摘要
目的探讨二甲双胍对晚期糖基化终末产物(AGEs)诱导牙周膜成纤维细胞(PDLCs)凋亡及p38丝裂原活化蛋白激酶(MAPK)/核因子κB(NF-κB)通路的影响。方法体外培养人PDLCs细胞,分别加入0、100、200、300μg/mL的AGEs培养24、48、72 h,采用CCK-8法检测不同浓度、不同时间点的细胞增殖情况,筛选AGEs的最佳作用浓度和作用时间,分别为300μg/mL和48 h。收集细胞,设对照组、AGEs组及2、4、8 mmol/L二甲双胍组,对照组仅加入培养基,AGEs组加入终浓度为300μg/mL的AGEs,2、4、8 mmol/L二甲双胍组加入终浓度为300μg/mL的AGEs及终浓度分别为2、4、8 mmol/L的二甲双胍,培养48 h。采用CCK-8法检测各组细胞增殖情况,流式细胞术检测细胞凋亡情况,ELISA法检测细胞中超氧化物歧化酶(SOD)、丙二醛(MDA)以及细胞上清液中白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)水平,Western blotting法检测细胞p38 MAPK/NF-κB通路相关蛋白p-p38 MAPK、NF-κB的相对表达量。结果与对照组相比,AGEs组细胞OD值降低,细胞凋亡率升高(P均<0.05);与AGEs组相比,2、4、8 mmol/L二甲双胍组细胞OD值升高,细胞凋亡率降低(P均<0.05),且二甲双胍高浓度组细胞OD值高于、细胞凋亡率低于低浓度组(P均<0.05)。与对照组相比,AGEs组细胞中SOD水平降低,MDA水平升高(P均<0.05);与AGEs组相比,2、4、8 mmol/L二甲双胍组细胞中SOD水平升高,MDA水平降低(P均<0.05),且二甲双胍高浓度组SOD高于、MDA低于低浓度组(P均<0.05)。与对照组相比,AGEs组细胞上清液中IL-6、TNF-α水平升高(P均<0.05);与AGEs组相比,2、4、8 mmol/L二甲双胍组细胞上清液中IL-6、TNF-α水平降低(P均<0.05),且二甲双胍高浓度组IL-6、TNF-α水平低于低浓度组(P均<0.05)。与对照组相比,AGEs组细胞p-p38 MAPK、NF-κB p65蛋白表达升高(P均<0.05);与AGEs组相比,2、4、8 mmol/L二甲双胍组细胞p-p38 MAPK、NF-κB p65蛋白表达降低(P均<0.05),且二甲双胍高浓度组p-p38 MAPK、NF-κB p65蛋白表达低于低浓度组(P均<0.05)。结论二甲双胍对AGEs诱导的氧化应激反应、p38 MAPK/NF-κB通路激活、炎症反应均具有抑制作用,可减少细胞凋亡并促进细胞增殖。
Objective To investigate the effects of metformin on p38 mitogen-activated protein kinase(MAPK)/nu⁃clear factorκB(NF-κB)pathway and apoptosis of periodontal ligament fibroblasts(PDLCs)induced by advanced glyca⁃tion end products(AGEs).Methods Human PDLCs were cultured in vitro with different concentrations of AGEs(0,100,200,300μg/mL AGEs)for 24,48,and 72 h.CCK-8 was used to detect the proliferation of PDLCs with different concentrations of AGEs and at different culture time points(24,48,72 h)to screen the optimal action concentration and time of AGEs,and finally 300μg/mL and 48 h were the best choice.In addition,we set up the control group,AGEs group(300μg/mL AGEs),2 mmol/L metformin group(300μg/mL AGEs+2 mmol/L metformin),4 mmol/L metfor⁃min group(300μg/mL AGEs+4 mmol/L metformin),and 8 mmol/L metformin group(300μg/mL AGEs+8 mmol/L metformin),and the cells were cultured for 48 h.The proliferation of PDLCs was detected by CCK-8,the apoptosis of PDLCs was detected by flow cytometry,the levels of superoxide dismutase(SOD),malondialdehyde(MDA)in PDLCs,and the levels of interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)in the supernatant of PDLCs were detected by enzyme-linked immunosorbent assay(ELISA),and the expression levels of p38 MAPK/NF-κB pathway-related proteins in PDLCs was detected by Western blotting.Results Compared with those in the control group,the OD value of PDLCs in the AGEs group was significantly lower(P<0.05),the apoptosis rate was significantly higher(P<0.05);compared with those in the AGEs group,with the increase of metformin concentrations,the OD values of PDLCs cells increased in turn,the apoptosis rates decreased in turn in the metformin groups,and the OD value of the high-concentration metformin group was higher,and the apotosis rate was lower than those of the low-concentration metformin group(both P<0.05).Com⁃pared with those in the control group,the SOD level of PDLCs in the AGEs group was significantly lower,the MDA level was significantly higher(both P<0.05);compared with those in the AGEs group,with the increase of metformin concen⁃trations,the SOD level in the PDLCs increased in turn,and the MDA level decreased in turn in the 2,4,and 8 mmol/L metformin groups(all P<0.05),and the SOD level of the high-concentration metformin group was higher,and the MDA level was lower than those of the low-concentration metformin group(both P<0.05).Compared with those in the control group,the IL-6 and TNF-αlevels in the cell supernatant were significantly higher in the AGEs group(all P<0.05);com⁃pared with those in the AGEs group,with the increase of metformin concentrations,the IL-6 and TNF-αlevels in cell su⁃pernatant decreased in turn in the 2,4,and 8 mmol/L metformin groups(all P<0.05),and the IL-6 and TNF-αlevels of the high-concentration metformin group were lower than those of the low-concentration metformin group(both P<0.05).Compared with those in the control group,the p-p38 MAPK/p38 MAPK and NF-κB p65 protein expression levels were sig⁃nificantly higher in the AGEs group(both P<0.05);compared with those in the AGEs group,with the increase of metfor⁃min concentrations,the p-p38 MAPK/p38 MAPK and NF-κB p65 protein expression levels in the cell supernatant de⁃creased in turn in the 2,4,and 8 mmol/L metformin groups(all P<0.05),and the protein expression levels of p-p38 MAPK and NF-κB p65 of the high-concentration metformin group were lower than those of the low-concentration metformin group(both P<0.05).Conclusion Metformin can inhibit AGEs-induced oxidative stress,p38 MAPK/NF-κB pathway activation and inflammatory response,and can reduce the apoptosis and promote cell proliferation of PDLCs.
作者
黄辉
于大海
黄炫赓
罗智杰
HUNAG Hui;YU Dahai;HUNAG Xuangeng;LUO Zhijie(Guangxi Medical University College of Stomatology,Nanning 530001,China)
出处
《山东医药》
CAS
2021年第14期19-23,共5页
Shandong Medical Journal
基金
广西医科大学青年科学基金项目(GXMUYSF201345)。
作者简介
第一作者:黄辉(1982-),男,硕士,副主任医师,主要研究方向为二甲双胍对糖尿病性牙周炎的影响。E-mail:jianhuang2002@163.com。
