西伯利亚远志乙醇提取物对脂多糖刺激的 RAW264.7 细胞的抗氧化和抗炎作用。

Anti-oxidant and Anti-inflammatory Effects of Ethanol Extract from Polygala sibirica L. var megalopha Fr. on Lipopolysaccharide-Stimulated RAW264.7 Cells.

机构信息

Key Laboratory of Biotechnology for Medicinal Plants of Jiangsu Province, School of Life Science, Jiangsu Normal University, Xuzhou, Jiangsu Province, 221116, China.

出版信息

Chin J Integr Med. 2023 Oct;29(10):905-913. doi: 10.1007/s11655-023-3602-7. Epub 2023 Jul 12.

DOI:10.1007/s11655-023-3602-7

PMID:37434032

Abstract

OBJECTIVE

To investigate the anti-oxidant and anti-inflammatory effects of ethanol extract of Polygala sibirica L. var megalopha Fr. (EEP) on RAW264.7 mouse macrophages.

METHODS

RAW264.7 cells were pretreated with 0-200 µg/mL EEP or vehicle for 2 h prior to exposure to 1 µg/mL lipopolysaccharide (LPS) for 24 h. Nitric oxide (NO) and prostaglandin (PGE) production were determined by Griess reagent and enzyme-linked immunosorbent assay (ELISA), respectively. The mRNA levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor α (TNF-α), interleukin-1beta (IL-1β), and IL-6 were determined using reverse transcription polymerase chain reaction (RT-PCR). Western blot assay was used to determine the protein expressions of iNOS, COX-2, phosphorylation of extracellular regulated protein kinases (ERK1/2), c-Jun N-terminal kinase (JNK), inhibitory subunit of nuclear factor Kappa B alpha (Iκ B-α) and p38. Immunofluorescence was used to observe the nuclear expression of nuclear factor-κ B p65 (NF-κ B p65). Additionally, the anti-oxidant potential of EEP was evaluated by reactive oxygen species (ROS) production and the activities of catalase (CAT) and superoxide dismutase (SOD). The 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl (OH), superoxide anion (O) radical and nitrite scavenging activity were also measured.

RESULTS

The total polyphenol and flavonoid contents of EEP were 23.50±2.16 mg gallic acid equivalent/100 g and 43.78±3.81 mg rutin equivalent/100 g. With EEP treatment (100 and 150 µg/mL), there was a notable decrease in NO and PGE production induced by LPS in RAW264.7 cells by downregulation of iNOS and COX-2 mRNA and protein expressions (P<0.01 or P<0.05). Furthermore, with EEP treatment (150 µg/mL), there was a decrease in the mRNA expression levels of TNF-α, IL-1β and IL-6, as well as in the phosphorylation of ERK, JNK and p38 mitogen-activated protein kinase (MAPK, P<0.01 or P<0.05), by blocking the nuclear translocation of NF-κ B p65 in LPS-stimulated cells. In addition, EEP (100 and 150 µg/mL) led to an increase in the anti-oxidant enzymes activity of SOD and CAT, with a concomitant decrease in ROS production (P<0.01 or P<0.05). EEP also indicated the DPPH, OH, O radical and nitrite scavenging activity.

CONCLUSION

EEP inhibited inflammatory responses in activated macrophages through blocking MAPK/NF-κ B pathway and protected against oxidative stress.

摘要

目的

研究西伯利亚远志乙醇提取物（EEP）对 RAW264.7 小鼠巨噬细胞的抗氧化和抗炎作用。

方法

用 0-200μg/ml EEP 或载体预处理 RAW264.7 细胞 2 h，然后用 1μg/ml 脂多糖（LPS）处理 24 h。用格里斯试剂和酶联免疫吸附试验（ELISA）分别测定一氧化氮（NO）和前列腺素（PGE）的产生。采用逆转录聚合酶链反应（RT-PCR）测定诱导型一氧化氮合酶（iNOS）、环氧化酶-2（COX-2）、肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）和白细胞介素-6（IL-6）的 mRNA 水平。采用 Western blot 检测 iNOS、COX-2、细胞外调节蛋白激酶（ERK1/2）磷酸化、c-Jun N-末端激酶（JNK）、核因子κB 抑制亚单位α（IκB-α）和 p38 的蛋白表达。免疫荧光法观察核因子-κB p65（NF-κB p65）的核表达。此外，通过测定活性氧（ROS）的产生以及过氧化氢酶（CAT）和超氧化物歧化酶（SOD）的活性来评估 EEP 的抗氧化潜力。还测量了 2,2-二苯基-1-苦基肼（DPPH）、羟基（OH）、超氧阴离子（O）自由基和亚硝酸盐清除活性。

结果

EEP 的总多酚和类黄酮含量分别为 23.50±2.16 mg 没食子酸当量/100 g 和 43.78±3.81 mg 芦丁当量/100 g。用 EEP（100 和 150μg/ml）处理时，通过下调 iNOS 和 COX-2 mRNA 和蛋白表达，可显著降低 LPS 诱导的 RAW264.7 细胞中 NO 和 PGE 的产生（P<0.01 或 P<0.05）。此外，用 EEP（150μg/ml）处理可降低 TNF-α、IL-1β和 IL-6 的 mRNA 表达水平，以及 LPS 刺激细胞中 ERK、JNK 和 p38 丝裂原活化蛋白激酶（MAPK，P<0.01 或 P<0.05）的磷酸化，从而阻断 NF-κB p65 的核易位。此外，EEP（100 和 150μg/ml）可增加 SOD 和 CAT 的抗氧化酶活性，同时降低 ROS 产生（P<0.01 或 P<0.05）。EEP 还表现出 DPPH、OH、O 自由基和亚硝酸盐清除活性。