Acinetobacter baumannii quorum-sensing signalling molecule induces the expression of drug-resistance genes.

Quorum-sensing signalling molecules such as N­acyl homoserine lactones (AHLs) enable certain Gram­negative bacteria to respond to environmental changes through behaviours, such as biofilm formation and flagellar movement. The present study aimed to identify Acinetobacter baumannii AHLs and assess their influence on antibiotic resistance. A clinical isolate of A. baumannii strain S (AbS) was collected from the wound of a burn patient and high­performance liquid chromatography and tandem quadrupole or quadrupole time­of­flight high­resolution mass spectrometry was used to identify AbS AHLs. Antibiotic sensitivity was assessed in an AHL­deficient AbS mutant (AbS­M), and the expression of drug-resistance genes in the presence of meropenem in AbS, AbS­M and AbS­M treated with the AHL N-3-hydroxy-dodecanoyl-homoserine lactone (N­3­OH­C12­HSL). AbS­M was more sensitive to meropenem and piperacillin than wild­type AbS, but resistance was restored by supplementation with N­3­OH­C12­HSL. In addition, meropenem­treated AbS­M expressed lower levels of the drug­resistance genes oxacillinase 51, AmpC, AdeA and AdeB; treatment with N­3­OH­C12­HSL also restored the expression of these genes. Overall, the results of the present study indicate that N­3­OH­C12­HSL may be involved in regulating the expression of drug­resistance genes in A. baumannii. Therefore, this quorum­sensing signalling molecule may be an important target for treating multidrug­resistant A. baumannii infections.

Melatonin Receptor Agonists as the "Perioceutics" Agents for Periodontal Disease through Modulation of Porphyromonas gingivalis Virulence and Inflammatory Response.

AIM: "Perioceutics" including antimicrobial therapy and host modulatory therapy has emerged as a vital adjunctive treatment of periodontal disease. Melatonin level was significantly reduced in patients with periodontal diseases suggesting melatonin could be applied as a potential "perioceutics" treatment of periodontal diseases. This study aims to investigate the effects of melatonin receptor agonists (melatonin and ramelteon) on Porphyromonas gingivalis virulence and Porphyromonas gingivalis-derived lipopolysaccharide (Pg-LPS)-induced inflammation. METHODS: Effects of melatonin receptor agonists on Porphyromonas gingivalis planktonic cultures were determined by microplate dilution assays. Formation, reduction, and viability of Porphyromonas gingivalis biofilms were detected by crystal violet staining and MTT assays, respectively. Meanwhile, biofilms formation was also observed by confocal laser scanning microscopy (CLSM). The effects on gingipains and hemolytic activities of Porphyromonas gingivalis were evaluated using chromogenic peptides and sheep erythrocytes. The mRNA expression of virulence and iron/heme utilization was assessed using RT-PCR. In addition, cell viability of melatonin receptor agonists on human gingival fibroblasts (HGFs) was evaluated by MTT assays. After pretreatment of melatonin receptor agonists, HGFs were stimulated with Pg-LPS and then release of cytokines (IL-6 and lL-8) was measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Melatonin and ramelteon did exhibit antimicrobial effects against planktonic culture. Importantly, they inhibited biofilm formation, reduced the established biofilms, and decreased biofilm viability of Porphyromonas gingivalis. Furthermore, they at sub-minimum inhibitory concentration (sub-MIC) concentrations markedly inhibited the proteinase activities of gingipains and hemolysis in a dose-dependent manner. They at sub-MIC concentrations significantly inhibited the mRNA expression of virulence factors (kgp, rgpA, rgpB, hagA, and ragA), while increasing the mRNA expression of ferritin (ftn) or hemolysin (hem). They did not show obvious cytotoxicity toward HGFs. They inhibited Pg-LPS-induced IL-6 and IL-8 secretion, which was reversed by luzindole, the melatonin receptor antagonist. CONCLUSION: Melatonin receptor agonists can inhibit planktonic and biofilm growth of Porphyromonas gingivalis by affecting the virulent properties, as well as Pg-LPS-induced inflammatory response. Our study provides new evidence that melatonin receptor agonists might be useful as novel "perioceutics" agents to prevent and treat Porphyromonas gingivalis-associated periodontal diseases.

Dentin tubule invasion by Enterococcus faecalis under stress conditions ex vivo.

