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

作　　者：陈海鹰 汪龙凤 林清凡 王伟德 付东杰[3] CHEN Haiying;WANG Longfeng;LIN Qingfan;WANG Weide;FU Dongjie(School of Stomatology,Quanzhou Medical College,Quanzhou Fujian 362000,China;Science and Technology Center,Quanzhou Medical College,Quanzhou Fujian 362000,China;Renmin Hospital of Wuhan University,Wuhan Hubei 430000,China)

机构地区：[1]泉州医学高等专科学校口腔医学院,福建泉州362000 [2]泉州医学高等专科学校科技中心,福建泉州362000 [3]武汉大学人民医院,湖北武汉430000

出　　处：《中医药导报》2023年第2期28-34,共7页Guiding Journal of Traditional Chinese Medicine and Pharmacy

基　　金：泉州市科技计划项目(2018Z172);泉州医学高等专科学校校级重点科技项目(XJK2001A)。

摘　　要：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(PObjective: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 s

关 键 词：慢性牙周炎 金龙胆草 醇提取物 牙龈卟啉单胞菌 体外抑菌 牙龈卟啉单胞菌脂多糖 牙龈成纤维细胞 炎症 NF-кB 

分 类 号：R285.5[医药卫生—中药学]