ABSTRACT Due to the complex microecology and microenvironment of dental plaque, novel caries prevention strategies require modulating the microbial communities ecologically and reducing the cariogenic properties effectively. Antimicrobial peptide GH12 reduced the lactic acid production and exopolysaccharide (EPS) synthesis of a Streptococcus mutans biofilm and a three-species biofilm in vitro in previous studies. However, the anticaries effects and microecological effects of GH12 remained to be investigated in a complex biofilm model in vitro and an animal caries model in vivo. In the present study, GH12 at 64 mg/liter showed the most effective inhibition of lactic acid production, EPS synthesis, pH decline, and biofilm integrity of human dental plaque-derived multispecies biofilms in vitro, and GH12 at 64 mg/liter was therefore chosen for use in subsequent in vitro and in vivo assays. When treated with 64-mg/liter GH12, the dental plaque-derived multispecies biofilms sampled from healthy volunteers maintained its microbial diversity and showed a microbial community structure similar to that of the control group. In the rat caries model with a caries-promoting diet, 64-mg/liter GH12 regulated the microbiota of dental plaque, in which the abundance of caries-associated bacteria was decreased and the abundance of commensal bacteria was increased. In addition, 64-mg/liter GH12 significantly reduced the caries scores of sulcal and smooth surface caries in all locations. In conclusion, GH12 inhibited the cariogenic properties of dental plaque without perturbing the dental plaque microbiota of healthy individuals and GH12 regulated the dysbiotic microbial ecology and arrested caries development under cariogenic conditions. IMPORTANCE The anticaries effects and microecological regulation effects of the antimicrobial peptide GH12 were evaluated systematically in vitro and in vivo. GH12 inhibited the cariogenic virulence of dental plaque without overintervening in the microbial ecology of healthy individuals in vitro. GH12 regulated the microbial ecology of dental plaque to a certain extent in vivo under cariogenic conditions, increased the proportion of commensal bacteria, and decreased the abundance of caries-associated bacteria. GH12 significantly suppressed the incidence and severity of dental caries in vivo. This study thus describes an alternative antimicrobial therapy for dental caries.

Dental caries is closely related to the acidification of the biofilms on the tooth surface, in which cariogenic bacteria bring about a dramatic pH decrease and disrupt remineralisation equilibrium upon the fermentation of dietary sugars. Thus, approaches targeting the acidified niches with enhanced anticaries activities at acidic pH are highly desirable. In our previous study, a cationic amphipathic α-helical antimicrobial peptide GH12 (Gly-Leu-Leu-Trp-His-Leu-Leu-His-His-Leu-Leu-His-NH₂) was designed with good stability, low cytotoxicity, and excellent antibacterial effects. Considering its potent antibacterial activity against the acidogenic bacteria and its histidine-rich sequence, it was speculated that GH12 might show enhanced antimicrobial effects at an acidic pH. In this study, the pH-responsive property of GH12 was determined to evaluate its potential as a smart acid-activated anticaries agent. GH12 possessed much lower minimal inhibitory concentrations and minimal bactericidal concentrations against various kinds of bacteria at pH 5.5 than at pH 7.2. Employing <i>Streptococcus mutans</i>, the principal caries pathogen, as the model system, it was found that GH12 showed much stronger bactericidal effects on both planktonic <i>S. mutans</i> and <i>S. mutans</i> embedded in the biofilm at pH 5.5. In addition, short-term treatment with GH12 showed much more effective inhibitory effects on water-insoluble exopolysaccharides synthesis and lactic acid production of the preformed <i>S. mutans</i> biofilm at pH 5.5. As for the mechanism exploration, it was found that the net positive charge of GH12 increased and the tryptophan fluorescence intensity heightened with the peak shifting towards the short wavelength at pH 5.5, which demonstrated that GH12 could be more easily attracted to the anionic microbial cell membranes and that GH12 showed stronger interactions with the lipid membranes. In conclusion, acidic pH enhanced the antibacterial and antibiofilm activities of GH12, and GH12 is a potential smart anticaries agent targeting the cariogenic acidic microenvironment.

Aim The objectives of this laboratory‐based study were to investigate the effects of GH12 on Enterococcus faecalis biofilm and virulence. Methodology Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of GH12 against E. faecalis were first determined. A time‐kill assay was further conducted. The effects of GH12 on the expression of virulence and stress genes in E. faecalis were evaluated by RT‐qPCR. Crystal violet stain was used to investigate the effects of GH12 on E. faecalis biofilm formation and 1‐day‐old biofilm. Finally, an ex vivo tooth model contaminated with E. faecalis was used to evaluate the antimicrobial activity of GH12 as an irrigant by CFU counting, SEM and CLSM. One‐way anova and Tukey’s multiple comparisons test were used to compare the differences amongst groups (α = 0.05). Results The MICs and MBCs of GH12 against E. faecalis were 8.0 ± 0.0 and 16.0 ± 0.0 mg L−1, respectively, and GH12 at 32.0 mg L−1 reduced the bacterial numbers by more than 99.9% within 1 min. Various virulence genes (efaA, esp and gelE) and stress genes (dnaK, groEL, ctsR and clpPBCEX) in E. faecalis were significantly downregulated by GH12 at sub‐MIC levels (P < 0.05). Additionally, both E. faecalis biofilm formation and the biomass of 1‐day‐old E. faecalis biofilm were significantly reduced by GH12 (P < 0.05). Elimination of E. faecalis in biofilms from root canal walls was achieved through irrigation with 64.0 mg L−1 GH12 for 30 min. CLSM analysis revealed that GH12 at 64.0 mg L−1 was most effective in eliminating bacteria within dentinal tubules (P < 0.05). Conclusion In a laboratory setting, and when used as an irrigant, GH12 suppressed E. faecalis, downregulated specific virulence and stress‐associated genes, eliminated intracanal E. faecalis protected by biofilms and killed bacteria in dentinal tubules. These results emphasize the need for preclinical and clinical studies to explore the potential of GH12 as an antimicrobial agent during root canal treatment.