Enterococcus faecalis is the species most frequently isolated from failed endodontic treatments because it can survive under stress conditions imposed by root canal treatment. The objective of this study was to determine the ability of E. faecalis to invade dentine tubules under alkaline and energy-starvation stress and to explore the potential mechanisms. Roots from single-rooted human teeth were infected with E. faecalis under alkaline and energy-starvation stress conditions. After 4 wk of culture, samples were processed to establish the tubule-penetration distance. In addition, the hydrophobicity of E. faecalis cells under these conditions was analysed and the expression of genes involved in adhesion was quantified by real-time quantitative PCR. Culture of E. faecalis under alkaline and energy-starvation stress conditions resulted in a marked reduction of tubule-penetration distance, a significant increase in hydrophobicity of the bacterial surface, and marked down-regulation of most adhesin genes compared with E. faecalis cultured in tryptic soy broth. The results indicate that the dentine tubule invasion ability of E. faecalis was markedly decreased under alkaline and glucose-starvation stress conditions, possibly because of the increase of hydrophobicity and down-regulation of some adhesion genes.

[Exploring the stem cell surface markers expressed in human dental pulp stem cells].

PURPOSE: To explore the stem cell surface markers expressed in human dental pulp stem cells which were selected and isolated by magnetic beads. METHODS: Human dental pulp cells (hDPCs) were separated and cultured from dental pulp of healthy third molars for orthodontic purpose. HDPSCs were isolated from cultured hDPCs by magnetic-activated cell sorting's (MACS) indirect magnetic cell labeling and positive selection strategy with antibody STRO-1 in the 2nd generation. Then the stem cell surface markers (CD73, CD90, CD105, CD166 and STRO-1) were respectively detected in 3, 4, 5, 6, 7 and 8 generation of dental pulp stem cells. HDPSCs were induced to differentiation by adipogenic medium and osteogenic medium in the 3rd generation. Adipogenic differentiation was assessed by oil red O staining in day 21, and osteogenic differentiation was assessed by alizarin red staining in day 21. RESULTS: HDPSCs could differentiate into adipocyte and osteoblasts. Oil red O staining and alizarin red staining were positively expressed after induction of HDPSCs. STRO-1's expression was decreased with the increase of generation. The expressions of CD73, CD90, CD105 and CD166 were relatively stable. CONCLUSIONS: The expression of STRO-1 is declined with the increase of generation, and the expressions of CD73, CD90, CD105 and CD166 are relatively stable with the changes of generation. Supported by National Natural Science Foundation of China (81070826/81371143) and Shanghai Rising-Star Program (12QH1401400).

[The efficacy of cold-light tooth bleaching on the formation of bacteria biofilm on the enamel surfaces].

PURPOSE: To evaluate the efficacy of Beyond cold-light tooth bleaching on the formation of main cariogenic bacteria biofilm on enamel surfaces. METHODS: Twenty enamel discs with the size of 4 mm×4 mm×1 mm in size, were made. The enamel discs were divided into 4 groups randomly: cold-light bleaching group, bleaching gel group, cold-light group and control group. Five discs were in each group. Cold-light bleaching group was whitened 3 times with bleaching gel and cold-light, and 12 min per session. Bleaching gel was smeared on the surface of enamel in bleaching gel group for 3 times and 12 min per session. Enamel discs of cold-light group were treated with cold-light for 12 min and 3 sessions. Control group was treated without any processing. The 4 groups were incubated in mixed bacteria liquid, including Streptococcus mutans(SM), Actinomyces viscosus (Av) and Fusobacterium nucleatum (Fn), within the artificial oral cavity model. After 36 h, the samples were observed under confocal laser scanning microscopy(CLSM). The data was analyzed with SAS8.2 software package. RESULTS: The biofilms in 3 experimental groups were sparser than the control group under CLSM, and the thickness significantly decreased after treatment (P<0.05), while no significant difference was found among 3 experimental groups (P>0.05).Compared with the control group, the percentage of vital bacteria in biofilm of the experimental groups decreased significantly after treatment (P<0.001). CONCLUSIONS: Cold-light tooth bleaching can inhibit the formation of mixed bacteria biofilm, damage the structure of biofilm and reduce the number of vital bacteria. Supported by Research Fund of Ninth People's Hospital of Shanghai Jiao Tong University School of Medicine (2013-06).

