葡萄籽原花青素对牙龈上皮细胞炎症介质表达的影响

Effects of grape seed proanthocyanidins on the expression of inflammatory mediators in gingival epithelial cells

目的研究葡萄籽原花青素(GSPs)预处理对脂多糖(LPS)诱导的人牙龈上皮细胞(HGECs)炎症介质表达的影响。方法用含不同浓度GSPs(0、1、5、10、20、40、60、80、100μg·mL^(-1))培养液培养HGECs 6、12、24、48 h后,CCK-8检测细胞增殖活性。不同浓度GSPs(0、10、20、40μg·mL^(-1))处理HGECs24 h后加入1.0μg·mL^(-1)LPS培养,酶联免疫吸附测定(ELISA)法检测促炎细胞因子肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β、IL-6及抗炎细胞因子IL-4、IL^(-1)0、转化生长因子(TGF-β)的表达水平,实时荧光定量聚合酶链反应(QRT-PCR)检测TNF-α、IL^(-1)、IL-6、IL-4、IL^(-1)0和TGF-βmRNA表达水平。结果GSPs浓度为0~40μg·mL^(-1)时细胞增殖无明显差异,且细胞增殖活性在24 h时最高。ELISA及QRT-PCR结果显示:与GSPs浓度为0μg·mL^(-1)相比,GSPs浓度为10、20、40μg·mL^(-1)时TNF-α、IL^(-1)β和IL-6的表达降低(P<0.05),IL-4、IL^(-1)0和TGF-β的表达升高(P<0.05)。结论GSPs浓度在0~40μg·mL^(-1)时对人HGECs增殖活性无明显影响,GSPs预处理HGECs能够抑制促炎因子的表达和促进抗炎因子的表达,对HGECs抵抗内毒素的刺激起到一定的预防作用。

Objective This study aims to determine the effect of grape seed proanthocyanidin(GSP)pretreatment on lipopolysaccharide(LPS)-induced inflammation of human gingival epithelial cells(HGECs).Methods HGECs were cultivated with different concentrations of GSPs(0,1,5,10,20,40,60,80,100μg·mL^(-1))for 6,12,24,and 48 h.CCK-8 was used to detect the proliferation activity of HGECs.HGECs were treated with different concentrations of GSPs(0,10,20,and 40μg·mL^(-1))for 24 h and then cultured with 1.0μg·mL^(-1) LPS.Enzyme-linked immunosorbent assay(ELISA)was used to detect the expression levels of pro-inflammatory cytokine tumor necrosis factor-α(TNF-α),interleukin(IL)-1β,and IL-6 and anti-inflammatory cytokines IL-4,IL^(-1)0,and transforming growth factor-β(TGF-β).Quantitative realtime polymerase chain reaction(QRT-PCR)was used to detect the mRNA expression levels of TNF-α,IL^(-1)β,IL-6,IL-4,IL^(-1)0,and TGF-β.Results When the GSP concentration was 0-40μg·mL^(-1),the cell proliferation had no significant difference.When the action time reached 24 h,the cell proliferation was the highest.The results of ELISA and QRT-PCR showed that 10,20,and 40μg·mL^(-1) GSPS decreased the expression levels of TNF-α,IL^(-1)β,and IL-6(P<0.05)and increased the expression levels of IL-4,IL^(-1)0,and TGF-βcompared with 0μg·mL^(-1) GSPS(P<0.05).Conclusion GSPS(0-40μg·mL^(-1))has no significant effect on the proliferation activity of HGECs.Pretreatment with GSPS can inhibit the expression of pro-inflammatory factors and enhance the expression of anti-inflammatory factors.Hence,GSPS has a certain preventive effect on the resistance of HGECs to the stimulation of endotoxin.