摘要
目的:探究圣草酚调节Yes相关蛋白(YAP)/转录共激活因子(TAZ)信号通路对脂多糖(LPS)诱导的牙周膜干细胞(hPDLSC)成骨分化的影响.方法:采用结扎法进行大鼠牙周炎造模,造模成功后随机分为模型组和圣草酚组,每组6只,另取6只正常大鼠作为对照组.体外培养hPDLSC,以10µg/mL的LPS诱导的同时采用0、40、80、160、320、480μmoL/L的圣草酚处理,采用试剂盒检测各处理组细胞碱性磷酸酶(ALP)活性后筛选圣草酚最佳作用浓度.将hPDLSC随机分为对照组、LPS组、LPS+圣草酚组、LPS+空载组、LPS+圣草酚+YAP敲低组,诱导其成骨分化并以LPS、圣草酚和质粒分组处理.采用HE染色观察牙周组织病理形态学变化;采用实时荧光定量PCR与免疫印迹实验检测各组牙周组织及细胞YAP、TAZ表达;采用ALP活性检测试剂盒、ALP染色及茜素红染色检测各组细胞成骨分化;采用酶联免疫吸附法(ELISA)检测各组血清及细胞炎症因子前列腺素E2(PGE2)、白细胞介素(IL)-6、IL-18水平;采用实时荧光定量PCR与免疫印迹实验检测各组牙周组织细胞成骨分化因子Runx2、OSX、骨桥蛋白(OPN)表达.结果:与对照组比较,模型组大鼠牙周组织呈现明显的病理损伤,血清PGE2、IL-6及与IL-18水平增高,牙周组织YAP、TAZ mRNA与蛋白表达降低(P＜0.05);与模型组相比,圣草酚组大鼠牙周组织病理损伤减轻,血清PGE2、IL-6及与IL-18水平下降,牙周组织YAP、TAZ mRNA与蛋白表达增高(P＜0.05).40、80、160、320、480μmoL/L的圣草酚均可升高LPS诱导的hP-DLSC中ALP活性(P＜0.05),其升高作用随着圣草酚浓度升高而增强并在320μmoL/L时达到平 台期,故选择320μmoL/L的圣草酚进行后续实验.与对照组相比,LPS组细胞 YAP、TAZ mRNA与蛋白表达、ALP阳性相对比例、ALP活性、矿化结节相对比例、Runx2及OSX、OPN mRNA与蛋白表达降低(P＜0.05),PGE2、IL-6及与IL-18水平升高(P＜0.05).与LPS组相比,LPS+圣草酚组细胞 YAP、TAZ mRNA与蛋白表达、ALP阳性相对比例、ALP活性、矿化结节相对比例、Runx2及OSX、OPN mRNA与蛋白表达升高(P＜0.05),PGE2、IL-6及与IL-18水平降低(P＜0.05);LPS+空载组细胞各指标无明显变化(P＞0.05).与LPS+圣草酚组相比,LPS+圣草酚+YAP敲低组细胞YAP、TAZ mRNA与蛋白表达、ALP阳性相对比例、ALP活性、矿化结节相对比例、Runx2及OSX、OPN mRNA与蛋白表达降低(P＜0.05),PGE2、IL-6及与IL-18水平升高(P＜0.05).结论:圣草酚可减轻牙周炎大鼠的炎症损伤,并通过上调YAP/TAZ通路蛋白表达抑制LPS诱导的hPDLSC炎症,从而促使其成骨分化.
Abstract
Objective:To investigate the effect of eriodictyol on the osteogenic differentiation of human periodontal ligament stem cells(hPDLSC)induced by lipopolysaccharide(LPS)by regulating the Yes-asso-ciated protein(YAP)/transcriptional co-activator(TAZ)signaling pathway.Methods:The Periodontitis model of rats was established by the ligation method.After successful modeling,the rats were randomly divid-ed into the model group and the eriodictyol group,with 6 rats in each group,and 6 normal rats as the control group.HPDLSC was cultured in vitro,induced with 10 μg/mL LPS,and treated with 0,40,80,160,320,and 480 μmol/L of eriodictyol,the alkaline phosphatase(ALP)activity of cells in each treatment group was detected with a kit to screen the optimal concentration of eriodictyol.hPDLSC were randomly divided into con-trol group,LPS group,LPS+eriodictyol group,LPS+empty group,LPS+eriodictyol+YAP knockdown group,after inducing osteogenic differentiation and treated with LPS,eriodictyol,and plasmids.The pathological changes of periodontal tissue were observed by HE staining.Real-time fluorescence quantitative PCR and im-munoblotting experiments were applied to detect the expression of YAP and TAZ in periodontal tissues and cells.ALP activity detection kit,ALP staining,and alizarin red staining were applied to detect osteogenic dif-ferentiation of cells.Enzyme linked immunosorbent assay(ELISA)was applied to detect the levels of inflam-matory cytokines prostaglandin E2(PGE2),interleukin(IL)-6 and IL-18 in periodontal tissues and cells.And real-time fluorescent quantitative PCR and Western blot were applied to detect the expression of osteo-genic differentiation factors Runx2,OSX,and osteopontin(OPN)of cells.Results:Compared with the con-trol group,the periodontal tissue of the model group showed obvious pathological damage,the serum levels of PGE2,IL-6 and IL-18 were increased,and the mRNA and protein expressions of YAP and TAZ were de-creased(P＜0.05).Compared with the model group,the pathological damage of periodontal tissue in the eri-odictyol group was alleviated,the levels of serum PGE2,IL-6,and IL-18 were decreased,and the mRNA and protein expressions of YAP and TAZ in periodontal tissue were increased(P＜0.05).The ALP activity in hPDLSC induced by LPS was increased after treatment with 40,80,160,320,and 480 μmol/L eriodictyol(P＜0.05),and the increasing effect was enhanced with the increase of the concentration of eriodictyol and reached a plateau at 320 μmol/L.Therefore,320μmol/L sericol was selected for subsequent experiments.Compared with the control group,the expression of YAP and TAZ mRNA and protein,the relative proportion of ALP positivity,ALP activity,the relative proportion of mineralized nodules,the expression of Runx2 and OSX,OPN mRNA and protein in the LPS group decreased(P＜0.05),the levels of PGE2,IL-6,and IL-18 increased(P＜0.05).Compared with the LPS group,the expression of YAP and TAZ mRNA and protein,the relative proportion of ALP positivity,ALP activity,the relative proportion of mineralized nodules,the ex-pression of Runx2 and OSX,OPN mRNA and protein in LPS+eriodictyol group increased(P＜0.05),the lev-els of PGE2,IL-6,and IL-18 decreased(P＜0.05);there was no obvious change in all indicators of cells in the LPS+empty group(P＞0.05).Compared with the LPS+eriodictyol group,the expression of YAP and TAZ mRNA and protein,the relative proportion of ALP positivity,ALP activity,the relative proportion of mineral-ized nodules,the expression of Runx2 and OSX,OPN mRNA and protein in LPS+eriodictyol+YAP knock-down group decreased(P＜0.05),the levels of PGE2,IL-6,and IL-18 increased(P＜0.05).Conclu-sion:Eriodictyol can reduce the inflammatory damage of periodontitis rats,and inhibit LPS-induced hPDLSC inflammation by up-regulating the expression of YAP/TAZ pathway proteins,thereby promoting its osteogenic differentiation.
维普
万方数据