Exploring the variation of oral microbiota in supragingival plaque during and after head-and-neck radiotherapy using pyrosequencing.

OBJECTIVE: The aim of this article was to study the variation in oral microflora of the subgingival plaque during and after radiotherapy. DESIGN: During and after radiotherapy, microbial samples were collected at seven time points (early stage, medium stage, and later stage of radiotherapy, and 1 month, 3 months, 6 months, and 1 year after radiotherapy) in three subjects for a total of 21 samples. Polymerase chain reaction (PCR) amplification was carried out on the 16S rDNA hypervariable V1-V3 region, and then the PCR products were determined by high-throughput pyrosequencing. RESULTS: The rarefaction curve indicating the richness of the microflora demonstrated that the number of operational taxonomic units (OTUs) was in decline from the early stage of radiotherapy to the time point 1 month after radiotherapy and then trended upward. The Shannon diversity index declined during radiotherapy (ranging from 4.59 to 3.73), and generally rose after radiotherapy, with the lowest value of 3.5 (1 month after radiotherapy) and highest value of 4.75 (6 months after radiotherapy). A total of 120 genera were found; five genera (Actinomyces, Veillonella, Prevotella, Streptococcus, Campylobacter) were found in all subjects across all time points. CONCLUSION: The richness and diversity of oral ecology decreased with increased radiation dose, and it was gradually restored with time.

[Immortalization of human dental pulp stem cells caused by transferring hTERT gene].

PURPOSE: To establish an immortalized human dental pulp stem cell line used for basic and clinical research of oral science. METHODS: Human telomerase reverse transcriptase (hTERT) cDNA was transferred into human dental pulp stem cells (hDPSCs) by lentivirus. The resultant stable clones reproduced successively and the expression of hTERT was identified. RESULTS: The hTERT gene was transferred into human dental pulp stem cells successfully. The transformed cells expressed telomerase activity and divided vigorously. p35 had been obtained so far. CONCLUSIONS: The hDPSCs can be immortalized by transferring exogenous hTERT gene to constitute telomerase activity.

The impact of various time intervals on the supragingival plaque dynamic core microbiome.

OBJECTIVE: The aim of this study was to examine the influence of various time intervals on the composition of the supragingival plaque microbiome, especially the dynamic core microbiome, and to find a suitable observation interval for further studies on oral microbiota. METHODS AND MATERIALS: Eight qualified volunteers whose respective age ranges from 25 to 28 years participated in the present study. The supragingival plaque was collected from the buccogingival surface of the maxillary first molar at eight time slots with different intervals (day 0, 1 day, 3 days, 1 week, 2 weeks, 3 weeks, 1 month, and 3 months). Bioinformatic analyses was performed based on 16S rDNA pyrosequencing (454 sequencing platform) targeting at the hypervariable V4-V5 region, in order to assess the diversity and variation of the supragingival plaque microbiome. RESULTS: A total of 359,565 qualified reads for 64 samples were generated for subsequent analyses, which represents 8,452 operational taxonomic units identified at 3% dissimilarity. The dynamic core microbiome detected in the current study included five phyla, 12 genera and 13 species. At the genus level, the relative abundance of bacterial communities under the "1 day," "1 month," and "3 months" intervals was clustered into sub-category. At the species level, the number of overlapping species remained stable between the "1 month" and "3 months" intervals, whereas the number of dynamic core species became stable within only 1 week. CONCLUSIONS: This study emphasized the impact of different time intervals (days, weeks and months) on the composition, commonality and diversity of the supragingival microbiome. The analyses found that for various types of studies, the time interval of a month is more suitable for observing the general composition of the supragingival microbiome, and that a week is better for observing the dynamic core microbiome.

Killing dental pathogens using antibacterial graphene oxide.

