黄芩苷促进少突胶质前体细胞分化及髓鞘再生的PPARγ/NFIL3信号转导机制

Demyelination is the common pathological features of several neurological disease. Oligodendrocyte precursor cell (OPC) differentiation is the cytological basis and therapeutic targets of remyelination. In the previous study, we reported that baicalin is effective in promoting myelin repair in an NFIL3-dependent manner, while the function of both baicalin and NFIL3 in myelination is unknown. Given the activation effect of baicalin on peroxisome proliferator-activated receptor gamma (PPARγ), which can bind multiple peroxisome proliferator elements (PPRE) in the promoter sequences of NFIL3, we thus hypothesize that the therapeutic effect of baicalin on remyelination is mediated by PPARγ/NFIL3 signal pathway. In this proposal, we will firstly elucidate the regulation mechanism and target genes underlying the induction of PPARγ/NFIL3 signal pathway by baicalin, using dual luciferase reporter assay, CHIP-PCR, site-directed mutagenesis and functional blocking/rescue assays. Then we will test the direct effects of baicalin on OPC differentiation and remyelination, and the role of NFIL3 in this process, based on the in vitro and in vivo demyelinating models. From the therapeutic angle, we will test the NFIL3-mediated therapeutic effects of baicalin on the chronic stage of chronic-progressive (CP) and relapsing-remitting (RR) EAE. This project, if successful, will provide findings confirming baicalin’s potential as a novel therapy for demyelinating disease and will elucidate the mechanisms underlying its action.

脱髓鞘性损伤是多种神经系统疾病共有的病理特征，少突胶质前体细胞(OPC)分化是髓鞘再生的细胞基础和治疗靶点。我们前期研究发现黄芩苷通过激活转录因子NFIL3有效促进髓鞘修复，但黄芩苷和NFIL3在OPC分化中的作用及信号转导机制未知。由于黄芩苷可靶向激活核受体PPARγ，而NFIL3启动子区有PPAR的结合位点，我们推测，黄芩苷通过调控PPARγ/NFIL3信号通路促进OPC分化。本项目拟采用萤光素酶报告基因法、CHIP-PCR、点突变和功能拮抗/恢复实验，明确黄芩苷对PPARγ/NFIL3的靶向调控机制；进而通过NFIL3过表达/缺失/条件型降低的脑片培养及脱髓鞘模型，揭示黄芩苷依赖NFIL3促进髓鞘再生的直接作用；最后利用CP-和RR-EAE模型，系统评估黄芩苷及其调控的NFIL3对脱髓鞘疾病的治疗效果。研究结果可为髓鞘损伤修复提供有效的候选药物(黄芩苷)和新的治疗靶点(NFIL3)。

脱髓鞘性损伤是多发性硬化症、脑白质营养不良、脊髓侧索硬化、阿尔兹海默症、帕金森、脑卒中、脑小血管病等多种中枢神经系统疾病共有的病理特征，少突胶质前体细胞(OPC)分化是髓鞘再生的细胞基础和治疗靶点。我们前期研究发现黄芩苷对髓鞘形成细胞OPC的发育具有调控作用，能有效促进髓鞘再生。但是，疾病严重程度的降低和对髓鞘的保护可能是黄芩苷免疫调节作用的次级效应，黄芩苷能否直接影响髓鞘形成或促进髓鞘再生尚不清楚；黄芩苷发挥作用的靶点和分子机制还并不明确。因此，本项目首先利用体内、体外OPC分化和髓鞘生成/再生模型，系统评估了黄芩苷在促进OPC分化和髓鞘生成/再生中的作用；然后基于计算机辅助模拟技术预测并确证了黄芩苷的直接作用靶点——PPARγ；进一步通过PPARγ缺失、选择性激活等方法，在多种脱髓鞘模型中明确了PPARγ调控OPC发育和髓鞘再生的功能，揭示了黄芩苷依赖PPARγ促进髓鞘再生的直接作用；最后结合RNA-seq和ChIP-seq分析，构建了OPC在黄芩苷刺激下的PPARγ靶基因谱及其调控网络，证实了PPARγ作为黄芩苷刺激的信号传递中心，靶向调控包括NFIL3在内的多个OPC及髓鞘发育相关基因的表达。研究结果为髓鞘损伤修复提供有效的候选药物（黄芩苷）和新的治疗靶点（PPARγ）。