Recent studies have demonstrated that the overexpression of H19 may contribute towards development of tumorigenesis in various types of cancer. To investigate the role of H19 in the development of non-small cell lung cancer (NSCLC), 76 NSCLC tissues samples and their adjacent normal tissue samples were collected. Expression level of H19, and its association with clinicopathological features and overall survival was analyzed. It was found that compared with normal adjacent tissues, H19 expression was elevated in NSCLC tissues along with a decreased miR-203 expression level. It was also found that patients who were in advanced clinical stages had a higher H19 and a lower miR-203 expression compared to normal tissues. The overall survival time of patients with higher H19 expression was shorter compared with the lower H19 expression group. Upregulation of A549 enhanced cell proliferation and promoted invasion. Overexpression of H19 stimulated the epithelial-mesenchymal transition (EMT) process in lung cancer cells and demonstrated typical morphological characteristics of EMT. The level of mesenchymal marker protein, such as Vimentin and SNAI1 increased; while CDH1 protein level decreased. Also, H19 negatively regulated miR-203. Inhibition of H19 attenuated miR-203 induced EMT process. Upregulation of H19 contributes to poor clinical features in patients with NSCLC, induces occurrence of EMT, promotes proliferation and stimulates cell invasion in NSCLC cell line through regulating miRNA-203 mediated EMT.

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Objectives:The aim of the study was to design and synthesise novel lactotransferrin-derived antimicrobial peptides (AMPs) with enhanced antibacterial activity against cariogenic bacteria. Methods: We obtained the LF-1 (WKLLRKAWKLLRKA) and LF-2 (GKLIWKLLRKAWKLLRKA) AMPs, based on the N-terminal functional sequence of lactotransferrin, and characterised their physicochemical properties and secondary structure. Their antibacterial activity against caries-associated bacteria was evaluated using bacterial susceptibility and time-killing assays, as well as transmission electron microscopy (TEM). The antibiofilm activity against Streptococcus mutans biofilms was determined using biofilm susceptibility assays and confocal laser scanning microscopy (CLSM). A rodent model of dental caries was adopted to evaluate their anticaries effectiveness in vivo. Results: Both peptides possessed an α-helical structure with excellent amphipathicity. LF-1 was effective against S. mutans and Actinomyces species, whereas LF-2 showed more potent antibacterial activity than LF-1 against a broader spectrum of tested strains. Both peptides inhibited the formation of S. mutans biofilm starting at 8 μmol/L and exerted effective eradication of S. mutans in preformed biofilms. Both peptides exhibited satisfactory biocompatibility and exerted significant anticaries effects in a rodent model. Conclusions: Both lactotransferrin-derived peptides displayed strong antimicrobial activity against cariogenic bacteria and S. mutans biofilm in vitro and effectively inhibited dental caries in vivo.

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Combining fluoride and antimicrobial agents enhances regulation of acid and exopolysaccharide production by biofilms. The combination also weakens the acidogenic and aciduric bacteria that contribute to caries, achieving stronger caries-controlling effects with lower concentrations of fluoride. In previous studies, antimicrobial peptide GH12 has been shown to inhibit lactic acid and exopolysaccharide synthesis in various cariogenic biofilm models, and reduce the proportion of acidogenic bacteria and Keyes caries scores in a rat caries model. The current study aimed to elucidate the effect of a combination of low concentrations of sodium fluoride (NaF) and GH12 and to determine the mechanism by which GH12/NaF combination controls caries. The GH12/NaF combination contained 8 mg/L GH12 and 250 ppm NaF. A rat caries model was built, and rat dental plaque was sampled and cultivated on bovine enamel slabs in vitro and subjected to short-term treatment (5 min, 3 times/day). The caries-controlling effects were evaluated using Keyes scoring and transverse microradiography. The results showed that the GH12/NaF combination significantly decreased the onset and development of dental caries, as well as mineral content loss and lesion depth in vitro (P< 0.05). For the caries-controlling mechanisms, 16S rRNA sequencing of in vivo dental plaque revealed that populations of commensal bacteria Rothia spp. and Streptococcus parasanguinis increased in the GH12/NaF group. In contrast, Veillonella, Lactobacillus, and Streptococcus mutans decreased. Furthermore, the GH12/NaF combination significantly reduced biomass, lactic acid, and exopolysaccharides production of in vitro biofilm (P< 0.05). Overall, fluoride and GH12 efficiently arrested caries development and demineralization by regulating the microbiota and suppressing acid and exopolysaccharide production in biofilms.