四甲基吡嗪通过激活NRF2/CLDN4通路改善肠道屏障功能抑制绝经后骨质疏松的机制研究

Abnormal immune response resulting from estrogen deficiency disturbs the RANKL-RANK-OPG axis, and this had been demonstrated as a key mechanism for the pathogenesis of postmenopausal osteoporosis (PMO). Most of research in this field have focused on the direct effects of estrogen on immune cells. Recent studies have shown that the intestinal barrier is involved in immune regulation, and estrogen deficiency can disrupt the intestinal barrier. Our previous study found that tetramethylpyrazine (TMP), an extract of Ligusticum chuanxiong Hort., can improve osteoporosis in OVX mice, and its mechanism is related to TMP improving intestinal barrier function and reducing IL-17, TNFα and other osteoclastogenesis inflammatory factors. Further studies showed that TMP could increase the expression of CLDN4 in intestinal epithelium of OVX mice, and bioinformatic analysis indicated that CLDN is a potential target molecule of NRF2. Combined with literature and preliminary experiments, we propose a hypothesis that TMP can promote the protective effect of PMO by activating NRF2 to promote CLDN4 expression and improve intestinal barrier function. We will use a combination of knockout mice and molecular biology experiments to validate this hypothesis in both phenotypic and molecular levels. This study will deepen the understanding of the pathogenesis of PMO and provide a theoretical basis for the development of relevant treatments.

雌激素缺乏导致异常免疫反应，进而扰乱RANKL-RANK-OPG轴是绝经后骨质疏松(PMO)的重要发病机制。目前相关研究均集中于雌激素对免疫细胞的直接作用。近来的研究显示肠道屏障参与机体免疫调节，而雌激素缺乏可以破坏肠道屏障。我们前期的研究发现川芎提取物四甲基吡嗪(TMP)可以改善OVX小鼠骨质疏松表现，其机制与TMP改善肠道屏障功能，降低体内IL17、TNFα等促破骨细胞炎症因子相关，进一步的研究显示TMP可以提高OVX小鼠肠道上皮CLDN4表达，生物信息分析提示CLDN是NRF2潜在靶分子。结合文献和预实验我们提出TMP通过激活NRF2促进CLDN4表达改善肠道屏障功能降低机体异常炎症反应对PMO发挥保护作用的研究假说。我们将综合应用基因敲除小鼠以及分子生物学实验等手段，从表型和分子机制等方面验证该假说。该课题的开展将加深对PMO发病机制的理解，并为研发相关治疗手段提供理论基础。

雌激素缺乏导致异常免疫反应进而扰乱RANKL-RANK-OPG轴是绝经后骨质疏松（PMO）的重要发病机制。目前相关研究主要集中于雌激素对免疫细胞的直接作用。近来的研究显示肠道屏障参与机体免疫调节，而雌激素缺乏可以破坏肠道屏障。结合文献和预实验我们提出四甲基吡嗪（TMP）通过激活NRF2促进肠道细胞间连接蛋白Claudin 4（CLDN4）表达改善肠道屏障功能，降低机体异常炎症反应从而对PMO发挥保护作用的研究假说。针对这一假说，我们采用CLDN4敲除小鼠去卵巢（OVX）模型、NRF2敲除小鼠OVX模型、FITC-Dextran、免疫组化等一系列技术方法验证了以下研究结果：1.TMP能够降低OVX小鼠促破骨细胞炎症因子水平，抑制OVX小鼠体内破骨细胞生成，对PMO发挥保护作用；2.TMP通过提高OVX小鼠肠道上皮CLDN4表达从而改善肠道屏障功能，而CLDN4敲除后，TMP无法改善雌激素缺乏导致的破骨细胞活性升高及骨量下降；3. TMP通过NRF2介导发挥对CLDN4的调控作用，NRF2是TMP改善PMO的作用靶点。通过上述研究我们阐明了TMP通过在肠道细胞中激活NRF2/CLDN4通路改善绝经后雌激素缺乏所致肠道屏障功能障碍，进而降低体内促破骨细胞炎症反应发挥对PMO保护作用的分子机制。本课题的成果加深了对PMO发病机制的理解，并为研发其治疗手段提供了理论基础，具有一定的转化及应用前景。