Toll样受体7抑制剂的高通量筛选模型建立及应用

Establishment and application of a high-throughput screening model for Toll-like receptor 7 inhibitors

目的:针对狼疮性肾炎(LN)的重要靶点Toll样受体7(TLR7),构建高通量筛选(HTS)体系从天然产物库中筛选TLR7抑制剂,为LN新药研发提供线索。方法:基于稳定表达人TLR7(hTLR7)基因的HEK-Blue^(TM)hTLR7细胞系构建HTS体系,从活性天然产物库中进行TLR7抑制剂筛选。分离并原代培养MRL/lpr狼疮小鼠脾脏单个核细胞,给予葫芦素B(CuB)处理,通过western blot和qPCR检测TLR7下游信号分子及炎症因子表达情况。结果:对TLR7配体瑞喹莫德(R848)及阳性药羟氯喹(HCQ)的最佳工作浓度进行优化后构建HTS体系,经初筛和次筛发现CuB可显著抑制TLR7活性且对细胞活力影响较小。体外实验证实CuB可抑制TLR7下游信号分子MyD88、p-p65表达,下调炎症因子白细胞介素6(IL-6)、干扰素γ(IFN-γ)的分泌以及I型干扰素签名基因(ISGs)表达。结论:本研究成功建立了TLR7抑制剂的HTS模型并筛选获得新TLR7抑制剂CuB,为治疗LN提供了先导化合物。

Objective:This study aimed to construct a high-throughput screening system to screen from natural product libraries targeting Toll-like receptor 7(TLR7), an important target of lupus nephritis(LN). Methodology:The screening system was constructed based on the HEK-Blue^(TM)hTLR7 cell line stably expressing hTLR7 and SEAP genes, the optimal working concentrations of TLR7 ligand resiquimod(R848) and positive drug hydroxychloroquine(HCQ) were optimized. And active natural product libraries were screened for TLR7 inhibitors in the primary and secondary screening.Results:Our results showed that cucurbitacin B(CuB) could significantly inhibit TLR7 activity with little effect on cell viability. Then the spleen mononuclear cells of MRL/lpr lupus mice were isolated and cultured in vitro and treated with CuB. The expressions of TLR7 downstream signaling molecules and inflammatory factors were detected by western blot and qPCR. In vitro experiments confirmed that CuB can inhibit the expression of TLR7 downstream signaling molecules MyD88 and p-p65, the secretion of inflammatory factors interleukin 6(IL-6)、interferon γ(IFN-γ) and the expression of type I interferon signature genes(ISGs). Conclusion:In summary, this study successfully established a high-throughput screening model with TLR7 as the target and a new TLR7 inhibitor CuB was screened, which provided a lead compound for the treatment of LN.