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


CONCLUSIONS
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.
Department of Periodontology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, 639 Zhi Zao Ju Road, Shanghai 200011, China.|Shanghai Research Institute of Stomatology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, 639 Zhi Zao Ju Road, Shanghai 200011, China.
Wei
W
Zhou
Department of Pharmacy, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, 639 Zhi Zao Ju Road, Shanghai 200011, China.
Xuan
X
Zhang
Shanghai Research Institute of Stomatology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, 639 Zhi Zao Ju Road, Shanghai 200011, China.
Cai-Lian
CL
Zhu
Shanghai Research Institute of Stomatology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, 639 Zhi Zao Ju Road, Shanghai 200011, China.
Zhi-Yan
ZY
He
Shanghai Research Institute of Stomatology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, 639 Zhi Zao Ju Road, Shanghai 200011, China.
Jing-Ping
JP
Liang
Department of Periodontology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, 639 Zhi Zao Ju Road, Shanghai 200011, China.
Zhong-Chen
ZC
Song
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D000893:Anti-Inflammatory Agents / Q000494:pharmacology*; D018441:Biofilms / Q000187:drug effects; D002478:Cells, Cultured; D005347:Fibroblasts / Q000187:drug effects / Q000276:immunology / Q000382:microbiology; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D007192:Indenes / Q000494:pharmacology*; D015850:Interleukin-6 / Q000276:immunology; D016209:Interleukin-8 / Q000276:immunology; D008550:Melatonin / Q000494:pharmacology*; D010510:Periodontal Diseases / Q000188:drug therapy* / Q000276:immunology; D016966:Porphyromonas gingivalis / Q000187:drug effects* / Q000276:immunology / Q000472:pathogenicity / Q000502:physiology; D044094:Receptors, Melatonin / Q000819:agonists*; D012756:Sheep
D000900:Anti-Bacterial Agents; D000893:Anti-Inflammatory Agents; D007192:Indenes; D015850:Interleukin-6; D016209:Interleukin-8; D044094:Receptors, Melatonin; C495910:ramelteon; D008550:Melatonin