基于补气固摄理论探讨芪参益气滴丸对db/db小鼠蛋白尿的治疗作用

Proteinuria is the major clinical manifestation of diabetic nephropathy.Traditional Chinese medicine believes that proteinuria is associated with QiBuGuShe. Our preliminary data showed that the expression of NDUFA10, Sirt1, ZO1, claudin5 were significantly down regulated but caveolin-1 was up regulated in renal cortex of db/db mice while QishenYiQi pills, a compound Chinese medicine for BuQiGuShe, could block these changes. These results indicated that BuQiGuShe was related to energy metabolism, cell junction and transcytosis. The proposal will use QishenYiQi pills treated db/db mice to observe the dynamic of glomerular albumin filtration and tubular reabsorption as well as renal microcirculation by two-photon microscopy, observe glomerular and tubular ultrastructure by transmission electron microscopy, check mitochondrial function and energy metabolism by assay kits and immunoblotting, detect albumin filtration and reabsorption related junction proteins and transcytosis proteins by immunofluorescence and immunoblotting, and use in vitro cell model to study the relationship among mitochondrial function, cytoskeleton and albumin filtration or reabsorption. New information resulted from this study is expected to offer new insight into the connotation of QiBuGuShe in diabetic nephropathy and to clear the scientific hypothesis that QiShenYiQi pills regulate mitochondrial function, stabilize cytoskeleton and then affect the albumin filtration related junction proteins and albumin reabsorption related transcytosis proteins to play a BuQiGuShe role in db/db mice.

蛋白尿是糖尿病肾病的主要临床表现，中医认为与气不固摄有关。预实验结果发现db/db小鼠肾脏皮质线粒体相关蛋白NDUFA10及Sirt1和细胞连接蛋白ZO1及claudin5的表达明显下调，微囊蛋白caveolin-1明显上调，而补气固摄中药芪参益气滴丸可阻断这些变化，提示补气固摄与能量代谢、细胞连接和胞吞转运有关。本研究拟利用db/db小鼠模型用双光子显微镜观察白蛋白滤过重吸收动态和肾脏微循环状态；用透射电镜观察肾小球和肾小管超微结构的改变；用试剂盒和免疫印迹方法检测线粒体功能和能量代谢；用免疫荧光和免疫印迹方法观察白蛋白滤过和重吸收相关连接蛋白及转运蛋白的影响；用体外细胞模型研究线粒体功能、细胞骨架蛋白和白蛋白滤过重吸收的关系；探讨糖尿病肾病气不固摄机理并明确芪参益气滴丸调节线粒体功能，稳定细胞骨架，进而影响肾小球滤过相关细胞连接蛋白和肾小管白蛋白重吸收相关转运蛋白的补气固摄科学假说。

本项目的主要目标是明确芪参益气滴丸对糖尿病db/db小鼠蛋白尿的改善作用并以线粒体功能、肾小球白蛋白漏出和肾小管白蛋白重吸收为切入点，阐明糖尿病气不固摄的本质，探讨补气固摄疗法治疗糖尿病肾病蛋白尿的机制。根据项目研究计划我们对芪参益气滴丸对db/db小鼠蛋白尿的治疗作用进行了药效学评价，结果显示芪参益气滴丸对db/db小鼠蛋白尿无明显改善作用。在与预期结果不符的基础上，我们延长了药物干预时间，在db/db小鼠病程晚期我们发现芪参益气滴丸治疗可明显提高糖尿病晚期db/db小鼠生存率并延缓体重的下降。系统性病理分析显示芪参益气滴丸可明显保护db/db小鼠胸腺萎缩和胰岛细胞死亡。同时给药组小鼠血清TNFα和IL6水平相比模型组显著降低，提示免疫调节可能是芪参益气滴丸提高糖尿病晚期db/db小鼠生存率的主要原因。脂肪组织慢性炎症是糖尿病胰岛素抵抗的重要原因。与模型组相比，给药组小鼠脂肪组织M1型巨噬细胞浸润减少同时CD206、CD25和Foxp3等基因表达上调，提示芪参益气滴丸可上调脂肪组织Foxp3阳性调节性T淋巴细胞的表达并促进组织浸润巨噬细胞向M2型转化。蛋白印迹结果显示给药组小鼠脂肪组织p-JNK表达显著下调而p-Akt和p-GSK3β显著上调。这些结果提示芪参益气滴丸可能通过保护胸腺萎缩，上调Foxp3阳性调节性T淋巴细胞的表达，促进脂肪组织M2型巨噬细胞转化，减轻脂肪组织慢性炎症和胰岛素抵抗，从而改善糖尿病晚期恶病质和生存率。.此外，本项目需要验证的科学假说是补气中药通过调节肾脏线粒体功能而改善肾损伤和蛋白尿。尽管在db/db小鼠模型中我们未观察到芪参益气滴丸的肾损伤保护作用，但在顺铂诱导的急性肾损伤模型中，芪参益气滴丸可以明显保护肾损伤和线粒体功能。同时为明确线粒体自噬和肾损伤之间的关系，我们利用PINK1基因敲除大鼠急性肾损伤模型发现PINK1基因敲除可明显保护顺铂诱导的肾损伤，提示线粒体自噬的“双刃剑”作用。在线粒体过度自噬情况下，芪参益气滴丸对线粒体自噬的调节可能是其发挥肾保护作用机制之一。