GHSR-1a-AMPK/Parkin通路介导的线粒体稳态调控在Silybin改善糖尿病肾病中的作用研究

Diabetic nephropathy (DKD) is the most common microvascular complications of diabetes, the study of effective prevention and treatment of DKD drugs for reducing the mortality of diabetic patients is of great significance. Silybin can reduce urinary protein in diabetic patients, but the mechanism of action is unknown. We found that Silybin can improve kidney dyslipidemia in obese mice and stimulate mitochondrial biosynthesis. Further study found that: in the renal tubular epithelial cells of DKD rats, the expression of GHSR-1a, AMPK and Parkin were inhibited, Silybin treatment increased expression, mitochondrial autophagy and synthesis was activated. Accordingly, we propose the hypothesis that Silybin acts on the tubular GHSR-1a receptor, activates the AMPK / Parkin pathway, initiates the autophagy clearance of damaged mitochondrion, promotes biosynthesis, maintains the homeostasis of quantity and quality, thus restoring energy metabolism and improving renal function. In this study, we will use DKD animal models, cell injury model, combined with RNA interference, plasmid expression vector, Tag-lite detection, immunofluorescence, transmission electron microscopy and other techniques, taking mitochondrial homeostasis regulation as a new entry point to explore the mechanism of Silybin’s renal activity, which could lay the foundation for the clinical prevention and treatment of DKD.

糖尿病肾病（DKD）是糖尿病最常见的微血管并发症，研究有效防治DKD的药物对于降低糖尿病患者死亡率有重要意义。Silybin可降低DKD患者尿蛋白含量，但作用机制不明。我们发现，Silybin可改善肥胖小鼠肾脏脂质代谢异常，并刺激线粒体生物合成。进一步研究发现：在DKD大鼠肾小管组织中，GHSR-1a、AMPK、Parkin表达被抑制，Silybin治疗后表达均增加，线粒体自噬与合成被激活。据此，我们提出假说：Silybin作用于肾小管GHSR-1a受体，激活AMPK/Parkin通路，启动受损线粒体自噬清除、促进生物合成，维持数量与质量稳态，从而恢复能量代谢、改善肾脏功能。本研究拟应用DKD动物模型、细胞损伤模型，结合RNA干扰、质粒载体表达、Tag-lite检测、免疫荧光、透射电镜等技术，以线粒体稳态调控为新切入点，探讨Silybin肾脏活性的产生机制，为应用于临床防治DKD奠定基础。

糖尿病肾病（DKD）是糖尿病最常见的微血管并发症，研究有效防治DKD的药物对于降低糖尿病患者死亡率有重要意义。Silybin可降低DKD患者尿蛋白含量，但作用机制不明。前期研究发现，Silybin可改善肥胖小鼠肾脏脂质代谢异常。在DKD大鼠肾小管组织中，GHSR-1a、AMPK、Parkin表达被抑制，Silybin治疗后表达均增加，线粒体自噬与合成被激活。因此，本研究应用DKD动物模型、细胞损伤模型，结合RNA干扰、质粒载体表达、Tag-lite检测、免疫荧光、透射电镜等技术，以线粒体稳态调控为新切入点，旨在明确Silybin通过恢复线粒体稳态从而发挥对DKD的治疗作用及其关键分子机制。.. 本项目通过评估Silybin处理前后，DKD动物模型肾小管及体外培养HK2细胞中线粒体结构与功能变化，线粒体自噬强度与生物合成水平，从体内、体外两个层面明确了Silybin对肾小管细胞线粒体稳态的改善作用。此外，我们一方面利用CRISPR/Cas9 质粒敲除HK2细胞GHSR-1a受体，及应用Tag-lite技术进行活细胞表面GHSR-1a与Silybin的结合分析，证实了Silybin与GHSR-1a的直接结合作用。另一方面，采用GHSR-1a拮抗剂、AMPK抑制剂/激动剂、干扰RNA降低AMPK/Parkin 表达、质粒载体过表达Parkin等手段，调节GHSR-1a-AMPK/Parkin 通路活性，反复验证了上述信号通路在肾小管细胞线粒体稳态平衡的重要作用。.. 本项目的研究结果，为阐明Silybin对糖尿病肾损害的保护作用机制提供了全新的分子生物学视角，为Silybin能够进一步应用于DKD的临床治疗以及开发新的同类型药物提供了可靠的理论基础。