参附注射液扶阳固脱调控核内高迁移率族蛋白B1与NF-κB结合的分子机制研究

Shenfu Injection with the function of rescuing Yang has certain curative effect in treating septic shock, which is an exogenous syncope syndrome in Chinese medicine. However, its pharmacological mechanism still needs further investigating. Inflammatory cascade reaction with NF-κB is the key mechanism of septic shock. Stimulated by endotoxin, HMGB1 with the function of protection in the nucleus may split cells which are lethal toxic factors at terminal sepsis. Previous studies have showed that Shenfu Injection could inhibit HMGB1 to run out of the nucleus, express highly in the nucleus, and inhibit activation of NF-kappa B (NF-κB). Meanwhile, HMGB1 could combine with the members of NF-κB family during the inflammatory response process. Therefore, in this project the hypothesis that Shenfu Injection may regulate the combination of HMGB1 and NF-κB in the nucleus and inhibit the transcription of inflammatory cytokines was put forward. The promoter of NF- kappa B domain and the promoter of NF-κB were constructed to study the effect of Shenfu Injection on the activity of the promoter and the intervention on HMGB1, and verified the protein binding with Mammalian two hybrid experiment and Biacore protein interaction apparatus and constructed over-expression of HMGB 1 cell line. Biochemical technologies such as Small interfering RNA (siRNA), Co-Immunoprecipitation (co-IP), Real-time reverse transcription polymerase chain reaction (RT-PCR) and western blot (WB) were applied to investigate the combination of HMGB1 and NF-κB, and the regulating effect of Shenfu Injection. This project studied the molecular mechanism of rescue-yang theory of Shenfu, which provided a theoretical basis for clinical treatment and a supplement for the molecular mechanisms of inflammation.

脓毒症休克属中医外感厥脱证,用扶阳固脱参附液疗效确切，但药理机制有待深入研究。以NF-κB为核心的炎症因子级联反应是脓毒症休克的关键机制。在核内起保护作用的HMGB1，经内毒素刺激分泌出胞为脓毒症晚期致死性毒性因子。前期研究发现参附液抑制HMGB1出核，高表达于核内，同时抑制NF-κB的活化，且两种蛋白有结合。因此提出“参附液通过调控HMGB 1与NF-κB核内结合，抑制炎症因子的转录”的假说。拟构建NF-κB结构域启动子及NF-κB启动子，研究参附液对启动子活性的影响及HMGB1干预作用；用哺乳动物双杂交实验及biacore蛋白质互作仪验证蛋白结合情况；构建过表达HMGB1细胞株，经siRNA技术、co-IP、qRT-PCR、western blot等方法探求HMGB1和NF-κB的结合情况及参附的调控作用。本项目探索中医扶阳固脱分子机制，为临床治法提供理论依据，也是炎症分子机制的补充。

内毒素休克属中医外感“厥脱”证，用扶阳固脱参附液疗效确切，但药理机制有待深入研究。项目在前期研究的基础上提出“参附液通过调控HMGB1与NF-κB核内结合，抑制炎症因子的转录”的假说，并通过体内外实验对假说进行验证。.项目基本按照计划书进行，实验结果如下：（1）SFI可显著抑制LPS诱导的小鼠巨噬细胞（RAW264.7细胞）中HMGB1核外迁移及炎症反应。（2）SFI对NF-κB结构域启动子及P50、P65、IL-1β启动子无抑制作用。（3）Tet-on-HMGB1+细胞（过表达HMGB1的RAW264.7细胞）对LPS的刺激产生了耐受现象，需将LPS的剂量提升到1μg/mL才能诱导HMGB1出核；SFI能抑制Tet-on-HMGB1+细胞中HMGB1的核移位。（4）哺乳动物双杂交实验证实SFI可抑制TNFα诱导的293T细胞中HMGB1与P65及P50的结合率，同时抑制P65与P50的结合。（5）CO-IP实验验证HMGB1、P65、P50在RAW264.7细胞中存在两两结合；在LPS诱导下，HMGB1与P65和P50结合率均降低，而P65与P50结合率升高，SFI可逆转LPS的上述诱导效应。Tet-on-HMGB1+细胞中，HMGB1与P65和P50结合比例与野生型细胞无明显差异，但LPS与参附注射液共同作用时，Tet-on-HMGB1+细胞中HMGB1与P65、HMGB1与P50的结合率较野生型细胞中显著升高。（6）SFI可显著抑制LPS诱导的RAW264.7细胞的吞噬作用。（7）动物体内实验发现SFI减少内毒素休克大鼠死亡率；升高平均动脉压；减轻肺组织损伤；抑制肺组织HMGB1转录、表达及核转位；抑制NF-κB信号通路关键蛋白的磷酸化。.综上所述，项目研究得出以下结论：SFI注射液的抗炎作用与NF-κB结构域启动子及P50、P65、IL-1β启动子无关；核内高表达HMGB1可导致RAW264.7细胞对LPS产生耐受；SFI通过上调HMGB1与P65、HMGB1与P50的结合，抑制P65-P50二聚体形成，从而抑制炎症因子的转录。.本项目从HMGB1与NF-κB蛋白互作的角度深入研究了SFI抗炎的作用机制，达到了预期研究目标。已发表论文5篇，其中SCI论文1篇，3篇为第一作者，1篇通讯作者,1篇为第二作者。培养硕士生3名（均为在读）。