Dihydrotanshinone exhibits an anti-inflammatory effect in vitro and in vivo through blocking TLR4 dimerization
Dihydrotanshinone exhibits an anti-inflammatory effect in vitro and in vivo through blocking TLR4 dimerization
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二氢丹参酮通过阻断 TLR4 二聚化在体外和体内表现出抗炎作用
DOI：
10.1016/j.phrs.2019.02.017
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发表时间：
2019-04-01
影响因子：
9.3
通讯作者：
Feng，Yu-Lin
中科院分区：
文献类型：
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作者：
Yuan，Renyikun;Huang，Liting;Feng，Yu-Lin
二氢丹参酮(DHT)是丹参的主要成分之一，具有多种生物活性。然而，双羟色胺的抗炎活性及其机制尚不清楚。本研究探讨了DHT的体内外抗炎作用及其分子机制。结果显示，双羟色胺显著降低脂多糖刺激的RAW264.7细胞、THP-1细胞和骨髓来源巨噬细胞的炎性细胞因子肿瘤坏死因子-α、白介素6和白介素1β的释放，并改变环氧合酶-2和诱导型一氧化氮合酶的表达。此外，流式细胞仪检测结果显示，DHT可降低脂多糖刺激的RAW264.7细胞的钙内流、活性氧(ROS)和一氧化氮(NO)的生成。此外，双羟色胺还可抑制核因子-κB(NF-κB)的转录，抑制核因子-κB蛋白的表达，抑制核转位，从而提示核因子-κB途径在双羟色胺的抗炎作用中起一定作用。此外，DHT通过干扰丝裂原活化蛋白激酶(MAPK)通路而减弱了内毒素激发的激活蛋白-1(AP-1)的活性。DHT与Toll样受体4(Toll-like Receptor 4，TLR4)的分子对接模拟表明，DHT与TLR4的活性部位结合，阻断TLR4的二聚化，细胞热位移实验和免疫共沉淀实验进一步证实了这一点。此外，TLR4二聚体的抑制还干扰了髓系分化主要反应基因88(MyD88)的募集和转化生长因子-b(TGF-b)激活的激酶1(p-TAK1)的表达。提示TLR4-MyD88-NF-κB/MAPK信号通路参与了双羟色胺的体外抗炎作用。在活体小鼠模型中，DHT能显著改善内毒素所致的急性肾损伤，抑制二甲苯所致的小鼠耳肿胀，并能挽救内毒素所致的小鼠脓毒症。综上所述，我们的结果表明DHT在体内外都具有显著的抗炎活性，提示DHT可能是一种潜在的炎症性疾病的治疗药物。
Dihydrotanshinone (DHT), one of the major ingredients ofSalvia miltiorrhizaBunge (Danshen), displays many bioactivities. However, the activity and underlying mechanism of DHT in anti-inflammation have not yet been elucidated. In this study, we investigated the anti-inflammatory activity and molecular mechanism of action of DHT bothin vitroandin vivo. Our data showed that DHT significantly decreased the release of inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1β in lipopolysaccharide (LPS)-stimulated RAW264.7 cells, THP-1 cells, and bone marrow-derived macrophages (BMDMs), and altered the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). In addition, flow cytometry results indicated that DHT reduced the calcium influx, and generation of reactive oxygen species (ROS), and nitric oxide (NO) generation in LPS-stimulated RAW264.7 cells. Moreover, DHT suppressed the transcription of nuclear factor-κB (NF-κB), the expressions of NF-κB proteins, and nuclear translocation of NF-κB/p65, thereby suggesting that the NF-κB pathway played a role in the anti-inflammatory action of DHT. In addition, DHT attenuated LPS-challenged activator protein-1 (AP-1) activity, resulting from interference of the mitogen-activated protein kinase (MAPK) pathway. The molecular docking simulation of DHT to toll-like receptor 4 (TLR4) suggested that DHT binds to the active sites of TLR4 to block TLR4 dimerization, which was further corroborated by cellular thermal shift assay and co-immunoprecipitation (Co-IP) experiments. Furthermore, the recruitment of myeloid differentiation primary response gene 88 (MyD88) and the expression of transforming growth factor-b (TGF-b)-activated kinase 1 (p-TAK1) were disturbed by the inhibition of TLR4 dimerization. Thus, investigating the molecular mechanism of DHT indicated that TLR4-MyD88-NF-κB/MAPK signaling cascades were involved in the anti-inflammatory activity of DHTin vitro. Inin vivomouse models, DHT significantly ameliorated LPS-challenged acute kidney injury, inhibited dimethylbenzene-induced mouse ear oedema, and rescued LPS-induced sepsis in mice. Taken together, our results indicated that DHT exhibited significant anti-inflammatory activity bothin vitroandin vivo, suggesting that DHT may be a potential therapeutic agent for inflammatory diseases.