华西口腔医学杂志 ›› 2021, Vol. 39 ›› Issue (2): 153-163.doi: 10.7518/hxkq.2021.02.005

• 基础研究 • 上一篇    

西格列汀通过阻断核因子-κB信号通路抑制脂多糖诱导的人牙龈成纤维细胞炎症反应

刘相(), 康文燕, 商玲玲, 葛少华()   

  1. 山东大学齐鲁医学院·口腔医学院·口腔医院牙周科 山东省口腔组织再生重点实验室 山东省口腔生物材料与组织再生工程实验室,济南 250012
  • 收稿日期:2020-07-19 修回日期:2020-11-27 出版日期:2021-04-01 发布日期:2021-04-09
  • 通讯作者: 葛少华 E-mail:492998461@qq.com;shaohuage@sdu.edu.cn
  • 作者简介:刘相,硕士,E-mail:492998461@qq.com
  • 基金资助:
    国家自然科学基金(81670993)

Sitagliptin inhibits lipopolysaccharide-induced inflammatory response in human gingival fibroblasts by blocking nuclear factor-κB signaling pathway

Liu Xiang(), Kang Wenyan, Shang Lingling, Ge Shaohua()   

  1. Dept. of Periodonto-logy, School and Hospital of Stomatology, Cheeloo College of Medicine, Shandong University & Shandong Key Laboratory of Oral Tissue Regeneration & Shandong Engineering Laboratory for Dental Materials and Oral Tissue Regeneration, Jinan 250012, China
  • Received:2020-07-19 Revised:2020-11-27 Online:2021-04-01 Published:2021-04-09
  • Contact: Ge Shaohua E-mail:492998461@qq.com;shaohuage@sdu.edu.cn
  • Supported by:
    The National Natural Science Foundation of China(81670993)

摘要: 目的

明确西格列汀对牙龈卟啉单胞菌脂多糖(LPS)诱导的人牙龈成纤维细胞(HGF)炎症反应的影响,并探讨其发挥作用的分子学机制,为未来西格列汀在牙周炎治疗中的应用提供初步的实验依据。

方法

收集临床患者健康牙龈组织标本,利用酶消化法分离培养HGF并进行细胞来源鉴定;采用细胞增殖-毒性检测试剂盒检测LPS和西格列汀与HGF共培养24、48 h细胞增殖变化;通过实时荧光定量聚合酶链式反应(qRT-PCR)检测炎性细胞因子白细胞介素(IL)-6、IL-8、趋化因子配体2(CCL2)、超氧化物歧化酶2(SOD2)的基因表达水平;酶联免疫吸附实验检测细胞培养上清液中IL-6、IL-8和CCL2的蛋白表达水平;应用蛋白免疫印迹法检测核因子-κB(NF-κB)信号通路的激活情况,通过qRT-PCR验证NF-κB通路抑制剂BAY11-7082对LPS诱导的HGF炎症细胞因子基因表达水平的影响。

结果

低浓度的西格列汀(0.1、0.25、0.5 μmol·L-1)作用24、48 h对HGF的生长无影响,而高浓度西格列汀(5~1 000 μmol·L-1)明显抑制了细胞增殖。西格列汀浓度依赖性抑制5 μg·mL-1 LPS诱导的HGF中IL-6、IL-8、CCL2和SOD2的基因表达水平。此外,西格列汀显著抑制LPS诱导的IL-6、IL-8和CCL2蛋白表达水平。蛋白免疫印迹结果表明:0.5 μmol·L-1西格列汀显著性抑制了LPS诱导的NF-κB信号通路激活,qRT-PCR结果表明0.5 μmol·L-1西格列汀与5 μmol·L-1BAY11-7082均显著抑制了LPS诱导的IL-6、IL-8、CCL2和SOD2的基因表达水平。

结论

西格列汀通过阻断NF-κB信号通路的激活来抑制LPS诱导的HGF炎症反应。

关键词: 西格列汀, 脂多糖, 核因子-κB, 炎症, 人牙龈成纤维细胞

Abstract: Objective

This study was performed to clarify the effects of sitagliptin on Porphyromonas gingivalis-lipopolysaccharide (LPS)-induced inflammatory response in human gingival fibroblasts (HGFs), explore the molecular mechanism of its roles, and provide a foundation for clinical therapeutics in periodontitis.

Methods

Healthy gingival samples were collected from the donors. HGFs were isolated with enzymic digestion method and identified. The effects of LPS and sitagliptin on cell viability were detected by cell-counting kit-8 (CCK8). The mRNA levels of inflammatory cytokines, namely, interleukin (IL)-6, IL-8, C-C motif ligand 2 (CCL2), and superoxide dismutase 2 (SOD2), were evaluated by quantity real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immune sorbent assay (ELISA) was used to measure the secretion protein levels of IL-6, IL-8, and CCL2. Western blot analysis was used to further investigate the activation of nuclear factor (NF)-κB signaling pathway. The effect of NF-κB pathway inhibitor BAY11-7082 on LPS-induced HGF inflammatory cytokines at the gene level was verified by qRT-PCR.

Results

Low concentrations of sitagliptin (0.1, 0.25, and 0.5 μmol·L-1) did not affect HGF growth in 24 and 48 h, whereas high concentrations of sitagliptin (5-1 000 μmol·L-1) significantly inhibited cell proliferation. Sitagliptin suppressed 5 μg·mL-1 of LPS-induced IL-6, IL-8, CCL2, and SOD2 gene expression levels in HGF in a concentration-dependent manner. Furthermore, sitagliptin significantly decreased the elevated secretion of IL-6, IL-8, and CCL2 protein induced by LPS. Western blot analysis showed that 0.5 μmol·L-1 of sitagliptin significantly inhibited LPS-induced NF-κB signaling pathway activation. Results of qRT-PCR analysis indicated that 0.5 μmol·L-1 of sitagliptin and 5 μmol·L-1 of BAY11-7082 significantly inhibited LPS-induced IL-6, IL-8, CCL2, and SOD2 gene expressions.

Conclusion

Sitagliptin could significantly inhibit LPS-induced HGF inflammatory response by blocking the NF-κB signaling pathway activation.

Key words: Sitagliptin, lipopolysaccharide, nuclear factor-κB, inflammation, human gingival fibroblasts

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