Ursodeoxycholic Acid Inhibits Inflammatory Cytokine Expression in THP-1 Cells Infected with Aggregatibacter actinomycetemcomitans

BACKGROUND: Periodontitis is an inflammatory disease characterized by the breakdown of tooth-supporting tissues, leading to tooth loss. Aggregatibacter actinomycetemcomitans are major etiologic bacterium causing aggressive periodontitis. Ursodeoxycholic acid (UDCA), a hydrophilic gall bladder acid, has been used as an effective drug for various diseases related to immunity. The aim of this study was to investigate the effect of UDCA on the inflammatory response induced by A. actinomycetemcomitans. METHODS: A human acute monocytic leukemia cell line (THP-1) was differentiated to macrophage- like cells by treatment with phorbol 12-mystristate 13-acetate (PMA) and used for all experiments. The cytotoxic effect of UDCA was examined by MTT assay. THP-1 cells were pretreated with UDCA for 30 min before A. actinomycetemcomitans infection and the culture supernatant was analyzed for various cytokine production by ELISA. The effect of UDCA on bacterial growth was examined by measuring optical densities using a spectrophotometer. RESULTS: UDCA showed no cytotoxic effect on THP-1 cells, up to 80 µM Ed highlight: Please confirm technical meaning. UDCA pretreatment inhibited the A. actinomycetemcomitans-induced IL-1β, TNF-α, and IL-17A secretion in a dosedependent manner. UDCA also inhibited IL-21 production at 60 µM. The production of IL-12 and IL-4 was not influenced by A. actinomycetemcomitans infection. CONCLUSION: These findings indicate that UDCA inhibits the production of inflammatory cytokines involved in innate and Th17 immune responses in A. actinomycetemcomitans-infected THP-1-derived macrophages, which suggests its possible use for the control of aggressive periodontitis.

Identification of Non-Aggregatibacter actinomycetemcomitans Bacteria Grown on the Tryptic soy-Serum-Bacitracin-Vancomycin Medium

The aim of this study was to identify the non-Aggregatibacter actinomycetemcomitans bacteria grown on the tryptic soy-serum-bacitracin-vancomycin (TSBV) medium, an A. actinomycetemcomitans selective medium. A total of 82 unidentified bacterial isolates from the oral cavities of a Korean population were kindly provide by the Korean Collection for Oral Microbiology. All the clinical isolates were grown on TSBV medium and bacterial DNA purified from each isolate was subjected to PCR with universal primers specific for bacterial 16S rRNA genes (16S rDNAs) sequence. The each bacterial 16S rDNA was amplified by PCR and the nucleotide sequences of it was determined by the dideoxynucleotide chain termination method. They were identified by 16S rDNA sequence comparison method at the specie-level. The data showed that Neisseria spp. (42 strains), Fusobacterium spp. (10 strains), Capnocytophaga spp. (8 strains), Propionibacterium acnes (5 strains), Aggregatibacter aprophilus (4 strains), Campylobacter spp. (5 strains), Veillonella dispar (3 strains), Streptococcus sp. (1 strain), Haemophilus parainfluenzae (1 strain), Leptotrichia wadei (1 strain), Morococcus sp./Neisseria sp. (1 strain), and Staphylococcus sp. (1 strain) were identified. These results could be used to develop a new A. actinomycetemcomitans-selective medium which is more effective than the TSBV medium in future studies.

Antimicrobial Activity of Berberine against Oral Bacteria Related to Endodontic Infections

It has been established that berberine has strong antimicrobial effects. Little is known however regarding the antimicrobial activity of berberine against endodontic pathogenic bacteria or its cytotoxicity in human oral tissue cells. The antibacterial properties of berberine were tested against 5 strains of Enterococcus faecalis and type strains of Aggregatibacter actinomycetemcomitans, Prevotella nigrescens, Prevotella intermedia, and Tannerella forsythia, which are involved in endodontic infections. Antimicrobial activity was evaluated through minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) measurements. The viability of normal human gingival fibroblast (NHGF) cells after exposure to berberine was measured using a methyl thiazolyl tetrazolium (MTT) assay. The data showed that berberine has antimicrobial effects against A. actinomycetemcomitans with an MIC and MBC of 12.5 microg/ml and 25 microg/ml, respectively. In the cytotoxicity studies, cell viability was maintained at 66.1% following exposure to 31.3 microg/ml berberine. Overall, these findings suggest that berberine has antimicrobial activity against the tested bacteria. Nevertheless, lower concentrations in combination with other reagents will need to be tested before these in vitro results can be translated to clinical use.

Antimicrobial Activity of Oleanolic Acid, Ursolic Acid, and Sophoraflavanone G against Periodontopathogens

In general, oleanolic acid (OA) and ursolic acid (UA) have antimicrobial effect against Gram-positive bacteria but not Gram-negative bacteria whereas sophoraflavanone G has antimicrobial activity against both bacterial types. However, the antimicrobial effects of OA, UA, and sophoraflavanone G against periodontopathogens have not been studied to any great extent. The aim of this study was to investigate antimicrobial effect of OA, UA, and sophoraflavanone G against 15 strains (5 species) of oral Gram-negative bacteria, which are the major causative bacteria of periodontal disease. The antimicrobial activity was evaluated by minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) determinations. OA and UA showed antimicrobial effects against all of the Porphyromonas gingivalis strains tested and also Prevotella intermedia ATCC 25611T. Interestingly, P. intermedia ATCC 49046 showed greater resistance to OA and UA than P. intermedia ATCC 25611T. In contrast, sophoraflavanone G had antimicrobial activity against all strains, with MIC and MBC values below 32 microg/ml, except Aggregatibacter actinomycetemcomitans. These results indicate that sophoraflavanone G may have potential for use in future oral hygiene products such as dentifrices and gargling solution to prevent periodontitis.

Induction of osteoclastogenesis-inducing cytokines and invasion by alive Aggregatibacter actinomycetemcomitans in osteoblasts / 대한치주과학회지

Osteoblasts regulate osteoclastogenesis by production of various cytokines. Aggregatibacter(A) actinomycetemcomitans is one of periodontopathogens which invades gingival tissue. Therefore, clarifying the effect of alive A. actinomycetemcomitans on osteoblasts is important to understand the mechanism of alveolar bone resorption in periodontitis. We investigated induction of osteoclastogenesis- inducing cytokines, adherence, and invasion by A. actinomycetemcomitans in osteoblasts. Osteoblasts were isolated from mouse calvaria and expression of cytokines was determined by RT-PCR. When the ratio of the number of A. actinomycetemcomtians to the number of osteoblasts was 10:1, 50:1 and 100:1, RANKL mRNA expression was increased. A. actinomycetemcomitans also increased expression of macrophage inflammatory protein (MIP)-1alpha, interleukin (IL)-1beta, and tumor necrosis factor (TNF)-alpha. A. actinomycetemcomitans attached to and invaded osteoblasts at ratio of 1000:1. These results suggest that A. actinomycetemcomitans increases osteoclastogenesis-inducing ability of osteoblasts by stimulating the expression of RANKL, MIP-1alpha, IL-1beta, and TNF-alpha and that invasion of A. actinomycetemcomitans provides a means by which the bacteria escape from immune system and antibiotic therapy.