Journal of Prevention and Treatment for Stomatological Diseases ›› 2021, Vol. 29 ›› Issue (5): 314-321.doi: 10.12016/j.issn.2096-1456.2021.05.004

• Basic Study • Previous Articles     Next Articles

Inhibitory effects of epigallocatechin-3-gallate on the pathogenic properties of P. gingivalis in vitro

QI Xia1(),KONG Lingxue2,LI Shujuan1,MA Siting1,QI Yali1,ZHAO Lei3()   

  1. 1. Department of Periodontics, School and Hospital of Stomatology, Hebei Medical University, Hebei Key Laboratory of Stomatology,Hebei Clinical Research Center for Oral Diseases, Shijiazhuang 050017, China
    2. Department of Periodontics and Oral Mucosa, Jinan Stomatologic Hospital, Jinan 250001, China
    3. State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases, Department of Periodontics, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China
  • Received:2020-11-13 Revised:2020-12-13 Online:2021-05-20 Published:2021-03-08
  • Contact: Lei ZHAO E-mail:summyly@163.com;jollyzldoc@163.com
  • Supported by:
    Medical Science Research Project in Hebei Province(20190096);Natural Science Foundation of Hebei Province(H2019206541);Senile Disease Prevention project of Hebei Provincial Department of Finance(2017068877-2)

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Abstract:

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.

Key words: epigallocatechin-3-gallate, Porphyromonas gingivalis, biofilm formation, biofilm reduction, arginine gingipain, lysine gingipain, antibacterial, anti-inflammatory, gingival fibroblasts, matrix metalloproteinase

CLC Number: 

  • R78

Table 1

The primers sequence of the genes used in qRT-PCR"

Target gene Primers sequence
MMP-1 F: 5′-GAAACCCTGAAGGTGATGAAGC-3′
R: 5′-TTGGCAAATCTGGCGTGTAAT-3′
MMP-2 F: 5′-TTCCGCTTCCAGGGCACAT-3′
R: 5′-GCACCTTCTGAGTTCCCACCAA-3′
β-actin F: 5′-CCACGAAACTACCTTCAACTCC-3′
R: 5′-GTGATCTCCTTCTGCATCCTGT-3′

Figure 1

Culture of P. gingivalis ATCC33277（×1 000）"

Figure 2

Inhibitory effect of different concentrations of EGCG on P. gingivalis biofilm a: the inhibitory effect of EGCG on P. gingivalis biofilm formation; 1 represents MBIC50 and 2 represents MBIC90; b: a marked reduction in the P. gingivalis biofilm biomass after exposure to EGCG ranging from 62.5 to 1 000 μg/mL; 3 represents MBRC50; c: the inhibitory effect of EGCG of pre-established P. gingivalis biofilm on the viability of P. gingivalis; 4 represents SMIC50; *: P < 0.05 vs. the control group; MBIC: minimum biofilm inhibition concentration; MBRC: minimum biofilm reduction concentration; SMIC: sessile minimal inhibitory concentration; EGCG: epi- gallocatechin-3-gallate"

Figure 3

Effect of the EGCG concentration on the mature biofilms of P. gingivalis using SEM（×1 000） a: untreated control group of P. gingivalis biofilm; b: a marked 50% reduction in P. gingivalis biofilm after 24 h of exposure to 250 μg/mL of EGCG compared with the control; c: disruption in the architecture of P. gingivalis biofilm after 24 h of exposure to 500 μg/mL of EGCG; d: no biofilm in 24 h pre-established P. gingivalis biofilms after 24 h of exposure to 1 000 μg/mL of EGCG; EGCG: epigallocate- chin-3-gallate"

Table 2

Effects of EGCG against P. gingivalis planktonic culture and biofilms"

Drug Planktonic P. gingivalis P.gingivalis biofilms
MIC MBC Formation Reduction Viability
MBIC50 MBIC90 MBRC50 MBRC90 SMIC50 SMIC90
EGCG（μg/mL） 62.5 500 7.8 31.25 250 > 1 000 125 > 1 000

Figure 4

Effect of EGCG on P. gingivalis Rgp and Kgp activities a-b: the inhibitory effect of EGCG on the proteolytic activity of P. gingivalis Rgp ; c-d: the inhibitory effect of EGCG on the proteolytic activity of P. gingivalis Kgp; *: P < 0.05 vs. the control group; EGCG: epigallocatechin-3-gallate"

Figure 5

Effect of P. gingivalis and EGCG on human gingival fibroblast viability using the MTT assay a: effects of P. gingivalis at different MOI values on HGFs viability; b: effects of EGCG on cell viability of HGFs; *: P < 0.05 vs. the control group; EGCG: epigallocatechin-3-gallate; HGFs: human gingival fibroblasts"

Figure 6

Effect of EGCG on the secretion of MMP-1 and MMP-2 by HGFs stimulated by P. gingivalis a-b: MMP-1 (a) and MMP-2 (b) in cell-free culture supernatants induced by P. gingivalis in the presence of EGCG for 24 h were quantified by ELISA; c-d: effects of EGCG on the expression of MMP-1 (c) and MMP-2 (d) mRNAs by HGFs for 24 h were measured by qRT-PCR analysis; *: P < 0.05 vs. the negative control [P. gingivalis (-), EGCG (-)]; #: P < 0.05 vs. the positive control [P. gingivalis(+), EGCG (-)]; EGCG: epigallocatechin-3-gallate; HGFs: human gingival fibroblasts"

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