香芹酚对成骨细胞及口腔常见致病菌的影响

Effects of carvacrol on osteoblasts and common oral pathogens

背景:天然植物提取物香芹酚具有广谱抗菌性、低耐药性和低细胞毒性的特点,是近年来抗生素替代物的研究热点,具有很好的前景。目的:测定香芹酚对口腔常见致病菌变异链球菌和金黄色葡萄球菌的最小抑菌浓度、最小杀菌浓度,探究香芹酚对这2种菌的抑菌活性及其对成骨细胞增殖的影响,以期在口腔疾病防治方面提供新的选择。方法:通过酶标仪检测吸光度值分别测定香芹酚对变异链球菌和金黄色葡萄球菌的最小抑菌浓度(MIC)、最小杀菌浓度,采用抑菌圈实验检测香芹酚的抑菌活性;CCK-8试剂盒检测1,4,7 d下不同浓度香芹酚诱导下的MC3T3-E1细胞增殖情况;碱性磷酸酶、实时荧光定量PCR试剂盒分别检测1,7,14 d细胞碱性磷酸酶活性以及Runx2、骨保护素基因表达水平。结果与结论:①香芹酚对变异链球菌和金黄色葡萄球菌的最小抑菌浓度值分别为0.20%,0.04%,最小杀菌浓度值分别为0.60%,0.12%;②抑菌圈实验中香芹酚实验组均显示出明显的抑菌圈,差异有显著性意义(P<0.05);③CCK-8实验显示,香芹酚实验组细胞增殖活性明显增强(P<0.05);④香芹酚各浓度组均使碱性磷酸酶活性增高;7 d时碱性磷酸酶活性增高更明显,1MIC组效果最突出(P<0.05);⑤实时荧光定量PCR检测结果显示,香芹酚实验组不同基因皆显示出mRNA高表达现象,且均出现在1MIC组(P<0.05);提示香芹酚对2种口腔常见致病菌变形链球菌和金黄色葡萄菌均具有较强的抑菌活性,并且可以提高MC3T3-E1细胞成骨活性,促进其增殖分化。

BACKGROUND:The natural plant extract carvacrol has the chara cteristics of broad-spectrum antibacterial activity,low drug resistance and low cytotoxicity,which has become a research hotspot of antibiotic substitutes with a good prospect in recent years.OBJECTIVE:To determine the minimum inhibitory concentration(MIC) and minimum bactericidal co ncentration(MBC) of carvac rol against Streptococcus mutons and Staphylococcus aureus,two common oral pathoge ns and to explore the antibacterial activity of carvacrol against these two bacte ria and its effect on osteoblast proliferation,thereby providing a new choice for oral disease prevention and treatment.METHODS:A microplate reader was used to determine the MIC and MBC values of carvacrol against Streptococcus mutons and Staphylococcus oureus.The antibacte rial activity of carvacrol was tested by a zone of inhibition test.Cell counting kit-8 was used to detect the prolife ration of MC3 T3-E1 cells induced by different concentrations of carvacrol at 1,4,and 7 days.Alkaline phosphatase activity and Runx2 and osteoprotegerin gene expression levels were detected by alkaline phosphatase and real-time fluorescence quantitative PCR kits at 1, 7,and 14 days,respectively.RESULTS AND CONCLUSION:The MIC values of carvacrol against Streptococcus mutons and Staphylococcus aureus were 0.20% and 0.04%,and the MBC values were 0.60% and 0.12%,respectively.In the inhibition zone test,carvacrol showed an obvious antibacterial zone,and the difference was statistically significant(P <0.05).Cell counting kit-8 assay showed that the proliferation activity of cells treated with carvacrol was significantly increased(P <0.05).The activity of alkaline phosphatase increased in all carvacrol concentration groups.The increase of alkaline phosphatase activity was more obvious on day 7 and the effect of 1 MIC group was the most obvious(P<0.05),Results from the real-time fluorescence quantitative PCR showed that different genes were highly expressed at the mRNA level after treated with carvacrol and all of them occurred in the 1 MIC group(P <0.05).To conclude,carvacrol has strong antibacterial activity against two common oral pathogens,Streptococcus mutons and Staphylococcus aureus,can improve the osteogenic activity of MC3 T3-E1 cells,and promote their proliferation and differentia tion.