Dental caries and periodontal diseases have a close relationship with microbes such as Streptococcus mutans, Porphyromonas gingivalis and Fusobacterium nucleatum. Graphene oxide (GO), as the derivative of graphene, plays an important role in many areas including biology and medicine. In particular, it has been known as a promising antimicrobial nanomaterial. In this study, we focused on the antimicrobial property of GO against dental pathogens. With the utilization of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduced test, colony forming units (CFU) counting, growth curve observation, live/dead fluorescent staining, and confocal laser scanning microscopy (CLSM), we found GO nanosheets were highly effective in inhibiting the growth of dental pathogens. Transmission electron microscopy (TEM) images revealed that the cell wall and membrane of bacteria lost their integrity and the intracellular contents leaked out after they were treated by GO. Therefore, GO nanosheets would be an effective antibacterial material against dental pathogens and the potential applications in dental care and therapy are promising.

[Methyl-metabolism contributes to the LuxS regulation of Streptococcus mutans].

OBJECTIVE: To investigate the predominant contribution of methyl-metabolism pathway to the regulation of LuxS of Strecptococcus mutans. METHODS: The differences in biofilm formation and aciduricity of Strecptococcus mutans among the methyl-metabolism-complementation strain (KO-S), the parental wide-type strain (WT) and the luxS null strain (KO) were observed by real-time PCR for monitoring the transcriptional level of genes related to biofilm formation (smu.238, gtfD) and aciduricity (smu.44, smu.46) of the studied strains, methyl thiazolyl tetrazolium (MTT) for quantifying the biofilm of the exhibited strains and confocal laser scanning microscopy for estimating the structure of the biofilm. RESULTS: The transcriptional level of smu.44, smu.46, smu.238, gtfD in WT were 1.289 ± 0.051, 1.694 ± 0.140, 1.565 ± 0.107, 1.667 ± 0.196 respectively; in KO were 1.001 ± 0.045, 1.007 ± 0.151, 1.000 ± 0.021, 1.012 ± 0.196 respectively, downregulated compared with WT (P < 0.05); in KO-S were 4.662 ± 0.091, 5.019 ± 0.258, 3.462±0.029, 3.071 ± 0.136 respectively, upregulated compared both with KO and with WT (P < 0.05). The quantity of biofilms formed by the studied strains were WT (1.592 ± 0.213), KO (0.939 ± 0.029), KO- S (2.177 ± 0.226), KO- P (1.020 ± 0.093), respectively, representing a less quantity by KO and KO-P than WT (P < 0.05) and a more quantity by KO-S than other three stains (P < 0.05). According to the observation of biofilms texture by confocal laser scanning microscopy, the WT biofilm was condensed and even. In contrast, fissures and gaps were found scattered in biofilms of KO, KO-P while lessened in that of KO-S, in which high-density bacterial aggregates were observed. The acid assay indicated a smaller biofilm decrease by WT and KO-S than that by KO and KO- P(P < 0.05). CONCLUSIONS: The methyl- metabolism pathway contributes to LuxS regulation on biofilm formation and auiduricity of Strecptococcus mutans.

[Complementation and function of methyl-metabolic pathway in Streptococcus mutans luxS null strain].

PURPOSE: To complement the activated methyl cycle (AMC) pathway at an AI-2 defect background in Streptococcus mutans (S. mutans) luxS null strain. METHODS: A sahH gene was amplified from Pseudomonas aeruginosa and introduced into the S. mutans luxS null strain to complement the methyl-metabolic disruption at an AI-2 defect background. Western blot, reverse-transcription PCR and AI-2 bioassay were performed to confirm the heterogenous expression of SahH in S. mutans luxS null strain. The data was statistically analyzed by SAS8.0 software package. RESULTS: LuxS and SahH were detected to express in Escherichia coli BL21 as well as their mRNA were confirmed to be successfully transcribed in S. mutans luxS null strain. AI-2 production was found in wide type S. mutans and its luxS-introduced luxS null strain but not found in the luxS null strain and its sahH and empty plasmid-introduced strains. CONCLUSIONS: A new S. mutans derivative with the AMC pathway complements while the AI-2 defect is constructed.

Development and evaluation of new primers for PCR-based identification of Prevotella intermedia.

The aim of this study was to develop new Prevotella intermedia-specific PCR primers based on the 16S rRNA. The new primer set, Pi-192 and Pi-468, increased the accuracy of PCR-based P. intermedia identification and could be useful in the detection of P. intermedia as well as epidemiological studies on periodontal disease.

[Effect of different stress conditions on growth and biofilm formation capability of Enterococcus faecalis].

