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

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

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(Por⁃phyromonas 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 gingi⁃val 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(argi⁃nine 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.gingi⁃valis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62.5μg/mL,最低杀菌浓度为500μg/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。10μg/mL及50μg/mL EGCG可有效抑制牙龈素的水解活性(P<0.05)。此外,50μg/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。

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 62.5μg/mL and 500μg/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,10μg/mL and 50μg/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 50μg/mL of EGCG(P<0.05).Conclusion EGCG exhibits antimicrobial effects against P.gingivalis and reduces the expression of MMPs by HGFs.