p38MAPK介导的Nrf2/ARE通路和CC趋化因子在对比剂急性肾损伤中的作用及制大黄-川芎对其调控的研究

As a refractory disease, the main clinical feature of Contrast-induced nephropathy (CIN) is acute kidney injury. Renal tubular epithelial cell apoptosis induced by oxidative stress is its important pathogenesis. Nrf2/ARE pathway is so far the most important endogenous antioxidant pathway. Recent data suggests that up-regulation of Nrf2 signal will increase the antioxidant and anti-inflammatory defense, and thus may be a target of blocking acute kidney injury. We first reported p38MAPK/iNOS pathway is the critical pathway in mediating CIN apoptotic mechanism, and then we found the effective drug pair in treating renal failure, prepared Dahuang-Chuanxiong, can regulate this pathway and thus prevent CIN. But the potential downstream targets after the activation of this pathway in CIN is unclear. P38MAPK signal was discovered to have the potential to regulate Nrf2 signal and mediate the secretion of CC Chemokine. Based on these observations, we hypothesized that Nrf2/ARE and CC chemokine are the potential down-streams targets of the activated p38MAPK signal in CIN, as well as the potential downstream of prepared Dahuang-Chuanxiong to mediate their efficacy in prohibiting CIN kidney damage. In order to ascertain and verify this hypothesis, CIN models both in vitro and in vivo will be rebuilt, and exogenous activators or blockers will be applied. We also combine siRNA and other molecular biology techniques to test the expressions of the related signaling molecules, and detect oxidative stress and apoptosis in renal epithelial cells. Meanwhile, the expressions of CC chemokine and their receptors should be measured. The successful completion of this project will provide new molecular mechanisms and new target in CIN prevention,especially the prevention with Chinese herbs.

难治性疾病CIN(对比剂肾病)主要表现为急性肾损伤，氧化应激诱使的肾小管上皮细胞凋亡是其重要发病机制。Nrf2/ARE是迄今发现最重要的内源性抗氧化通路。新近资料认为上调Nrf2能增加抗氧化及抗炎防御从而可能是阻断急性肾损伤的靶标。我们在国际上首先报道p38MAPK/iNOS是CIN凋亡机制的关键通路，后发现治疗肾衰竭经验药对制大黄-川芎能调控该通路防治CIN，但该通路激活后的下游作用途径还不清楚。p38MAPK被发现能调控Nrf2信号及介导CC趋化因子分泌。故推测"Nrf2/ARE、CC趋化因子是CIN中p38MAPK激活后作用的下游途径，也是制大黄-川芎疗效机制的关键下游途径"。建立CIN体内外模型，应用外源性激活物或阻断剂诱导、siRNA等技术，并通过对氧化应激、细胞凋亡、相关信号分子表达、CC趋化因子及其受体表达等检测以验证假说，为CIN分子机制及中医药防治提供新思路和新靶点。

本项目针对发病机制不清、临床难治的疾病——对比剂肾病（CIN）的中医药防治途径以及CIN发生的分子机制开展了探索性的理论及基础研究。率先对CIN的中医病机进行了归纳，拟定了以制大黄-川芎药对为主的解毒化瘀中医治法，并立足于肾小管上皮细胞凋亡研究了调控凋亡的信号通路机制及制大黄-川芎的作用机制。本项目率先发现并报道 CIN 中调控p38MAPK/iNOS 通路的上游信号是Trx/ASK1，从而为CI-AKI 的发病机制提供了一条更为完整的信号通路机制，即： ROS-Trx/ASK1-p38MAPK-iNOS-FoxO1-Bcl2 family-Caspase；另外，还进一步发现 CIN 中肾小管上皮细胞凋亡的调控机制中有Nrf2/Kep1通路和炎症信号CCL2/CCR2的参与，并且该通路接受p38MAPK信号的调控；外源性失活Nrf2进一步加重对比剂（CM）诱使的肾小管上皮细胞凋亡；我们还进一步揭示了制大黄-川芎药对解毒化瘀防治 CIN的分子机制是通过调控p38 MAPK通路进而调控Nrf2/Keap1通路和CCL2/CCR2炎症信号。上述发现为我们提出的CIN中医主要病机是“毒瘀互结”、以及倡导以制大黄-川芎为主的“解毒化瘀”中医治法提供了坚实的实验室基础。同时，为了进一步获得AKI 标志物在中国人各类人群中的临床应用价值，项目组新增了对健康人、AKI 患者、慢性肾脏病(CKD)患者中AKI 标志物的研究内容，结果提示AKI 标志物临床检测的价值不仅仅局限于诊断AKI，还具有其他的生物学意义。.研究成果已被多个国际同行引用和证实，并在国内多家医院进行了推广应用。已发表论文SCI论文4篇（影响因子合计13.47分）、国内核心期刊论文8篇，引用的著名期刊包括molecular therapy、Archives of toxicology等。以第一完成人获得上海市科技进步奖三等奖、中华中医药学会科学技术奖三等奖、第七届上海中医药科技奖成果推广奖、第五届上海中医药科技奖二等奖各1次；第一负责人入选人才项目3项，包括第四批全国中医（临床）优秀人才培养项目、2017年上海市卫计委“优秀学科带头人”培养项目、上海市“浦江人才”培养项目；相关成果在中华医学会肾脏病学分会2014年全国学术年会进行壁报交流。共培养4名硕士研究生毕业。