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

doi:10.12016/j.issn.2096-1456.2021.05.004

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

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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. 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
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)

表没食子儿茶素没食子酸酯对牙龈卟啉单胞菌细菌毒力抑制作用的体外研究

齐霞¹(),孔令雪²,李淑娟¹,马思婷¹,齐雅丽¹,赵蕾³()

1.河北医科大学口腔医学院·口腔医院牙周一科,河北省口腔医学重点实验室,河北省口腔疾病临床医学研究中心,河北石家庄（050017）
2.济南口腔医院牙周黏膜科,山东济南（250001）
3.口腔疾病研究国家重点实验室国家口腔疾病临床医学研究中心四川大学华西口腔医院牙周科,四川成都（610041）

通讯作者: 赵蕾
作者简介:齐霞,主治医师,硕士,Email： summyly@163.com
基金资助:
河北省医学科学研究课题计划(20190096);河北省自然科学基金项目(H2019206541);河北省财政厅老年病防治项目(2017068877-2)

Abstract

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

摘要：

目的研究表没食子儿茶素没食子酸酯（epigallocatechin-3-gallate,EGCG）对牙龈卟啉单胞菌（Porphyromonas gingivalis,P. gingivalis）体外抑菌活性及EGCG对P. gingivalis诱导人牙龈成纤维细胞（human gingival fibroblasts,HGFs）表达基质金属蛋白酶（matrix metalloproteinases,MMPs）的抑制作用。方法采用微量稀释法分析EGCG对P. gingivalis浮游菌和生物膜的抑制作用。扫描电镜检测EGCG作用下P. gingivalis生物膜微观结构的变化。特异性人工合成显色多肽底物评价EGCG对牙龈素精氨酸特异性半胱氨酸蛋白酶（arginine gingipain, Rgp）和赖氨酸特异性半胱氨酸蛋白酶（lysine gingipain, Kgp）蛋白水解活性的影响。P. gingivalis刺激HGFs,与EGCG共培养,ELISA检测MMP-1和MMP-2的蛋白分泌水平,qRT-PCR检测MMP-1及MMP-2 mRNA的表达。结果 EGCG对P. gingivalis浮游菌的最低抑菌浓度为62.5 μg/mL,最低杀菌浓度为500 μg/mL。EGCG可抑制P. gingivalis生物膜形成,并对成熟生物膜具有清除作用,且抑制成熟生物膜的代谢活性。10 μg/mL及50 μg/mL EGCG可有效抑制牙龈素的水解活性（P < 0.05）。此外,50 μg/mL EGCG对P. gingivalis刺激HGFs表达及分泌MMP-1、MMP-2均有抑制作用（P < 0.05）。结论 EGCG对P. gingivalis有抑菌作用,且可抑制HGFs表达MMPs。

关键词: 表没食子儿茶素没食子酸酯, 牙龈卟啉单胞菌, 生物膜形成, 生物膜清除, 精氨酸特异性半胱氨酸蛋白酶, 赖氨酸特异性半胱氨酸蛋白酶, 抗菌, 抗炎, 牙龈成纤维细胞, 基质金属蛋白酶

CLC Number:

QI Xia,KONG Lingxue,LI Shujuan,MA Siting,QI Yali,ZHAO Lei. Inhibitory effects of epigallocatechin-3-gallate on the pathogenic properties of P. gingivalis in vitro[J]. Journal of Prevention and Treatment for Stomatological Diseases, 2021, 29(5): 314-321.

齐霞,孔令雪,李淑娟,马思婷,齐雅丽,赵蕾. 表没食子儿茶素没食子酸酯对牙龈卟啉单胞菌细菌毒力抑制作用的体外研究[J]. 口腔疾病防治, 2021, 29(5): 314-321.

Figures/Tables 8

Table 1 The primers sequence of the genes used in qRT-PCR

Target gene	Primers sequence
MMP-1	F: 5′-GAAACCCTGAAGGTGATGAAGC-3′
MMP-1	R: 5′-TTGGCAAATCTGGCGTGTAAT-3′
MMP-2	F: 5′-TTCCGCTTCCAGGGCACAT-3′
MMP-2	R: 5′-GCACCTTCTGAGTTCCCACCAA-3′
β-actin	F: 5′-CCACGAAACTACCTTCAACTCC-3′
β-actin	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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Inhibitory effects of epigallocatechin-3-gallate on the pathogenic properties of P. gingivalis in vitro

表没食子儿茶素没食子酸酯对牙龈卟啉单胞菌细菌毒力抑制作用的体外研究

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