丹参中甘西鼠尾新酮A通过干扰LPS与TLR4/MD2的结合发挥抗炎作用

Neoprzewaquinone A from Salvia miltiorrhiza Bunge exerts anti-inflammatory activity by disrupting LPS binding to TLR4/MD2

研究丹参中是否存在能与Toll样受体4/髓样分化蛋白2(Toll like receptor 4/myeloid differentiation protein 2,TLR4/MD2)结合发挥抗炎活性的化合物。本文以TLR4/MD2为靶蛋白虚拟筛选了丹参已报道的化学成分,并检测了筛选出的甘西鼠尾新酮A(neoprzewaquinone A,Neo A)对脂多糖(lipopolysaccharide,LPS)与细胞膜上受体结合的影响、与蛋白的亲和力作用、对LPS介导细胞中TLR4与MD2二聚化的影响、对LPS所致细胞核转录因子-κB(nuclear factor-κB,NF-κB)p65蛋白磷酸化及细胞炎性因子分泌的影响。研究结果表明,丹参中Neo A与TLR4/MD2虚拟结合能最高,为−12.8 kcal·mol^(-1);Neo A可极显著抑制LPS与细胞膜上的受体结合(P<0.01),且Neo A能与rhTLR4/MD2、rhTLR4、r hMD2结合,亲和力分别为267、534、228 nmol·L^(-1);Neo A与TLR4/MD2的氨基酸残基TYR131、PHE121等位点可能存在烷基及π-烷基疏水作用;Neo A能极显著抑制LPS介导细胞中TLR4与MD2的二聚化(P<0.01),显著抑制NF-κBp65蛋白的磷酸化水平(P<0.05);Neo A能显著或极显著抑制LPS所致细胞一氧化氮(nitric oxide,NO)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-6(interleukin-6,IL-6)和白细胞介素-1β(interleukin-1β,IL-1β)的分泌(P<0.05,P<0.01)。综上,Neo A可通过与TLR4/MD2结合干扰LPS与TLR4/MD2结合发挥抗炎作用,可能是一种TLR4/MD2抑制剂,可用于TLR4/MD2为靶点的炎症相关疾病治疗。

This study investigates whether compounds in Salvia miltiorrhiza Bunge can bind to the Toll like receptor 4/myeloid differentiation protein 2(TLR4/MD2)protein complex and exhibit anti-inflammatory activity.Virtual screening of reported chemical components of Salvia miltiorrhiza Bunge against TLR4/MD2 was conducted in this study.The selected compound,neoprzewaquinone A(Neo A),was tested for its impact on the binding of lipopolysaccharide(LPS)to receptors on the cell membrane,its affinity for the protein,its influence on the dimerization of TLR4 and MD2 in LPS-induced cells,and its effects on the phosphorylation of nuclear factor-κB(NF-κB)p65 protein and the secretion of inflammatory cytokines in cells.Results indicate that Neo A in Salvia miltiorrhiza Bunge exhibited the highest virtual binding affinity with TLR4/MD2,with a value of−12.8 kcal·mol^(-1).Neo A significantly inhibited the binding of LPS to receptors on the cell membrane(P<0.01).Moreover,Neo A demonstrated affinity for rhTLR4/MD2,rhTLR4,and rhMD2,with KD values of 267,534,and 228 nmol·L^(-1),respectively.Amino acid residues like TYR131 and PHE121 in TLR4/MD2 might play a role in the alkyl andπ-alkyl hydrophobic interactions with Neo A.Neo A also significantly inhibited the dimerization of TLR4 and MD2 in LPS-mediated cells(P<0.01)and markedly suppressed the phosphorylation of NF-κBp65 protein(P<0.05).Furthermore,Neo A significantly or markedly inhibited the secretion of nitric oxide(NO),tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and interleukin-1β(IL-1β)in LPS-induced cells(P<0.05,P<0.01).In conclusion,Neo A exerts its anti-inflammatory effects by binding TLR4/MD2 then disrupting the binding of LPS to TLR4/MD2.It may serve as a TLR4/MD2 inhibitor with the potential to treat inflammation-related diseases targeting TLR4/MD2.