OBJECTIVE: To study the changes of growth and biofilm formation capability of Enterococcus faecalis (Ef) in different stress conditions. METHODS: The changes of growth of Ef in stress conditions were observed by measuring the A600 value with ultraviolet spectrophotometer. Ef was incubated on glass slide in stress conditions, biofilm formation capability of cells was investigated by colony-forming unit (CFU) counting of the culturable bacteria and fluorescence confocal laser scanning microscopy. RESULTS: Ef couldn't growth under the conditions of 2%, 5%NaClO, pH = 11 and 12, the A600 value was unchanged in 96 hours. But the growth curve changed at different levels in other stress conditions: under 1%NaClO, the A600 value peaked at 1.461 at 16 hour (the peaked level was 1.238 at 6 hours in control group) ; under 0,0.05%,0.15% glucose, it peaked at 0.645,0.890, 1.173, respectively, at 6 hour (it was maximized to 1.195 at 6 hours in control group); the A600 value peaked at 1.704 at 6 hours at pH = 9 and 1.225 at 10 hours at pH = 10 (the peak level was 1.732 at 6 hours at pH = 7) . Biofilm assay showed that Ef were able to form biofilm in these stress conditions except 5%NaClO and pH = 12. CONCLUSIONS: Ef could growth and form biofilms in energy starvation, low concentrations of sodium hypochlorite and weak alkaline stress.

Bacterial flora and extraradicular biofilm associated with the apical segment of teeth with post-treatment apical periodontitis.

INTRODUCTION: Microorganisms are able to survive and cause persistent infection in the extraradicular area. The aims of this study were to investigate the primary bacterial flora and the localization of extraradicular biofilm in persistent apical periodontitis lesions. METHODS: Apical root samples from root-end surgery were collected from 23 root-filled teeth with apical periodontitis. Five samples were examined for the presence of biofilm by scanning electron microscopy. Another 5 samples were examined for the presence of biofilm by Brown and Brenn-modified Gram staining. The DNA from 13 samples was processed for amplification via polymerase chain reaction and separated with denaturing gradient gel electrophoresis. Selected bands were excised from the gel and sequenced for identification. RESULTS: The extraradicular biofilm present on the external root surface of treated teeth consisted of abundant, amorphous extracellular material and multiple bacterial species. The following species were detected in the microbial community from the apical samples: Actinomyces sp. oral, Propionibacterium, Prevotella sp. oral, Streptococcus, Porphyromonas endodontalis, and Burkholderia. The prevalence of Actinomyces sp. oral and Propionibacterium were highest (84.6% and 61.5%, respectively). CONCLUSIONS: Extraradicular biofilm was present on the external root surface of treated teeth with persistent periapical lesions. Actinomyces sp. oral and Propionibacterium are likely important contributors to extraradicular biofilm formation and persistent periapical infection.

[Effect of compound Chinese traditional medicine on periapical periodontitis in dogs and cytotoxic assay].

PURPOSE: To assess the efficacy of compound Chinese traditional medicine(CTM), which is composed of gallic acid, magnolol and polysaccharide of Bletilla, against apical periodontitis in dogs and cytotoxic assay. METHODS: A animal model of apical periodontitis was built, CTM was then used to disinfect the root canal. The effect of the restoration of periapical bone in dogs was investigated after regular root canal filling. SAS6.12 software package was used for statistical analysis, and MTT was used to test cell toxicity of CTM. RESULTS: CTM can cure inflammation effectively, and CTM had no cytotoxic effect on periodontal ligament cells at 5-week. CONCLUSIONS: The compound Chinese traditional medicine may be an effective disinfecting drug for root canal disinfection.

[Observation of genetic diversity in dental plaque of elder people with root caries].

PURPOSE: Bacterial community in dental plaque of elder people was analyzed to learn about the microhabitat composition and diversity. METHODS: Dental plaque samples were collected from 25 elders. PCR-based denaturing gradient gel electrophoresis (PCR-DGGE) was used to evaluate the microbial diversity by displaying PCR-generated 16SrDNA fragments that migrate at different distances, reflecting the different sequence of fragment. SPSS12.0 software was used to analyze the variance of genotypes between different groups of bacteria. RESULTS: Genotypes of bacteria in dental plaques in the root caries group was significantly more than the other two groups. Crown caries group and caries-free group had no significant difference. CONCLUSIONS: The genetic diversity of the dental plaque microflora in the root caries group is significantly higher than coronal caries group and caries-free group.

