularly pseudaminic acid in ATCC 43037, contribute to dampening the response of epithelial tissues to initial infection and hence play a pivotal role in orchestrating the bacterium's virulence.