Table2_The interaction of MD-2 with small molecules in huanglian jiedu decoction play a critical role in the treatment of sepsis.XLSX

Huanglian Jiedu Decoction (HJD) is used for treating sepsis in China. Active components from HJD refer to various active ingredients of HJD, while active component formulation (ACF) refers to the combination of palmatine, berberine, baicalin, and geniposide from HJD according to the quantity of HJD. The detailed mechanisms of the active components from HJD and ACF in sepsis treatment are unclear. Molecular docking, surface plasmon resonance (SPR), ELISA, RT-qPCR, and Western blotting were used to assay the possible mechanism in vitro. The efficacy and mechanism of ACF and HJD were assessed by pharmacodynamics and metabolomics analyses, respectively. The results revealed that palmatine, berberine, baicalin, and geniposide showed good binding capacity to MD-2; decreased the release of NO, TNF-α, IL-6, and IL-1β; inhibited the mRNA expression of iNOS, TNF-α, IL-6, IL-1β, and COX-2; and downregulated the protein expressions of MD-2, MyD88, p-p65, and iNOS induced by LPS; which indicated that they can inactivate the LPS-TLR4/MD-2-NF-κB pathway. Thus, ACF was formed, and the pharmacodynamics assay suggested that ACF can reduce inflammatory cell infiltration and organ damage in accordance with HJD. Furthermore, 39 metabolites were selected and identified and the regulatory effect of these metabolites by ACF and HJD was almost consistent, but ACF might alleviate physical damage caused by HJD through regulating metabolites, such as 3-hydroxyanthranilic acid. ACF could represent HJD as a new formulation to treat sepsis.

黄连解毒汤（Huanglian Jiedu Decoction，HJD）在我国常用于脓毒症的临床治疗。其中，HJD活性成分指该方剂中的各类有效单体组分；而活性组分配伍（active component formulation，ACF）则是依据HJD的原方投料量，选取其中巴马汀、小檗碱、黄芩苷与京尼平苷组合而成的制剂。目前，HJD活性成分及ACF治疗脓毒症的具体分子机制尚未明确。本研究通过分子对接、表面等离子体共振（surface plasmon resonance，SPR）、酶联免疫吸附实验（enzyme-linked immunosorbent assay，ELISA）、实时定量聚合酶链反应（reverse transcription quantitative polymerase chain reaction，RT-qPCR）以及蛋白质印迹（Western blotting）等技术，体外探究二者潜在的作用通路。同时分别采用药效学与代谢组学分析手段，评估ACF与HJD的疗效及作用机制。
实验结果显示：巴马汀、小檗碱、黄芩苷及京尼平苷可与MD-2蛋白产生良好结合能力；能够抑制脂多糖（lipopolysaccharide，LPS）诱导的一氧化氮（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）及白细胞介素-1β（interleukin-1β，IL-1β）的释放；下调诱导型一氧化氮合酶（inducible nitric oxide synthase，iNOS）、TNF-α、IL-6、IL-1β及环氧合酶-2（cyclooxygenase-2，COX-2）的mRNA转录水平；同时抑制LPS诱导的MD-2、髓系分化因子88（myeloid differentiation primary response 88，MyD88）、磷酸化p65（p-p65）及iNOS的蛋白表达。上述结果表明，该四类活性组分可有效阻断LPS-Toll样受体4（Toll-like receptor 4，TLR4）/MD-2-核因子κB（nuclear factor kappa-B，NF-κB）信号通路。
据此构建了ACF制剂，药效学实验证实其可如HJD一般，显著减轻炎症细胞浸润与器官损伤。此外，本研究共筛选并鉴定出39种代谢物，ACF与HJD对上述代谢物的调控效果基本一致；且ACF可通过调控3-羟基邻氨基苯甲酸（3-hydroxyanthranilic acid）等代谢物水平，缓解HJD可能带来的机体损伤。综上，ACF可作为HJD的新型替代制剂，用于脓毒症的治疗。