睡莲花总多酚对脂多糖诱导A549细胞炎性损伤的保护作用研究

Protective effects of total polyphenols from Nymphaea candida on lipopolysaccharide induced inflammatory injury in A549 cells

中文关键词:  雪白睡莲  多酚  A549 细胞  急性肺损伤  抗氧化作用  Nrf2/HO-1通路

英文关键词:Nymphaea candida  polyphenol  A549 cells  acute lung injury  antioxidant effect  nuclear factor erythroid 2-related factor 2/heme oxygenase-1 signal pathway

中文摘要:

      目的 研究睡莲花总多酚(total polyphenols from Nymphaea candid, NCTP)对脂多糖(lipopolysaccharide, LPS)诱导肺泡上皮细胞A549炎性损伤的保护作用。方法 采用福林酚法测定NCTP中的多酚含量, 高效液相色谱法(high performance liquid chromatography, HPLC)法测定其特征性成分含量。以1.0 μg/mL LPS干预A549细胞12 h造成炎性损伤模型, CCK-8 (cell counting kit-8)法检测细胞毒性, 以筛选出合适的NCTP干预浓度; 进一步将细胞分为空白组、脂多糖模型组、阳性对照地塞米松(0.039 μg/mL dexamethasone, DXM)组、NCTP低中高剂量(6.0、15.0、30.0 μg/mL)组。生化法检测一氧化氮(nitric oxide, NO)及前列素E2 (prostaglandin E2, PGE2)、丙二醛(malondialdehyde, MDA)、超氧化物歧化酶(superoxide dismutase, SOD)、谷胱甘肽(glutathione, GSH)活性; 酶联免疫法(enzyme-linked immunosorbent assay, ELISA)检测白细胞介素-1β (interleukin-1β, IL-1β)、白细胞介素-6 (interleukin-6, IL-6)、肿瘤坏死因子-α (tumor necrosis factor-α, TNF-α)水平; 蛋白免疫印迹(Western Blot)法检测核转录因子E2相关因子2/血红素加氧酶-1 (nuclear factor erythroid-2-related factor 2/heme oxygenase 1, Nrf2/HO-1)信号通路的相关蛋白[Kelch样ECH结合蛋白1 (Kelch-like ECH-associating protein 1, Keap1)、核因子(红细胞衍生2)相关因子2 (nuclear factor erythroid 2-related factor 2, Nrf2)、血红素加氧酶1 (heme oxygenase-1, HO-1)]表达。结果 NCTP中多酚含量为(90.15±0.17)%, 所含成分睡莲酚、鞣花酸和烟花苷含量分别为(28.05±0.02)%、(2.15±0.03)%和(3.50±0.01)%, CCK-8法筛选出NCTP的干预质量浓度为6.0、15.0、30.0 μg/mL。LPS刺激A549细胞后, 细胞内炎症介质NO、PGE2和炎症因子IL-1β、IL-6、TNF-α表达显著升高(P<0.01), 说明A549细胞炎性损伤模型成功构建。与模型组比较, NCTP能显著抑制LPS诱导A549细胞上清中NO、PGE2、IL-1β、IL-6、TNF-α、MDA水平, 明显提高细胞中SOD和GSH活力。与模型组相比, NCTP还能显著增加受损细胞中Nrf2、HO-1的蛋白表达, 降低Keap1蛋白表达水平。结论 NCTP对LPS诱导A549细胞炎性损伤具有显著的保护作用, 机制与其抗氧化、对Nrf2/HO-1信号通路的调控相关。研究结果可为该有效部位预防呼吸系统疾病的功能性食品研发提供依据。

英文摘要:

      Objective To study protective effects of total polyphenols from Nymphaea candida (NCTP) on lipopolysaccharide (LPS) induced inflammatory injury in A549 cells. Methods Polyphenol content in NCTP was determined by Folin-Ciocalte method, and its characteristic components were detected by high performance liquid chromatography (HPLC). A549 cells were treated with 1.0 μg/mL LPS for 12 h to induce inflammatory damage model, and cell counting kit-8 (CCK-8) method was used to screen out the optimal intervention concentration of NCTP. The experiment was divided into control group, lipopolysaccharide model group, dexamethasone (DXM 0.039 μg/mL) group, and NCTP (6.0, 15.0, 30.0 μg/mL) groups. The levels of nitric oxide (NO), prostaglandin E2 (PGE2), malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione (GSH) were detected by biochemical method. Enzyme-linked immunosorbent assay (ELISA) was used to detect the expression of interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in A549 cells. The expression of related proteins [Kelch-like ECH-associating protein 1 (Keap1), nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1)] in nuclear factor erythroid-2-related factor 2/heme oxygenase 1 (Nrf2/HO-1) signaling pathway was detected by Western Blot. Results The content of total polyphenols in NCTP was (90.15±0.17)%, and the content of isostrictiniin, ellagic acid and nicotiflorin was (28.05±0.02)%, (2.15±0.03)% and (3.50±0.01)%, respectively. The mass concentrations of NCTP screened by CCK-8 were 6.0, 15.0, 30.0 μg/mL. After LPS stimulation of A549 cells, the expressions of intracellular inflammatory mediators NO, PGE2 and inflammatory factors IL-1β, IL-6 and TNF-α were significantly increased (P<0.01), indicating that the inflammatory damage model of A549 cells was successfully constructed. Compared with the model group, NCTP significantly inhibited the expression of NO, PGE2, IL-1β, IL-6, TNF-α and MDA and increased the activities of SOD and GSH in LPS-induced A549 cells. Compared with the model group, NCTP also significantly increased the expression of Nrf2 and HO-1 proteins in the damaged cells, and decreased the expression level of Keap1 proteins. Conclusion NCTP has the better protective effect on LPS-induced inflammatory injury in A549 cells, and its mechanism is relative to the regulation of the Nrf2/HO-1 signal pathway. The results of this study can provide a basis for the research and development of functional foods with this effective part to prevent respiratory diseases.