肾炎防衰液通过激活AMPK通路调控线粒体稳态对抗糖尿病肾病足细胞损伤的机制研究

The imbalance of accumulation and elimination of “shenluozhenjia” is the core pathogenesis of podocyte injury in diabetic nephropathy (DN), and the imbalance of accumulation and elimination of mitochondrial homeostasis is an important internal factors in podocyte injury. So the hypothesis “Shen-Yan-Fang-Shuai formula can restore mitochondrial homeostasis by promoting mitochondrial biogenesis and mitophagy through regulating AMPK pathway, thereby protect podocytes against injury in DN” is proposed. In this study, first, by interfering with Shen-Yan-Fang-Shuai formula and its disassembled prescriptions in rats with DN (induced by unilateral nephrectomy, plus high fat diet, and STZ injection), we will investigate whether the podocyte protection effect of Shen-Yan-Fang-Shuai formula has a relationship with AMPK pathway activation, mitochondrial biogenesis promotion and mitophagy increase. Then, in high glucose-induced podocytes in vitro, siRNA will be used to silence the gene that makes AMPK to explore whether the serum containing Shen-Yan-Fang-Shuai formula and its disassembled prescriptions promote mitochondrial biogenesis and mitophagy by activating AMPK pathway. Ultimately, the pathway of mitochondrial biogenesis and mitophagy will be blocked by silencing the gene that makes PGC-1α and ULK1,respectively, to study whether Shen-Yan-Fang-Shuai formula protect podocytes by promoting mitochondrial homeostasis. It is expected to further clarify the material basis and scientific connotation of “shenluozhenjia” from the perspective of the balance between vital energy and pathogenic factor in our study.

肾络癥瘕聚散消长失衡是糖尿病肾病（DN）足细胞损伤的核心病机，线粒体稳态聚散失衡是DN足细胞损伤的重要内因，据此提出假说“肾炎防衰液通过激活AMPK通路，促进新生线粒体生物合成、增加受损线粒体自噬降解，恢复线粒体稳态，从而对抗DN足细胞损伤”。本研究首先借单侧肾摘除+高脂+STZ诱导的DN大鼠模型，以肾炎防衰液全方及其聚、散拆方进行干预，探讨其对抗足细胞损伤是否与激活AMPK通路、促进线粒体生物合成及增加线粒体自噬有关；进而借高糖诱导的足细胞模型，以siRNA沉默AMPK基因，探讨该方及拆方含药血清是否通过激活AMPK通路促进线粒体生物合成、增加线粒体自噬；最后以siRNA分别沉默PGC-1α（阻断线粒体生物合成通路）及ULK1基因（阻断线粒体自噬通路），探讨肾炎防衰液是否通过调控线粒体稳态对抗足细胞损伤。本研究有望从正邪聚散平衡角度进一步阐明DN“肾络癥瘕”的物质基础和科学内涵。

足细胞损伤是糖尿病肾病（DN）发生发展的关键病理环节，基于前期研究肾炎防衰液能够有效降低DN患者的尿蛋白，延缓肾功能进展，本研究从调节线粒体聚散失衡入手探讨肾炎防衰液改善DN足细胞损伤的作用机制。研究首先以自发2型糖尿病db/db小鼠为研究模型，应用肾炎防衰液进行干预，发现其可以显著减少db/db小鼠尿白蛋白肌酐比（UACR），改善其肾脏病理损伤，恢复足细胞标志蛋白nephrin、podocin的表达，下调凋亡蛋白cleaved caspase3表达，验证肾炎防衰液可以有效改善DN足细胞损伤；并通过检测肾组织pAMPKa、AMPKa的表达，线粒体生物合成指标如线粒体DNA（mtDNA）及PGC-1a、TFAM等，线粒体自噬相关蛋白如ULK1、LC3II等，明确肾炎防衰液改善DN足细胞损伤可能与其改善AMPKa磷酸化，促进线粒体生物合成及线粒体自噬有关。体外实验使用高糖培养的小鼠肾足细胞MPC5建立足细胞损伤模型，应用肾炎防衰液含药血清干预，同样发现肾炎防衰液含药血清可以有效改善DN足细胞损伤，减少足细胞凋亡，同时增加足细胞AMPK磷酸化，改善线粒体功能，增加mtDNA，恢复PGC-1 a、NRF-1、TFAM等线粒体生物合成蛋白及ULK1、LC3II等线粒体自噬诱导蛋白的表达。最后在体外实验中，以siRNA沉默AMPKa基因，发现基因沉默后，肾炎防衰液含药血清减轻足细胞凋亡的作用减弱，且促进线粒体生物合成相关蛋白的效果减低，但对线粒体自噬蛋白的表达无显著影响。本研究的科学意义在于揭示了肾炎防衰液可以通过调控AMPKa改善线粒体生物合成从而减轻足细胞损伤，其改善足细胞损伤也可能与促进线粒体自噬有关，但具体机制仍需要进一步探讨。