[Influence of Streptococcus mutans luxS gene on polysaccharide matrix metabolism].

PURPOSE: To investigate the effect of Streptococcus mutans luxS gene on polysaccharide matrix metabolism. METHODS: Based on the immobilization of magnetic beads by adherent cells,an assay of biofilm quantitative analysis was developed for the kinetic quantification of biofilm formation. S.mutans luxS gene mutant strain and wild-type strain were compared for their ability of utilizing exogenous carbohydrate to form extracellular polysaccharide matrix. SPSS 10.0 software package was used for statistical analysis. Dunnet t two-side test of one factor analysis of variance was performed. RESULTS: Both luxS mutant strain and wild-type strain could use exogenous carbohydrate to form polysaccharide matrix.With 1% sucrose added ,both strains completed their biofilm formation within one hour.When adding 1% glucose,these strains also accelerated the formation of biofilm,and this was more significant in the mutant strain. CONCLUSIONS: The luxS gene of S. mutans can regulate its extracellular polysaccharide matrix metabolism. Moreover, the regulation of this gene on biofilm formation is more probably via polysaccharide matrix pathway. Supported by National Natural Science Foundation of China(30872886), Research Fund of Science and Technology Commission of Shanghai Municipality(08DZ2271100),Shanghai Leading Academic Discipline Project(S30206) and Youth Phosphor Program of Science and Technology Commission of Shanghai Municipality (09QA1403700).

[Analysis of community composition in dental plaque of elder people with root caries].

OBJECTIVE: To analyze the community in dental plaque of elder people with root caries. METHODS: Total DNAs were extracted from the root caries dental plaques of nine elders over 60 years of age. Polymerase chaid reaction-based denaturing gradient gel electrophoresis (PCR-DGGE) was used to analyze the microbial composition, DGGE bands were excised from the gels for sequencing and identification. RESULTS: The dominant genus in root caries dental plaque of elder people were: Acinetobacte [0.9% (1/114)], Actinobaculum [1.8% (2/114)], Actinomyces [15.8% (18/114)], Aggregatibacter [0.9% (1/114)], Capnocytophaga [14.0% (16/114)], Corynebacterium [0.9% (1/114)], Haemophilus [0.9% (1/114)], Mobiluncus [0.9% (1/114)], Naxibacter [0.9% (1/114)], Neisseriaceae [10.5% (12/114)], Porphyromonas [0.9% (1/114)], Prevotella [12.3% (14/114)], Selenomonas [6.1% (7/114)], Staphylococcus [1.8% (2/114)], Oralis streptococcus [6.1% (7/114)], Mutans streptococcu [7.9% (9/114)], Tannerella [0.9% (1/114)], Treponema [1.8% (2/114)], Veillonella [10.5% (12/114)] and two uncultured unknown genus [1.8% (2/114)]. Uncultred genotypes accounted for 19.30% of the total. Gram-positive bacteria genotype accounted for 31.6% (36/114), and Gram-negative bacteria genotype accounted for 66.7% (76/114). CONCLUSIONS: There were many bacteria genotypes in root caries dental plaque in the elderly, which were widely distributed. Gram-negative bacteria accounted for the majority. Genotype-specific pathogenic bacteria were not found.

[Effect of compound Chinese traditional medicine on infected root canal bacteria biofilm].

PURPOSE: To assess the efficacy of compound Chinese traditional medicine(CTM), which composed of gallic acid, magnolol and polysaccharide of Blettila striata, against the infected root canal bacterial biofilm. METHODS: Actinomyces viscosus (Av), Enterococcus faecalis (Ef), Fusobacterium nucleatum (Fn) were composed to form biofilm, then confocal laser scan microscope (CLSM) was used to observe and study the bacterial activity. SAS6.12 software package was used for statistical analysis. RESULTS: The biofilm thickness reduced after treatment by both CTM and ZnO (P>0.05),while there was a significant decrease of the percentage of vital bacterias after treatment by CTM (P<0.01). CONCLUSIONS: The compound Chinese traditional medicine is effective on biofilm control, so that it would be an effective disinfecting drug for root canal sealers. Supported by Research Fund of Bureau of Traditional Chinese Medicine of Shanghai Municipality (Grant No.2008L008A).