葡萄籽原花青素对牙龈上皮细胞炎症介质表达的影响

doi:10.7518/hxkq.2022.01.006

摘要/Abstract

摘要： 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、100 μg·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、40 μg·mL^-1）处理HGECs 24 h后加入1.0 μg·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-10、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-10和TGF-β mRNA表达水平。

结果

GSPs浓度为0~40 μg·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0 μg·mL^-1相比，GSPs浓度为10、20、40 μg·mL^-1时TNF-α、IL-1β和IL-6的表达降低（P<0.05），IL-4、IL-10和TGF-β的表达升高（P<0.05）。

结论

GSPs浓度在0~40 μg·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。

关键词: 葡萄籽原花青素, 牙龈上皮细胞, 增殖活性, 脂多糖, 炎症因子

Abstract: 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, 100 μg·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 40 μg·mL^-1) for 24 h and then cultured with 1.0 μg·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-10, and transforming growth factor-β (TGF-β). Quantitative real-time polymerase chain reaction (QRT-PCR) was used to detect the mRNA expression levels of TNF-α, IL-1β, IL-6, IL-4, IL-10, and TGF-β.

Results

When the GSP concentration was 0-40 μg·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 40 μg·mL^-1 GSPS decreased the expression levels of TNF-α, IL-1β, and IL-6 (P<0.05) and increased the expression levels of IL-4, IL-10, and TGF-β compared with 0 μg·mL^-1 GSPS (P<0.05).

Conclusion

GSPS (0-40 μg·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.

Key words: grape seed proanthocyanidins, gingival epithelial cells, proliferative activity, lipopolysaccharide, inflammatory factors

中图分类号:

R 781.4

张秀娟, 吴伟, 陈欣, 房芷纯, 叶金香, 欧晓艳. 葡萄籽原花青素对牙龈上皮细胞炎症介质表达的影响[J]. 华西口腔医学杂志, 2022, 40(1): 39-44.

Zhang Xiujuan, Wu Wei, Chen Xin, Fang Zhichun, Ye Jinxiang, Ou Xiaoyan.. Effects of grape seed proanthocyanidins on the expression of inflammatory mediators in gingival epithelial cells[J]. West China Journal of Stomatology, 2022, 40(1): 39-44.

图/表 4

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引物	引物序列	产物大小/bp
IL-1β	上游：5’-AGCTACGAATCTCCGACCAC-3’	186
	下游：5’-CGTTATCCCATGTGTCGAAGAA-3’
IL-4	上游：5’-ATGGGTCTCACCTCCCAACT-3’	168
	下游：5’-GATGTCTGTTACGGTCAACTCG-3’
IL-6	上游：5’-ACTCACCTCTTCAGAACGAATTG-3’	149
	下游：5’-CCATCTTTGGAAGGTTCAGGTTG-3’
IL-10	上游：5’-TCAAGGCGCATGTGAACTCC-3’	176
	下游：5’-GATGTCAAACTCACTCATGGCT-3’
TNF-α	上游：5’-CCTCTCTCTAATCAGCCCTCTG-3’	220
	下游：5’-GAGGACCTGGGAGTAGATGAG-3’
TGF-β	上游：5’-GGCCAGATCCTGTCCAAGC-3’	201
	下游：5’-GTGGGTTTCCACCATTAGCAC-3’
β-actin	上游：5’-ATGGCCTCCCTGTACCACATC-3’	151
	下游：5’-TGTTGCGCTCAATCTCCTCCT-3’

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