金龙胆草醇提取物对慢性牙周炎的体外抑菌抗炎作用研究

Study on the Antibacterial and Anti-inflammatory Effects of Conyza Blinii Lévl.Ethanol Extract on Chronic Periodontitis In Vitro

目的:探讨金龙胆草醇提取物对慢性牙周炎常见致病菌牙龈卟啉单胞菌(Pg)、具核梭杆菌(Fn)的体外抑菌效果,观察金龙胆草醇提取物对牙龈卟啉单胞菌脂多糖(Pg-LPS)诱导人牙龈成纤维细胞(HGF)炎症反应的影响。方法:以乙醇为提取剂,采用索式提取法、热水浴浸提法和超声辅助提取法对金龙胆草进行提取,冷冻干燥后计算提取率;使用滤纸片扩散法观察提取物对Pg、Fn的抑菌效果;采用细胞计数试剂-8(CCK-8)检测金龙胆草醇提取物和Pg-LPS、HGF共同培养24 h、48 h对细胞增殖的影响;通过酶联免疫吸附试验检测细胞上清液中肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、前列腺素E_(2)(PGE_(2))分泌水平;应用蛋白免疫印迹法(Western blotting)检测HGF炎症模型中核因子-кB(NF-кB)、基质金属蛋白酶-1(MMP-1)蛋白表达水平,分析金龙胆草醇提取物对NF-кB信号通路的抑制情况。结果:3种提取方式的金龙胆草提取率比较,超声辅助提取法显著高于索式提取法和热水浴浸提法(P<0.05)。金龙胆草醇提取物对Pg、Fn的最小抑菌浓度分别为20 mg/mL和40 mg/mL,索式提取法的抑菌效果优于超声辅助提取法和热水浴浸提法。干预24、48 h后,10、20、50μg/mL药液组HGF活性与对照组比较,差异无统计学意义(P>0.05);100、200、500、1000μg/mL药液组HGF活性显著低于对照组(P<0.05)。干预12、24 h后,Pg-LPS组HGF培养液上清液中TNF-α、IL-1β、PGE_(2)水平均高于对照组(P<0.05);干预12、24 h后,Pg-LPS+20μg/mL药液组、Pg-LPS+50μg/mL药液组TNF-α水平均低于Pg-LPS组(P<0.05);干预12 h后,Pg-LPS+10μg/mL药液组、Pg-LPS+20μg/mL药液组、Pg-LPS+50μg/mL药液组IL-1β水平均明显低于Pg-LPS组(P<0.05);干预12、24 h后,Pg-LPS+10μg/mL药液组、Pg-LPS+20μg/mL药液组、Pg-LPS+50μg/mL药液组PGE_(2)水平均低于Pg-LPS组(P<0.05)。干预12、24 h后,Pg-LPS组HGF中NF-κB、MMP-1蛋白相对表达量均高于对照组(P<0.05);干预12、24 h后,Pg-LPS+10μg/mL药液组、Pg-LPS+20μg/mL药液组、Pg-LPS+50μg/mL药液组NF-κB、MMP-1蛋白相对表达量均低于Pg-LPS组(P<0.05)。结论:金龙胆草醇提取物对Pg、Fn有一定的抑菌作用,索式提取法可提高抑菌效果;金龙胆草醇提取物可抑制Pg-LPS诱导HGF的TNF-α、IL-1β、PGE_(2)分泌,阻断NF-кB信号通路的激活,这可能是其发挥抗炎作用的机制之一。

Objective:To explore the in vitro antibacterial effect of Conyza blinii Lévl.ethanol extract on porphyromonas gingivalis(Pg)and fusobacterium nucleatum(Fn),and to observe the effects of Conyza blinii Lévl.ethanol extract on the inflammatory response of human gingival fibroblasts(HGF)induced by Porphyromonas gingival lipopolysaccharide(Pg-LPS).Methods:Conyza blinii Lévl.was extracted by soxhlet extraction,hot water bath extraction and ultrasonic-assisted extraction with ethanol as extraction agent.The extraction rate was calculated after freeze drying.The antibacterial effect of extract on Pg and Fn was observed by filter paper diffusion method.Cell counting kit-8(CCK-8)was used to detect the effects of Conyza blinii Lévl.ethanol extract and Pg-LPS co-cultured with HGF for 24h and 48h on cell proliferation.The secretion levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and prostaglandin E_(2)(PGE_(2))in cell culture supernatant were detected by enzyme-linked immunosorbent assay.Western blotting was used to detect the expression of nuclear factor-кb(NF-кB)and matrix metalloproteinase-1(MMP-1)in HGF inflammatory model,and the inhibition of NF-кB signaling pathway by Conyza blinii Lévl.ethanol extract was analyzed.Results:Compared with three extraction methods,ultrasonic-assisted extraction method was significantly higher than soxhle extraction method and hot water bath extraction method(P<0.05).The minimum inhibitory concentrations of Pg and Fn were 20 mg/mL and 40 mg/mL,respectively.The antibacterial effect of soxhlet extraction method was better than that of ultrasonic assisted extraction method and hot water bath extraction method.After 24 and 48 h of intervention,there was no significant difference in HGF activity between the 10,20 and 50μg/mL groups and the control group(P>0.05),but the HGF activity in 100,200,500 and 1000μg/mL groups were significantly lower than that in the control group(P<0.05).After 12 and 24 h of intervention,the levels of TNF-α,IL-1βand PGE_(2) in HGF culture supernatant of Pg-LPS group were higher than those of control group(P<0.05).After 12 and 24 h of intervention,the TNF-αlevel in Pg-LPS+20μg/mL and Pg-LPS+50μg/mL groups were lower than that in Pg-LPS group(P<0.05).After 12 h of intervention,the IL-1βlevel in Pg-LPS+10μg/mL,Pg-LPS+20μg/mL and Pg-LPS+50μg/mL groups were significantly lower than that in Pg-LPS group(P<0.05).After 12 and 24 h of intervention,PGE_(2) levels in Pg-LPS+10μg/mL,Pg-LPS+20μg/mL and Pg-LPS+50μg/mL groups were lower than those in Pg-LPS group(P<0.05).After 12 and 24 h of intervention,the relative expression levels of NF-κB and MMP-1 in HGF of Pg-LPS group were higher than those of control group(P<0.05).After 12 and 24 h of intervention,the relative expressions of NF-κB and MMP-1 in Pg-LPS+10μg/mL,Pg-LPS+20μg/mL and Pg-LPS+50μg/mL groups were lower than those in Pg-LPS group(P<0.05).Conclusion:The Conyza blinii Lévl.ethanol extract has certain antibacterial effect on Pg and Fn,and the soxhlet extraction can improve the antibacterial effect.The Conyza blinii Lévl.ethanol extract could inhibit the secretion of TNF-α,IL-1βand PGE_(2) in HGF induced by Pg-LPS,and block the activation of NF-кB signaling pathway,which may be one of the mechanisms of its anti-inflammatory effect.