槐糖脂通过 NF-κB 信号通路抑制 LPS 诱导的 RAW264.7 细胞炎症反应。
Sophorolipid Suppresses LPS-Induced Inflammation in RAW264.7 Cells through the NF-κB Signaling Pathway.
机构信息
Key Laboratory of Synthetic and Biological Colloids, Ministry of Education, School of Chemical and Material Engineering, Jiangnan University, Wuxi 214122, China.
Adolph Innovation Laboratory, Guangzhou Degu Personal Care Products Co., Ltd., Guangzhou 510000, China.
出版信息
Molecules. 2022 Aug 8;27(15):5037. doi: 10.3390/molecules27155037.
OBJECTIVES
Biosurfactants with anti-inflammatory activity may alleviate skin irritation caused by synthetic surfactants in cleaning products. Sophorolipid (SL) is a promising alternative to synthetic surfactants. However, there are few reports on the anti-inflammatory activity of SL and the underlying mechanism. The purpose of this work is to verify that lipopolysaccharide (LPS)-induced inflammation could be inhibited through targeting the pathway of nuclear factor-κB (NF-κB) in RAW264.7 cells.
METHODS
The influence of SL on cytokine release was investigated by LPS-induced RAW264.7 cells using ELISA. The quantification of the protein expression of corresponding molecular markers was realized by Western blot analysis. Flow cytometry was employed to determine the levels of Ca and reactive oxygen species (ROS). The relative expression of inducible nitric oxide synthase (INOS) and cyclooxygenase-2 (COX-2) was determined by RT-PCR. An immunofluorescence assay and confocal microscope were used to observe the NF-κB/p65 translocation from the cytoplasm into the nucleus. The likely targets of SL were predicted by molecular docking analysis.
RESULTS
SL showed anti-inflammatory activity and reduced the release of inflammatory cytokines including interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and nitric oxide (NO). The experimental results show that SL suppressed the Ca and ROS levels influx in the LPS-induced RAW264.7 cells and alleviated the LPS-induced expression of iNOS and COX-2, the LPS-induced translocation of NF-κB (p65) from the cytoplasm into the nucleus, and the expression of phosphorylated proteins such as p65 and IκBα. Furthermore, molecular docking analysis showed that SL may inhibit inflammatory signaling by competing with LPS to bind TLR4/MD-2 through hydrophobic interactions and by inhibiting IKKβ activation through the hydrogen bonding and hydrophobic interactions.
CONCLUSION
This study demonstrated that SL exerted anti-inflammatory activity via the pathway of NF-κB in RAW264.7 cells.
目的
具有抗炎活性的生物表面活性剂可以减轻清洁产品中合成表面活性剂引起的皮肤刺激。槐糖脂（SL）是合成表面活性剂的一种很有前途的替代品。然而，关于 SL 的抗炎活性及其潜在机制的报道很少。本工作的目的是验证 LPS 诱导的 RAW264.7 细胞中 NF-κB 通路的靶向作用可以抑制炎症。
方法
通过 LPS 诱导的 RAW264.7 细胞，用 ELISA 法研究 SL 对细胞因子释放的影响。用 Western blot 分析测定相应分子标志物的蛋白表达量。用流式细胞术测定 Ca 和活性氧（ROS）的水平。通过 RT-PCR 测定诱导型一氧化氮合酶（iNOS）和环氧化酶-2（COX-2）的相对表达。用免疫荧光法和共聚焦显微镜观察 NF-κB/p65 从细胞质向细胞核的易位。通过分子对接分析预测 SL 的可能靶点。
结果
SL 表现出抗炎活性，减少了包括白细胞介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）和一氧化氮（NO）在内的炎症细胞因子的释放。实验结果表明，SL 抑制了 LPS 诱导的 RAW264.7 细胞中 Ca 和 ROS 水平的流入，减轻了 LPS 诱导的 iNOS 和 COX-2 的表达，LPS 诱导的 NF-κB（p65）从细胞质向细胞核的易位，以及磷酸化蛋白如 p65 和 IκBα的表达。此外，分子对接分析表明，SL 可能通过与 LPS 竞争与 TLR4/MD-2 结合，通过疏水相互作用，以及通过氢键和疏水相互作用抑制 IKKβ 的激活，从而抑制炎症信号。
结论
本研究表明，SL 通过 RAW264.7 细胞中的 NF-κB 通路发挥抗炎活性。
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