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Objective·To observe the effects of gingipain extract on the biological characteristics of oral squamous cell carcinoma cell HN6.Methods·The HN6 cell line was selected,cultivated,and divided into different groups based on the protein concentration of gingipain extract from Porphyromonas gingivalis:control group,3.125 μg/mL group,6.25 μg/mL group,12.5 μg/mL group,25 μg/mL group,50 μg/mL group,and 100 μg/mL group.After 24 and 48 h of cultivation,CCK-8 assay was used to detect the effects of gingipain extract on HN6 cell proliferation activity.Subsequent experiments were divided into control group,25 μg/mL group and 50 μg/mL group.Flow cytometry was used to examine the effects of gingipain extract on cell cycle.Scratch assay and Transwell assay were performed to evaluate cell migration and invasion ability.Real-time PCR(RT-PCR)and Western blotting were used to measure the expression of E-cadherin and N-cadherin proteins and genes in cells.Results·Stimulated with gingipain extract for 24 h,the HN6 cells showed significantly increased proliferation activity in the 25 μg/mL(P=0.025),50 μg/mL(P=0.000),and 100 μg/mL(P=0.049)groups compared to the control group.After 48 h,proliferation activity was significantly higher in the 6.25 μg/mL(P=0.024),12.5 μg/mL(P=0.006),25 μg/mL(P=0.000),50 μg/mL(P=0.000),and 100 μg/mL(P=0.000)groups compared to the control group.Cell cycle analysis revealed that,after 24 h of gingipain stimulation,the proportion of HN6 cells in the G1 phase decreased,while the proportion in the S+G2 phase significantly increased compared to the control group(25 μg/mL group:P=0.024;50 μg/mL group:P=0.001).Compared to the control group,the scratch assay demonstrated a significant increase in the percentage of scratch closure as the concentration of gingipain extract increased(P=0.001).Compared to the control group,the Transwell invasion assay showed a significant increase in the number of cells passing through the bottom of the chamber as the concentration of gingipain extract increased.RT-PCR and Western blotting results indicated that as the concentration of gingipain extract increased,the expression levels of N-cadherin mRNA and protein in HN6 cells significantly increased,while the expression levels of E-cadherin mRNA and protein significantly decreased compared to the control group.Conclusion·Gingipain extract could promote proliferation,migration,and invasion of oral squamous cell carcinoma HN6 cells.
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Objective @#To explore the antibacterial activity of epigallocatechin-3-gallate (EGCG) on P. gingivalis and the inhibitory effects on matrix metalloproteinases (MMPs) production induced by P. gingivalis.@*Methods@# The antimicrobial effect of EGCG against planktonic cultures and biofilms of P. gingivalis was evaluated using microplate dilution assays. The microstructural changes in biofilms were studied using scanning electron microscopy (SEM). The inhibitory effect of EGCG on arginine gingipain (Rgp) and lysine gingipain (Kgp) activity of P. gingivalis was evaluated using synthetic chromogenic peptides and fluorogenic substrates. Enzyme-linked immunosorbent assay (ELISA) and qRT-PCR analysis were used to assess MMP-1 and MMP-2 mRNA expression and secretion by human gingival fibroblasts (HGFs) stimulated with P. gingivalis in the presence or absence of EGCG, respectively. @*Results @# The MIC and MBC of EGCG against P. gingivalis were 62.5 μg/mL and 500 μg/mL, respectively. EGCG can not only inhibit the biofilm formation of P. gingivalis but also has a scavenging effect on mature biofilms and can affect their viability. Additionally, 10 μg/mL and 50 μg/mL of EGCG inhibited the proteinase activities of Rgp and Kgp, respectively (P < 0.05). Finally, the mRNA expression and secretion of MMP-1 and MMP-2 by HGFs stimulated by P. gingivalis were significantly inhibited by 50 μg/mL of EGCG (P < 0.05). @*Conclusion@#EGCG exhibits antimicrobial effects against P. gingivalis and reduces the expression of MMPs by HGFs.
