从BNIP3和PINK1/Parkin介导的线粒体自噬和线粒体质量控制研究对比剂急性肾损伤的发病机制及“制大黄-川芎”药对的肾保护机制

As a refractory disease, the main clinical feature of Contrast-induced nephropathy (CIN) is acute kidney injury. Recent data show that mitophagy and mitochondrial quality control (MQC) may be a new target for AKI prevention and treatment. We have first reported that p38MAPK is the critical pathway of mediating CIN apoptotic mechanism, and the following signals, FoxO1, Mcl-1 and Nrf2, also were involved in the pathogenesis of CIN and acted as the downstream of p38MAPK. Furthermore, we found the effective drug pair in treating renal failure, prepared Dahuang-Chuanxiong, can regulate this pathway and thus prevent CIN. Recently, we have discovered that coexistence of apoptosis and autophagy in CIN, mitophagy and MQC are involved in CIN pathogenesis, but it is not clear by which intracellular pathways regulate mitophagy and MQC in CIN. There are several signaling pathways that can mediate the mitophagy in the body. In these pathways, BNIP3 was found to be a downstream target of FoxO family, PINKl could be activated by FoxO1 and Nrf2, and Parkin could bind with Mcl-1 directly. Thus, we hypothesized that “the mitophagy in CIN is mediated by BNIP3 and PINK1/Parkin pathway, and the renoprotective mechanism of prepared Dahuang-Chuanxiong against CIN is by modulating the mitophagy mediated by BINP3 and PINK1/Parkin as well as MQC”. In order to ascertain and verify this hypothesis, CIN models both in vitro and in vivo will be rebuilt, and exogenous blockers, siRNA, and Laser confocal microscope will be applied. We also combine other molecular biology techniques to test the expressions of the related signaling molecules, and detect apoptosis and autophagy, mitophagy and mitochondrial potential, mitochondrial fission and fusion in renal epithelial cells. The successful completion of this project will provide new molecular mechanisms and new target in CIN prevention, especially the prevention with Chinese herbs.

CIN(对比剂肾病)表现为急性肾损伤(AKI)，线粒体自噬和线粒体质量控制（MQC）是防治AKI新靶点。我们曾率先报道p38MAPK是CIN分子机制关键通路，其下游信号有FoxO1、Mcl-1、Nrf2等，制大黄川芎药对经调控该通路能防治CIN。我们新发现肾小管上皮细胞线粒体自噬和MQC参与了CIN病变，但其胞内调控机制未知。在被报道能介导线粒体自噬的途径中，BNIP3被发现是FoxO1下游靶点，PINK1可被FoxO1、Nrf2激活，Parkin能特异性结合Mcl-1，推测“CIN中线粒体自噬由BNIP3、PINK1/Parkin介导，对该线粒体自噬途径和MQC的调控是制大黄川芎肾保护的机制”。建立大鼠和细胞模型，应用siRNA、抑制物诱导、激光共聚焦等技术，检测肾小管上皮细胞自噬、线粒体自噬、线粒体膜电位、MQC相关蛋白及信号分子表达等验证假说，阐释本病发病机制及药对肾保护的科学内涵。

CIN(对比剂肾病)表现为急性肾损伤(AKI)，也被称为CI-AKI，是临床上AKI常见病因，其发病机制仍未完全清楚，并且有效的防治药物缺乏。前期研究证实具有解毒化瘀功效的制大黄-川芎药对能有效防治CI-AKI，我们在本项目中着眼BNIP3和PINK1/Parkin对线粒体自噬和MQC的调控来进一步探究该药对防治CI-AKI的机制。本研究建立CI-AKI大鼠和细胞模型，发现肾小管上皮细胞线粒体自噬和MQC确实是该肾损伤的重要机制，表现为模型组(CI-AKI)与正常组(Con)相比，肾小管上皮细胞凋亡增多的同时，伴随肾小管上皮细胞自噬和线粒体自噬增多，自噬相关基因LC3 II/I、Becline等表达上调；同时，线粒体融合标志蛋白表达降低（Mfn2等）、线粒体分裂标志蛋白表达上调（Drp1等）。制大黄-川芎药对（CI-AKI+DCH）及其有效成分TMP在有效降低模型大鼠（CI-AKI）Scr、BUN、AKI标志物（如尿NAG、IL-18、γ-GT等）的同时，肾小管上皮细胞自噬、线粒体自噬均有效下调。另外与正常组相比，模型组存在BNIP3和PINK1/Parkin通路激活，分别阻断BNIP3和PINK1加重了对比剂（CM）诱使的肾小管上皮细胞损伤。与CI-AKI组相比，制大黄-川芎药对组（CI-AKI+DCH）肾组织BNIP3和PINK1/Parkin通路相关信号（如BNIP3、FoxO1、PINK1、Parkin、Mcl-1等）表达下调。立足大鼠模型，我们还探索了CI-AKI肾外器官损伤的可能性及CI-AKI炎症机制。我们发现CM在诱使AKI同时，能诱使小肠损伤和小肠绒毛毛细血管内皮细胞凋亡；制大黄-川芎药对（CI-AKI+DCH）在保护肾功能的同时，有效抑制了CM诱使的小肠损伤和小肠绒毛毛细胞内皮细胞凋亡；模型鼠肾组织和血清存在炎症因子（IL-6、TNF-α等）分泌增加，但被制大黄-川芎抑制。综合起来，本研究除证实BNIP3和PINK1/Parkin介导的线粒体自噬和MQC是CI-AKI的重要发病机制外，还为CI-AKI为多系统损伤性疾病提供佐证，同时制大黄-川芎是通过调控BNIP3和PINK1/Parkin介导的线粒体自噬和MQC发挥肾保护效应，对CI-AKI肾外累及的器官如小肠有良好保护效应，并抑制CM诱使的炎症因子分泌。上述发现非常值得进一步深入研究。