锌转运体与锌离子转运调控自噬影响肾间质纤维化的机制和虫草益肾方的保护作用

Renal interstitial fibrosis is decreased kidney function in patients with kidney disease, a major cause of its pathogenesis is unclear.Autophagy in renal fibrosis can relieve renal tubular atrophy and degradation of the ECM deposition protection.Zinc is a trace elements necessary for human body, zinc deficiency can increase the fibrosis.At the same time, a growing number of studies have shown that zinc concentration can improve the level of autophagy, zinc deficiency can inhibit autophagy.Autophagy of zinc in kidney disease and there is no report of the role of research.The applicant to participate in the kingdom of nature on the project, the study of differences associated with fibrosis of PI3K protein - Akt pathway and the relationship between autophagy has been a large number of reports, but the result also shows that after UUO Znt7 was found in the kidney tissues, Zfp512 expression;Chinese caterpillar fungus prognosis of kidney Fang Gan Znt1 expression.So we speculated that caterpillar fungus kidney may by influencing the transporter of zinc and zinc, and zinc finger protein expression, regulating cell autophagy level so as to delay the role of renal interstitial fibrosis.This study as a continuation of the project, national natural fund surface fitting by in vivo and in vitro experiment, dynamic observing autophagy changes, to further study the mechanism of renal interstitial fibrosis and Chinese caterpillar fungus kidney delay the mechanisms of renal fibrosis, to provide the effective prevention and control of new theory and new drug targets.

肾间质纤维化（RIF）贯穿多种肾脏疾病的全过程，其发病机制尚不明确。在RIF中自噬可减轻肾小管的萎缩和降解细胞内过多的胶原纤维沉积起到肾保护作用。虫草益肾方治疗慢性肾衰竭临床应用20余年，前期诸多实验证实虫草益肾方可延缓RIF进展。其中蛋白质组学研究提示其疗效与PI3k/Akt通路相关，其下游分子mTOR是公认的自噬抑制剂；研究显示UUO大鼠肾中锌转运体7、锌指蛋白512上调，虫草益肾方干预后锌转运体1上调。有报道锌可以调控自噬。因此我们推测虫草益肾方可能通过上调锌转运体1，从而调节锌离子在细胞内外的水平和分布以及减少锌指蛋白512的合成，促进细胞自噬达到延缓RIF的作用。本研究拟建立UUO大鼠动物模型以及观察高糖诱导下肾小管上皮细胞，动态研究锌转运蛋白、锌离子、锌指蛋白和自噬的变化，进一步探讨RIF机制及虫草益肾方延缓RIF的机制，为其有效防治提供新理论和药物新机制。

肾间质纤维化（RIF）贯穿多种肾脏疾病的全过程，其发病机制尚不明确。在RIF中自噬可减轻肾小管的萎缩和降解细胞内过多的胶原纤维沉积起到肾保护作用。虫草益肾方应用于慢性肾衰竭的治疗20余年，前期诸多实验证实虫草益肾方可延缓RIF进展。蛋白质组学研究提示其疗效与PI3k/Akt通路相关，其下游分子mTOR是公认的自噬抑制剂；并提示UUO大鼠肾中锌转运体7、锌指蛋白512上调，虫草益肾方干预后锌转运体1上调。有报道锌可以调控自噬，结合网络药理学研究，发现调节自噬可能是虫草益肾方的作用机制之一。因此我们推测虫草益肾方可能通过上调锌转运体1，从而调节锌离子在细胞内外的水平和分布，促进细胞自噬达到延缓RIF的作用。本研究首先通过建立UUO大鼠动物模型，发现虫草益肾方可显著改善RIF大鼠一般状态，增加体重和尿量；虫草益肾方可降低RIF大鼠尿蛋白，改善肾脏功能，并可增强细胞自噬，调节肾小管上皮细胞转分化标志蛋白α-SMA、E-cadherin表达，从而抑制肾小管上皮细胞转分化，减轻肾纤维化病理损伤；虫草益肾方可降低RIF大鼠血清锌水平，提高肾组织中锌含量；虫草益肾方干预可以显著纠正RIF大鼠血清中TNF-α、IL-1β、IL-6、IL-10、IL-12等炎症因子与MDA、SOD、还原型GSH等氧化损伤相关分子水平的异常；虫草益肾方可减少RIF大鼠肾组织Col-I、Col-Ⅲ、FN等细胞外基质成分表达水平，增强LC3B、LC3II/I、Atg5、Atg7等自噬相关蛋白表达，调节锌转运体蛋白（Znt1、Znt7）表达；提示虫草益肾方可能通过增强细胞自噬，调节锌及锌转运体减少细胞外基质积聚，最终发挥延缓RIF的作用。在进一步的体外实验中，我们发现虫草益肾方可减轻高糖诱导的肾小管上皮细胞细胞外基质积聚及肾小管上皮细胞转分化；虫草益肾方可增强高糖诱导下的肾小管上皮细胞LC3B、Atg5表达，从而增强自噬，其作用与锌转运体相关。本研究进一步揭示RIF的作用机制和潜在药物治疗靶点，具有极大的应用潜力和广阔的前景。同时，进一步阐明中药复方防治肾间质纤维化的机制，对揭示中药复方复杂药效的作用机制提供了新思路。