壮药龙钻通痹方调控类风湿性关节炎HMGB1- TLR4/ RANKL-NF-κB 信号通路的时序性研究

According to our previous studies, Zhuang medicine Longzuantongbi Formula was clinically effective in treating rheumatoid arthritis (RA) by regulating the level of IL-4, IFN-γ, MMP-13 and other factors. For the purpose of learning whether the treatment of RA by the formula shows a trend that effect values change with time in terms of improvement of RA's common symptoms and whether the underlying mechanism is related to HMGB1- TLR4/ RANKL -NF-κB signaling pathways, this project aims to find out the change in anti-inflammation effect with time from onset of action and peak effect values by using RA rat models which are to be subject to different doses of Longzuantongbi Formula, different administration time and observation of appearance manifestation and pathological morphology; this project also explore the mechanism for the formula intervening the interaction between HMGB1-TLR4 and RANKL-NF-κB signals in RA by detecting the expression variation of HMGB1-TLR4/RANKL-NF-κB and downstream proteins such as IL-6, IL-1β and c-Fos in the synovium, cartilaginous tissue, plasma and culture medium of RA rats using Western Blot, ELISA and similar technologies; the ultrastructure of osteoclast was observed under electron microscope. This project tries to disclose the best time for starting treatment of RA and thus offers a scientific basis for effectively preventing and treating RA.

课题前期研究发现：壮药龙钻通痹方治疗RA临床疗效肯定，可通过调控IL-4、IFN-γ、MMP-13等因子水平而发挥抗炎作用，为了探讨该方在治疗中对是否存在效应值的时序变化规律，其机制是否与HMGB1- TLR4/ RANKL -NF-κB信号通路相关？由此，本项目以RA大鼠模型为研究对象，以不同剂量龙钻通痹方，以不同给药时段为观察点，通过外观表征及病理形态学观察相结合的药效学观测，从起效时间、效应峰值探寻其抗炎时序变化规律；应用Western Blot、ELISA等技术与方法，检测RA大鼠滑膜、软骨组织、血浆及其培养液中HMGB1-TLR4/RANKL-NF-κB通路及下游IL-6、IL-1β及c-Fos等蛋白表达变化，电镜观察破骨细胞超微结构，探讨该方对RA HMGB1-TLR4/ RANKL-NF-κB信号交互作用的干预机制，力图揭示该方最佳治疗时机，为RA的有效防治提供科学依据。

本项目以RA大鼠模型为研究对象，以不同剂量壮药龙钻通痹方，以不同给药时段为观察点，通过外观表征及病理形态学观察相结合的药效学观测，从起效时间、效应峰值探寻其抗炎时序变化规律；应用Western Blot、ELISA等技术与方法，检测RA大鼠滑膜、软骨组织、血清及其培养液中HMGB1-TLR4/RANKL-NF-κB通路及下游IL-6、IL-1β等蛋白表达变化，证实了壮药龙钻通痹方对RA的治疗机制是通过抑制炎性细胞因子IL-1β、IL-6、TNF-α等因子的释放，减少炎症反应；抑制HMGB1-TLR4-NF- κB信号通路的表达，下调RANK、RANKL等蛋白质表达水平，减少RANKL与其受体RANK的结合，抑制NF-κB的激活，进一步减少下游炎症因子的释放，揭示了疗效、HMGB1-TLR4/ RANKL-NF-κB通路调控与用药时间和用药浓度呈正相关。. 同时以C57BL/6小鼠破骨细胞为研究对象，以壮药龙钻通痹方含药血清干预后，通过细胞形态学观察，应用ELISA、 RT-PCR、免疫荧光、Western Blot等技术与方法，检查破骨细胞的TLR4、MYD88、TNF-α、NFAT-c1、c-Fos的表达，证实了壮药龙钻通痹方能升高破骨细胞凋亡率，降低破骨细胞活性，能降低破骨细胞TLR4、MYD88蛋白表达量，能下调破骨细胞TNF-α、NFAT-c1、c-Fos基因的表达，说明壮药龙钻通痹方对破骨细胞HMGB1-TLR4/RANKL-NF-κB信号通路关键因子具有明显的调控作用，进而提示该方能阻断TLR4/NF-kB信号转导网络导致破骨细胞活性降低，并调控TNF-α、NFAT-c1、c-Fos表达影响破骨细胞的分化。. 综上所述，揭示了该方对RA的最佳治疗时机，证明了该方对RA的 HMGB1-TLR4/ RANKL-NF-κB信号交互作用与时序性变化的机制，为进一步深入研究奠定了基础。