Li Xin-Xing, Zheng Xiaoting, Liu Zhenjie, Xu Qiongming, Tang Hongzhen, Feng Jianfang, Yang Shilin, Vong Chi Teng, Gao Hongwei, Wang Yitao
1College of Pharmacy, Guangxi University of Chinese Medicine, Nanning, 530000 China.
Guangxi Engineering Technology Research Center of Advantage Chinese Patent Drug and Ethnic Drug Development, Nanning, 530020 China.
Chin Med. 2020 Mar 2;15:20. doi: 10.1186/s13020-020-00303-3. eCollection 2020.
Cryptotanshinone (CPT), as a major component of Bunge (Danshen), displays many pharmacological activities including anti-inflammatory effects. However, the exact cellular and molecular mechanisms of the anti-inflammatory activities of CPT remain to be elucidated. The present study was aimed to clarify its mechanisms on lipopolysaccharide (LPS)-induced inflammatory responses in mouse macrophages, RAW264.7 cells.
In the current study, the anti-inflammatory properties of CPT were evaluated using LPS-stimulated RAW264.7 cell model. MTT assay was used to determine the viability of RAW264.7 cells. The anti-inflammatory effects of CPT were measured based on the detection of nitric oxide (NO) production (Griess and flow cytometry assay), and tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) release (ELISA). Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) enzyme expressions were also determined by western blotting. Besides, by using flow cytometry, we also evaluated the effect of CPT on LPS-induced calcium influx. Finally, the underlying anti-inflammatory mechanisms of CPT were investigated using western blotting to assess the protein levels of toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), phosphatidylinositol 3-kinase (PI3K)/AKT, nuclear factor erythroid 2 related factor 2 (Nrf2), mitogen-activated protein kinase (MAPK), and nuclear factor-kappa B (NF-κB) pathways.
Our data showed that CPT inhibited LPS-induced pro-inflammatory cytokine release like IL-6, and TNF-α, as well as NO production. It displayed a significant inhibitory effect on the protein expressions such as iNOS, COX-2, NF-κB pathway like inhibitor of kappa B kinase (IKK)α/β, inhibitor of kappa B (IκB)-α and NF-κB/p65, PI3K/AKT pathway like PI3K and AKT, and MAPK pathway like c-Jun N-terminal kinase (JNK)1/2, extracellular signal-regulated kinase (ERK)1/2, and p38, in LPS-stimulated RAW264.7 macrophages. Moreover, the immunofluorescence results indicated that CPT suppressed NF-κB/p65 translocation from the cytoplasm into the nucleus. Further investigations showed that CPT treatment increased NAD(P)H quinone oxidoreductase-1 (NQO1) and heme oxygenase-1 (HO-1) expressions together with its upstream mediator, Nrf2. In addition, CPT inhibited LPS-induced toll-like receptor 4 (TLR4) and MyD88 expressions in RAW264.7 macrophages.
Collectively, we suggested that CPT exerted significant anti-inflammatory effects via modulating TLR4-MyD88/PI3K/Nrf2 and TLR4-MyD88/NF-κB/MAPK pathways.
隐丹参酮（CPT）作为丹参的主要成分，具有多种药理活性，包括抗炎作用。然而，CPT抗炎活性的确切细胞和分子机制仍有待阐明。本研究旨在阐明其对脂多糖（LPS）诱导的小鼠巨噬细胞RAW264.7细胞炎症反应的作用机制。
在本研究中，使用LPS刺激的RAW264.7细胞模型评估CPT的抗炎特性。MTT法用于测定RAW264.7细胞的活力。基于一氧化氮（NO）产生（Griess法和流式细胞术检测）、肿瘤坏死因子-α（TNF-α）和白细胞介素-6（IL-6）释放（ELISA）检测来测定CPT的抗炎作用。还通过蛋白质印迹法测定环氧合酶-2（COX-2）和诱导型一氧化氮合酶（iNOS）的酶表达。此外，通过流式细胞术，我们还评估了CPT对LPS诱导的钙内流的影响。最后，使用蛋白质印迹法研究CPT潜在的抗炎机制，以评估Toll样受体4（TLR4）、髓样分化因子8（MyD88）、磷脂酰肌醇3-激酶（PI3K）/蛋白激酶B（AKT）、核因子红细胞2相关因子2（Nrf2）、丝裂原活化蛋白激酶（MAPK）和核因子κB（NF-κB）信号通路的蛋白质水平。
我们的数据表明，CPT抑制LPS诱导的促炎细胞因子如IL-6、TNF-α的释放以及NO的产生。它对LPS刺激的RAW264.7巨噬细胞中的iNOS、COX-2、NF-κB信号通路（如κB激酶（IKK）α/β、κB抑制蛋白（IκB）-α和NF-κB/p65）、PI3K/AKT信号通路（如PI3K和AKT）以及MAPK信号通路（如c-Jun氨基末端激酶（JNK）1/2、细胞外信号调节激酶（ERK）1/2和p38）的蛋白质表达具有显著抑制作用。此外，免疫荧光结果表明CPT抑制NF-κB/p65从细胞质向细胞核的转位。进一步研究表明，CPT处理增加了NAD（P）H醌氧化还原酶-1（NQO1）和血红素加氧酶-1（HO-1）的表达及其上游介质Nrf2。此外，CPT抑制RAW264.7巨噬细胞中LPS诱导的Toll样受体4（TLR4）和MyD88表达。
总体而言，我们认为CPT通过调节TLR4-MyD88/PI3K/Nrf2和TLR4-MyD88/NF-κB/MAPK信号通路发挥显著的抗炎作用。
