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. 2021 Apr 1;39(2):153-163.
doi: 10.7518/hxkq.2021.02.005.

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

[Article in Chinese]
Xiang Liu  1 , Wen-Yan Kang  1 , Ling-Ling Shang  1 , Shao-Hua Ge  1
Affiliations

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

[Article in Chinese]
Xiang Liu et al. Hua Xi Kou Qiang Yi Xue Za Zhi. .

Abstract
in English, Chinese

Objectives: 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.

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

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。.

Keywords: Sitagliptin; human gingival fibroblasts; inflammation; lipopolysaccharide; nuclear factor-κB.

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Conflict of interest statement

利益冲突声明：作者声明本文无利益冲突。

Figures

图 1
图 1. 分离和培养HGF
Fig 1 Isolation and culture of HGFs A：原代培养HGF × 40；B：第4代HGF × 40；C：第4代HGF × 100。
图 2
图 2. HGF的鉴定 荧光显微镜 × 400
Fig 2 Identification of HGFs fluorescence microscope × 400 上：波形丝蛋白染色阳性；下：角蛋白17染色阴性；左：DAPI；中上：波形丝蛋白；中下：角蛋白；右：图层合并。
图 3
图 3. 不同浓度西格列汀对HGF活性的影响
Fig 3 Effects of sitagliptin at different concentrations on HGFs viability A：24 h；B：48 h。与对照组相比，***P<0.001，****P<0.000; ns: 差异无统计学意义。
图 4
图 4. 不同浓度西格列汀和5 µg·mL−1 LPS共同作用对HGF活性的影响
Fig 4 Effects of sitagliptin at different concentrations and 5 µg·mL−1 LPS on the viability of HGFs A：24 h；B：48 h。
图 5
图 5. 西格列汀对LPS诱导的HGF炎症细胞因子mRNA表达水平的影响
Fig 5 Effects of sitagliptin on the mRNA expression levels of LPS-induced HGFs inflammatory cytokines A：CCL2 mRNA表达水平；B：IL-6 mRNA表达水平；C：IL-8 mRNA表达水平；D：SOD2 mRNA表达水平。与对照组相比，####P<0.000 1；与LPS组相比，*P<0.05，**P<0.01，***P<0.001，****P<0.000 1; ns：差异无统计学意义。
图 6
图 6. 西格列汀对LPS诱导的HGF炎症细胞因子蛋白表达水平的影响
Fig 6 Effects of sitagliptin on the protein expression levels of LPS-induced HGFs inflammatory cytokines A：CCL2蛋白表达水平；B：IL-6蛋白表达水平；C：IL-8蛋白表达水平。与对照组相比，##P<0.01，####P<0.000 1；与LPS组相比，*P<0.05，**P<0.01，***P<0.001，****P<0.000 1；ns: 差异无统计学意义。
图 7
图 7. 西格列汀和BAY11-7082对LPS诱导的NF-κB通路活化的影响
Fig 7 Effects of sitagliptin and BAY11-7082 on LPS-induced activation of NF-κB pathway A：西格列汀和BAY11-7082对HGF中p-p65蛋白的表达影响及灰度分析p-p65/p65相对表达量；B：西格列汀和BAY11-7082对HGF中IκBα和p-IκBα蛋白的表达影响及灰度分析IκBα/GAPDH和p-IκBα/IκBα相对表达量。与对照组相比，####P<0.000 1；与LPS组相比，***P<0.001，****P<0.000 1。
图 8
图 8. 西格列汀和BAY11-7082对LPS诱导的炎症因子表达的影响
Fig 8 Effects of sitagliptin and BAY11-7082 on LPS-induced inflammatory cytokines A：CCL2 mRNA表达水平；B：IL-6 mRNA表达水平；C：IL-8 mRNA表达水平；D：SOD2 mRNA表达水平。与对照组相比，####P<0.000 1；与LPS组相比，***P<0.001，****P<0.000 1。
图 9
图 9. 西格列汀在LPS诱导的NF-κB信号通路中的作用机制
Fig 9 Putative mechanism for the role of sitagliptin in LPS-induced NF-κB signaling pathway
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