甘草总黄酮通过调控MAPK/NF-κB信号通路对脂多糖诱导的人牙龈成纤维细胞炎症反应的抑制作用

Inhibitory Effect of Total Flavonoids of Licorice on Lipopolysaccharide-induced Inflammatory Response of Human Gingival Fibroblasts by Regulating MAPK/NF-κB Signaling Pathway

目的:探讨甘草总黄酮通过调控丝裂原活化蛋白激酶(MAPK)/核转录因子κB(NF-κB)信号通路对脂多糖(LPS)诱导的人牙龈成纤维细胞(HGF)炎症反应的抑制作用。方法:体外分离培养原代HGF,以5μg·ml^(-1)LPS处理后,采用CCK-8实验检测0,1.5,3,6,12,24μg·ml^(-1)甘草总黄酮对HGF细胞活力的影响,筛选出甘草总黄酮适合的剂量和作用时间。体外培养原代HGF,随机分为对照组(不进行任何药物处理)、LPS组(5μg·ml^(-1))、甘草总黄酮低剂量(6μg·ml^(-1))+LPS(5μg·ml^(-1))组、甘草总黄酮高剂量(12μg·ml^(-1))+LPS(5μg·ml^(-1))组、甘草总黄酮高剂量(12μg·ml^(-1))+LPS(5μg·ml^(-1))+C16-PAF(MAPK激活剂,4μmol·L-1)组。采用CCK-8实验、划痕实验分别检测各组细胞活力和迁移率;酶联免疫吸附(ELISA)试剂盒检测各组细胞炎性因子白细胞介素6(IL-6)、白细胞介素8(IL-8)、环氧合酶-2(COX-2)释放水平;免疫印迹法检测各组细胞IL-6、IL-8、COX-2、细胞间黏附分子-1(ICAM-1)、趋化因子2(CCL2)、基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶-8(MMP-8)及MAPK/NF-κB通路相关蛋白表达。结果:甘草总黄酮可提高LPS诱导的HGF细胞活力,与药物剂量及作用时间呈正相关。与对照组相比,LPS组细胞活力和迁移率显著降低(P<0.05),IL-6、IL-8及COX-2的释放量、IL-6、IL-8、COX-2、ICAM-1、CCL2、MMP-2及MMP-8蛋白表达、p-细胞外调节蛋白激酶1/2(ERK1/2)/ERK1/2、p-c-Jun N-末端激酶(JNK)/JNK、p-p38 MAPK/p38 MAPK、p-NF-κB p65/NF-κB p65及肿瘤坏死因子α(TNF-α)表达显著升高(P<0.05)。与LPS组相比,甘草总黄酮低、高剂量+LPS组细胞活力和迁移率均显著升高(P<0.05),IL-6、IL-8及COX-2的释放量、IL-6、IL-8、COX-2、ICAM-1、CCL2、MMP-2及MMP-8蛋白表达、p-ERK1/2/ERK1/2、p-JNK/JNK、p-p38 MAPK/p38 MAPK、p-NF-κB p65/NF-κB p65及TNF-α表达均显著降低(P<0.05),且甘草总黄酮低、高剂量组间差异有统计学意义(P<0.05)。与甘草总黄酮高剂量+LPS组相比,甘草总黄酮高剂量+LPS+C16-PAF组细胞活力和迁移率显著降低(P<0.05),IL-6、IL-8及COX-2的释放量、IL-6、IL-8、COX-2、ICAM-1、CCL2、MMP-2及MMP-8蛋白表达、p-ERK1/2/ERK1/2、p-JNK/JNK、p-p38 MAPK/p38 MAPK、p-NF-κB p65/NF-κB p65及TNF-α显著升高(P<0.05)。结论:甘草总黄酮可通过抑制MAPK/NF-κB信号通路而降低炎性因子表达,并下调ICAM-1、CCL2、MMP-2、MMP-8蛋白表达,抑制LPS诱导的炎症,促使HGF生长和迁移。

Objective: To investigate the inhibitory effect of total flavonoids of licorice on lipopolysaccharide(LPS)-induced inflammation in human gingival fibroblasts(HGF) by regulating MAPK/NF-κB signaling pathway.Methods: Primary HGF was isolated and cultured in vitro, and treated with 5 μg·ml^(-1)LPS. The impacts of 0, 1.5, 3, 6, 12 and 24 μg·ml^(-1)total flavonoids of licorice on the viability of HGF cells were detected by CCK-8 assay, and the appropriate dose and action time of total flavonoids of licorice were screened. Primary HGF was cultured in vitro and randomly grouped into control group(no drug treatment), LPS(5 μg·ml^(-1)) group, low-dose total flavonoids of licorice(6 μg·ml^(-1)) + LPS(5 μg·ml^(-1)) group, high-dose total flavonoids of licorice(12 μg·ml^(-1)) + LPS(5 μg·ml^(-1)) group, and high-dose total flavonoids of licorice(12 μg·ml^(-1)) + LPS(5 μg·ml^(-1)) + C16-PAF(MAPK activator, 4 μmol·L-1) group. The cell viability and migration rate of each group were detected respectively by CCK-8 test and scratch test;the release levels of inflammatory factors IL-6, IL-8 and COX-2 in each group were detected by enzyme-linked immunosorbent assay(ELISA) kit;the expressions of IL-6, IL-8, COX-2, ICAM-1, CCL2, MMP-2, MMP-8 and MAPK/NF-κB pathway related proteins were detected by Western blot. Results: Total flavonoids of licorice could increase the viability of HGF cells induced by LPS, which was positively correlated with the drug dosage and action time. Compared with those in the control group, the cell viability and migration rate of the LPS group were significantly decreased(P<0.05), and the release levels of IL-6, IL-8 and COX-2, the protein expression of IL-6, IL-8, COX-2, ICAM-1, CCL2, MMP-2 and MMP-8, and the expression of p-ERK1/2/ERK1/2, p-JNK/JNK, p-p38 MAPK/p38 MAPK, p-NF-κB p65/NF-κB p65 and TNF-α were significantly increased(P<0.05). Compared with those in the LPS group, the cell viability and migration rate of the low-and high-dose of total flavonoids of licorice+LPS groups were significantly increased(P<0.05), the release levels of IL-6, IL-8 and COX-2, the protein expression of IL-6, IL-8, COX-2, ICAM-1, CCL2, MMP-2 and MMP-8, and the expression of p-ERK1/2/ERK1/2, p-JNK/JNK, p-p38 MAPK/p38 MAPK, p-NF-κB p65/NF-κB p65 and TNF-α were significantly decreased(P<0.05), and the differences between low-and high-dose of total flavonoids of licorice groups were statistically significant(P<0.05). Compared with those in the high-dose total flavonoids of licorice+LPS group, the cell viability and migration rate of the high-dose total flavonoids of licorice+LPS+C16-PAF group were significantly decreased(P<0.05), the release levels of IL-6, IL-8 and COX-2, and the protein expression of IL-6, IL-8, COX-2, ICAM-1, CCL2, MMP-2 and MMP-8, and the expression of p-ERK1/2/ERK1/2, p-JNK/JNK, p-p38 MAPK/p38 MAPK, p-NF-κB p65/NF-κB p65 and TNF-α were significantly increased(P<0.05). Conclusion: Total flavonoids of licorice can reduce the expression of inflammatory factors by inhibiting MAPK/NF-κB signaling pathway, and down-regulate the protein expression of ICAM-1, CCL2, MMP-2 and MMP-8, inhibit LPS-induced inflammation, and promote the growth and development of HGF.