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Objective@#To investigate the distribution of fimA and kgp genotypes as well as the common genotype combination of Porphyromonas gingivalis (Pg) in infected root canals of primary apical periodontitis for virulent isolates screening in future.@*Methods@#Thirty-four samples harboring Pg were selected from infected root canals of primary apical periodontitis from patients of the Department of Endodontics, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine from June 2013 to September 2015. FimA type-specific primers were used to amplify the samples, revealing the distribution of various fimA genotypes. The genotypes of kgp were obtained by using Mse Ⅰ restriction endonuclease. The prevalence of each genotype and common genotype combinations were then calculated. Pearson's chi-squared test was performed to analyze the correlation between genotype combinations and clinical symptoms and major signs of apical periodontitis. In addition, the bioflim architectures between Pg isolates with different fimA and kgp genotype combinations were observed compared using confocal laser scanning microscope.@*Results@#Among the 34 Pg-positive samples, fimA Ⅱ was the most prevalent genotype [47% (16/34)] followed by fimA Ⅰ [26% (9/34)], while fimA Ⅴ was detected in only one sample. The prevalence of kgp Ⅰ [56% (19/34)] was slightly higher than that of kgp Ⅱ [44% (15/34)]. Both fimA Ⅱ+kgp Ⅰ and fimAⅡ+kgp Ⅱ were the most prevalent genotype combinations [24% (8/34) each]. No significant correlation was found between specific genotype combination and such major clinical manifestations as gingival swelling and sinus tract of dental origin (P>0.05). Three Pg isolates with different genotype combinations were acquired. Isolate A (fimAⅠ+kgpⅠ) formed densest biofilm, while the biofilm of isolate C (fimAⅤ+kgp Ⅰ) was much looser. The biofilm feature of isolate B (fimAⅢ+kgp Ⅱ) fell in between A and C.@*Conclusions@#Pg with fimA Ⅱ was most frequently detected in infected root canals of primary apical periodontitis. The prevalence of Pg with kgp Ⅰ was slightly higher than that with kgp Ⅱ, and fimAⅡ+kgp Ⅰ as well as fimA Ⅱ+kgp Ⅱ were the commonest genotype combinations. According to the comparison of Pg biofilms formed by clinical isolates, it might be possible that different genotype combinations may lead to distinct biofilm architectures.
ABSTRACT
Oral infection with Porphyromonas (P.) gingivalis causes periodontitis that is manifested by the destruction of gingival connective tissues. Although a few types of antibiotics are effective against the infection, its use induces the appearance of drug-resistant bacteria. The present study shows that the fermented product of Aspergillus (A.) oryzae S-03, cultivated on the fat-removed soybean, inhibits the cell growth of the P. gingivalis. Likewise, the fermented product of the S-03 strain cultured for 26~42 h displays an inhibitory activity to gingipain as a virulence factor of P. gingivalis. The activity is not lost even with heat treatment at 100degrees C for 15 min. We also demonstrate that the S-03 strain exhibits high protease activity. In addition, the strain does not produce aflatoxin because of the loss of a regulatory gene, aflR, necessary for the toxin biosynthesis.
Subject(s)
Aflatoxins , Anti-Bacterial Agents , Aspergillus , Aspergillus oryzae , Bacteria , Connective Tissue , Genes, Regulator , Hot Temperature , Oryza , Periodontitis , Porphyromonas , Porphyromonas gingivalis , Glycine max , VirulenceABSTRACT
The desired DNA product of KGPcd and KGP-hag was obtained from the total DNA of Porphyromonas gingivalis by PCR with two pairs of gene specific primers. The segment of KGPcd and KGP-hag (about 1.5kb and 1.6kb) was inserted into pGEM-T easy Vector. The double-stranded DNA of the postitive clone was analyzed by restriction endonuclease mapping and DNA sequenceing. The sequences of KGPcd and KGP-hag were consistent with those of the references appeared. The proteins of KGPcd and KGP-hag will be obtained for further study.