基于miRNA-145调控系膜细胞自噬探讨丹酚酸B改善慢性肾炎的分子机制

It have been proved that salvianolic acid B(Sal B) had effect of improving chronic glomerulonephritis (CGN) and up-regulating the expression of miR-145 in glomerular mesangial cells. Further predicted by bioinformatic method，Rab 14, which was closely related to autophagy, may be miR-145 target genes. So, were there some relationships between autophagy and the mechanism of CGN improvied by Sal B? What’s more, we found that the proliferation of mesangial cell and the inflammatory transcription factors could be suppressed by miR-145 via regulating PI3K/AKT signaling. Thus, a brand new mechanism of improved CGN by salvianolic acid B were born based on the above researches. That is, by up-regulating miR-145, both Rab14 expression and PI3K/Akt/AS160 pathways which has an effect on the activation of Rab14 could be suppressed by salvianolic acid B. And then improving of CGN was accomplished on account of autophagy from mesangial cell which was controlled by the changes mentioned above. On the basic of our preliminary studies, to explore the inner relation of miR-145 and autophagy in the CGN treatment of Sal B, the rat model of CGN and human mesangial cell line were established and adenovirus transfection and western blot technique were carried out. The mechanism of CGN improved by Sal B is made clear through the perspectives of regulation of miRNA on autophagy, which developing new vision for the pathogenesis of CGN and providing new ideas for prevention and treatment of CGN.

我们已证明丹酚酸B可改善慢性肾炎(CGN)，上调肾系膜细胞miR-145表达，进一步生物信息学预测发现，与自噬密切相关的Rab 14可能为miR-145靶基因。那么，丹酚酸B改善CGN的机制是否与调控自噬有关？此外，我们还发现，miR-145可调控PI3K/AKT信号抑制肾系膜细胞增殖和炎性转录因子。由此，我们首次提出丹酚酸B改善CGN新机制，即：丹酚酸B通过上调miR-145，既可靶向抑制Rab14表达，又可调节PI3K/Akt/AS160通路抑制Rab 14在吞噬体膜聚集，继而促进肾系膜细胞的自噬来改善CGN。故本课题拟在前期研究基础上，建立CGN模型和人系膜细胞系，应用腺病毒转染、Western Blot等手段，旨在获得miR-145与自噬在丹酚酸B改善CGN中的内在联系。从miRNA调控自噬的角度，诠释丹酚酸B改善CGN的机制，以期为CGN的病机拓展新视野，为CGN防治提供新思路。

慢性肾小球肾炎（CGN）是由多种原因引起的免疫炎症性疾病，自噬、miRNAs广泛参与各类肾脏疾病发生、发展；因此，本研究从动物、细胞水平探讨CGN中丹酚酸B通过miR-145-5p调节肾脏自噬活动的情况，阐释miR-145-5p对系膜自噬活动的分子调控机制及丹酚酸B的影响。方法与结果：采用阳离子化小牛血清白蛋白（C-BSA）建立肾小球肾炎（CGN）大鼠动物模型，经尾静脉注射miR-145-5p antagomir后检测大鼠肾功能、肾脏病理、炎症因子以及肾脏自噬水平的变化情况；利用脂多糖刺激诱导人肾小球系膜细胞（HMC）炎性增殖模型，采用miR-145-5p抑制剂、模拟物进行干预，测定系膜细胞炎症、自噬及PI3K/AKT/AS160信号通路的变化情况。结果与结论：丹酚酸B可通过抑制肾脏免疫复合物沉积，减少肾脏炎性浸润，减轻C-BSA导致的病理损伤，保护CGN大鼠肾功能；丹酚酸B还可通过上调miR-145-5p的表达进而促进肾脏的自噬。利用miR-145-5p mimic或inhibitor、LY294002干预后的进一步研究表明，丹酚酸B可通过上调miR-145-5p，抑制PI3K/AKT信号，进而抑制AS160的磷酸化抑制Rab14蛋白的表达，从而促进肾脏自噬改善慢性肾炎预后，进一步揭示慢性肾炎中miR-145-5p调节自噬的具体调控分子机制，拓展了丹酚酸B改善CGN的机制。