【作者】 康文燕； 王婷； 胡哲凯； 刘峰； 孙允东； 葛少华；

【Author】 Wenyan Kang;Ting Wang;Zhekai Hu;Feng Liu;Yundong Sun;Shaohua Ge;Shandong Provincial Key Laboratory of Oral Tissue Regeneration,School of Stomatology,Shandong University;Department of Periodontology,School of Stomatology,Shandong University;Department of Oral and maxillofacial surgery,School of Stomatology,Shandong University;Department of Microbiology,Key Laboratory for Experimental Teratology of Chinese Ministry of Education,School of Medicine,Shandong University;

【机构】 山东大学口腔医学院山东省组织再生重点实验室； 山东大学口腔医学院牙周科； 山东大学口腔医学院口腔颌面外科； 山东大学基础医学院病原微生物学重点实验室；

【摘要】 背景与目的:慢性牙周炎是口腔流行性疾病,牙龈卟啉单胞菌是其主要致病菌。其发生与发展与II型糖尿病具有密切关系。二甲基双胍是常用降糖药,已证实该药可以在多种细胞内发挥抗炎作用。本文旨在研究二甲基双胍对牙龈成纤维细胞炎性反应的影响,并对其作用机制进行进一步探讨。材料及方法:通过LPS(5ug/ml)刺激牙龈成纤维细胞,构建体外牙周炎症模型。对照组细胞采用完全培养基培养,实验组细胞用不同浓度二甲基双胍(0-4mM)预处理。利用细胞增值-毒性检测试剂盒检测二甲基双胍及LPS药物对细胞毒性影响。实时荧光定量PCR及酶联免疫吸附(ELISA)实验检测不同处理组细胞炎症因子IL-6,IL-1β,TNF-α表达。转录激活因子3(ATF3)小干扰转染细胞进一步研究二甲基双胍对牙龈成纤维细胞炎症反应的作用机制。通过荧光显微镜、PCR及Westernblot检测干扰效率。PCR及ELISA进一步检测ATF3敲低后不同处理组细胞炎症因子IL-6,IL-1β,TNF-α表达变化.结果:CCK8实验表明5ug/ml LPS及低浓度二甲基双胍(0、0.1、0.5mM)对牙龈成纤维细胞无毒性。二甲基双胍以剂量依赖性方式抑制LPS诱导的牙龈成纤维细胞IL-6,IL-1β,TNF-α的产生。二甲基双胍与LPS可以协同促进ATF3的表达,ATF3敲低减弱了二甲基双胍对LPS诱导的炎症反应的抑制作用。结论:二甲基双胍通过ATF3表达来抑制LPS诱导的牙龈成纤维细胞炎症反应。低剂量的二甲基双胍有望成为控制牙周炎发生发展的临床药物。更多还原

【Abstract】 Background:Chronic periodontitis is one of the most prevalent oral diseases,which is associated with Porphyromonas gingivalis(P.gingivalis) Lipopolysaccharide(LPS) infection,and has profound effects on type 2 diabetes mellitus(DM2).Metformin,a well-known antidiabetic agent,has been reported to exert anti-inflammatory effects on various cells.This study aimed to investigate the role of metformin on LPS-induced inflammatory response in gingival fibroblasts(GFs).Methods:Dose-dependent additive effects of metformin on LPS-induced GFs were detected.Cell-counting kit-8(CCK-8) was used to determine the effects of metformin and LPS on the viability of GFs.Enzyme linked immunosorbent assay(ELISA) and quantitative Real-time polymerase chain reaction(qRT-PCR) were applied to detect the level of interleukin(IL)-1β,IL-6 and tumor necrosis factor(TNF)-α in different treated cells.ATF3 siRNA transfection was used to determine the mechanism of metformin action and the transfection efficiency was observed by fluorescence microscope.The effects of ATF3 knockdown were determined by qRT-PCR and Western blot.Results:The results showed that 5 μg/ml P.gingivalis LPS and 0.1,0.5,1 mM metformin exhibited no toxicity to GFs and metformin inhibited LPS-induced IL-1β,IL-6 and TNF-α production in a dose-dependent manner.Metformin and LPS could synergistically facilitate ATF3 expression and ATF3 knockdown abolished the inhibitory effects of metformin on LPS-induced inflammatory cytokines production in GFs.Conclusions:Our study confirmed that metformin inhibited LPS-induced inflammatory response in GFs via ATF3 induction and low dose of metformin could act as a therapeutic agent in the controlling of periodontal disease.更多还原