中国科学院昆明动物研究所机构知识库

固醇调控元件结合蛋白（SREBPs）是激活参与胆固醇，脂肪酸，三酰甘油等生物合成相关基因表达的转录因子。SREBP信号通路的异常表达会导致体内脂质稳态平衡发生改变，造成多种脂质代谢综合征，例如肥胖，动脉粥样硬化等疾病。目前，基于SREBP信号通路的脂质代谢药物种类多种多样，其中使用最为广泛的是他啶类药物，其作用机理主要是抑制甲基戊二酰辅酶A还原酶。除此之外，多种活性分子也展现出降脂潜力。有研究表明，天然分子化合物RA-V具有调控SREBP信号通路的功能。该研究发现，RA-V能够抑制显著下调抑制胆固醇合成通路绝大多数酶的表达，改变胆固醇分布等。基于此，我们对RA-V调控SREBP信号通路进行深入研究，探索其具体的作用机理。我们应用线虫模型进行实验，结果显示RA-V具有抑制SREBP-1a入核的效应。使用RA-V对HUH-7细胞进行处理，结果显示核型SREBP-1a的总量显著下降。基于上述结果，我们对RA-V调控SREBP-1a蛋白的机制进行了研究。首先，我们对S1P,S2P酶的活性经行检测，发现RA-V对二者没有影响。随后，在对内质网进行激光共聚焦拍照后，发现RA-V明显破坏内质网的完整性。随后应用透射电镜对内质网进行观察，发现低浓度RA-V会破坏内质网扁平囊状结构，造成内质网出现肿胀，并且核糖体分布出现混乱；而在高浓度时，内质网结构完全破坏，核糖体处于弥散分布状态。根据相关报道，Sec-23蛋白有作为靶蛋白的可能，但通过Western Blot实验并没有发现Sec-23蛋白总量出现变化。根据已有的证据，我们设定RA-V可能的靶蛋白，构建了其质粒，通过氚标RA-VII进行靶点验证。我们通过替代实验以及毒性实验证明了氚标探针的正确性。通过转染质粒，我们进行了时间梯度，浓度梯度实验，分别得到其结合平衡时间以及饱和浓度等参数。通过不同的质粒表达时间，我们发现设定的可疑靶蛋白都没有出现与探针特异性的结合。除此外，通过相应文献报道，eEF2蛋白具有成为RA-V靶蛋白的可能。我们通过小分子相互作用实验发现，无论是单扣除比较还是特异性结合分析，都没有发现eEF2与RA-V存在特异性结合证据。本次研究证实，RA-V能够降低核型SREBP-1a的总量，减少SREBP-1a的入核，并且对内质网完整性具有破坏作用。在靶点验证中，我们证实了氚标探针的正确，并且完成对可疑靶蛋白的验证。在后续的试验中，小鼠模型试验以及其他靶蛋白的验证将是下一阶段重要的工作，这将为RA-V作为潜在降脂药物提供有力证据。

Sterol regulatory element-binding proteins (SREBPs) are major transcription factors activating the expression of genes involved in biosynthesis of cholesterol, fatty acid and triglyceride. Abnormal expression of SREBP signaling pathway can lead to changes in lipid homeostasis in the body, resulting in a variety of lipid metabolic syndrome, such as obesity, atherosclerosis and other diseases. At present, there are many kinds of drugs for treating lipid metabolism based on SREBP signaling pathway. The most widely used is statins, and the mechanism of action is mainly to inhibit the HMG-CoA reductase. In addition, a variety of active molecules also show the potential of lipid-lowering.The research had shown that the natural molecular compound RA-V has the function of regulating SREBP signaling pathway. it was found that RA-V could inhibit the expression of most enzymes in the cholesterol synthesis pathway and change the cholesterol distribution. Based on this, we study the regulation of SREBP signaling pathway by RA-V and explore its mechanism.We used the model of elegans to test RA-V and the results showed that RA-V has the effect of inhibiting SREBP-1a entring into the nucleus. Treated HUH-7 cells with RA-V, and the results showed that the amount of nSREBP-1a decreased significantly. Based on the above results, we investigated the mechanism of RA-V regulating SREBP-1a protein. We tested the activity of S1P, S2P, and found that RA-V has no effect on the activity of S1P, S2P. Subsequently, by means of laser confocal microscopy to test the endoplasmic reticulum, RA-V was found to significantly damage the integrity of the endoplasmic reticulum. Then we used transmission electron microscopy to test the endoplasmic reticulum, and we found that low concentrations of RA-V destroyed the endoplasmic reticulum flattened vesicles, and caused swelling of endoplasmic reticulum, and destroyed the distribution of ribosome; while at higher concentration, the structure of the endoplasmic reticulum was completely destroyed, and ribosome dispersively distributed. According to the relevant reports, we viewed Sec-23 protein as a possible target. However, we found that no change appeared in the total Sec-23 protein by Western Blot.According to the available evidence, we set up the possible target protein of RA-V. We constructed the plasmid, and verified the target by tritium labeled RA-VII. We have proved the correctness of the tritium labeled probe by substitution test and toxicity test. By means of the transfection of the plasmid, we performed the time gradient and concentration gradient test to obtain the equilibrium time and the saturation concentration. Through the different plasmid expression time, we found that these possible target protein did not appear to bind with the probe specifically. In addition, we find the eEF2 protein has the potential to be a target protein of RA-V by reading the relevant literature. We tested eEF2 by small molecule interaction, and the results showed that there was no evidence that eEF2 and RA-V had specific binding by single deduction comparison or specific binding assay.This study confirmed that RA-V can reduce the total amount of SREBP-1a, inhibite SREBP-1a entring into the nucleus, and damage the integrity of endoplasmic reticulum. In the validation of the target, we confirmed the correctness of the tritium labeled probe and the verification of the possible target protein. In subsequent trials, mouse model testing and validation of other target proteins will be the next phase of the work, which will provide strong evidence for RA-V as a potential lipid-lowering drugs.