基于HIF/JMJD1A/MALAT1信号通路探讨肾康丸干预糖尿病肾病内皮细胞损伤的机制研究

Renal glomerular endothelial cell (RGEC) injury accelerates the progression of diabetic nephropathy (DN). Hypoxia is an important reason for RGEC injury. Our previous works found that hypoxia can upregulate HIF-1α expression and enhance RGEC inflammatory injury, which did not pass through the NF-κB signal pathway. Furthermore, previous gene microarray data demonstrated that the expression of lncRNA MALAT1 increased in RGEC stimulated by high glucose. During the process of MALAT1-mediated inflammatory cascade induced by HIF-1α, histone demethylase JMJD1A might be targeted to modulated by HIF-1α. Therefore, We hypothesize that HIF-1α activates JMJD1A, which regulates MALAT1 expression and induces RGEC inflammatory damage. There has no relative research reported in this field. We will adopt JMJD1A knockout mouse model and gene silencing cell model to study the role of HIF/JMJD1A/ MALAT1 signaling pathway in pathogenesis of DN by Co-IP, CHIP, Western-Blot and qRT-PCR. We will administrate Shen-Kang pill for DN mouse, which has good effect for improving DN in human, to demonstrate whether Shen-Kang pill alleviate RGEC injury via HIF / JMJD1A / MALAT1 signaling pathway. We expect that our research can provide scientific evidences to explain the treatment mechanism and target of Traditional Chinese Medicine in diabetic nephropathy.

肾小球内皮细胞（RGEC）损伤是糖尿病肾病（DN）发生发展的重要机制，缺氧是RGEC损伤的重要环节。我们前期研究发现，缺氧可上调HIF-1α表达，增强RGEC炎性损伤，其并非通过NF-κB通路实现。进一步通过基因芯片筛选,发现lncRNA MALAT1在高糖刺激下RGEC中表达增高，HIF-1α可诱导增强MALAT1介导的炎症级联反应，此过程可能通过靶向调控组蛋白去甲基化酶JMJD1A而实现。据此我们提出假说：HIF-1α可激活JMJD1A，进而调控MALAT1表达，诱导发生RGEC炎症损伤。目前此方面研究未见报道。本项目拟通过利用JMJD1A敲除小鼠模型及基因沉默细胞模型，采用Co-IP、CHIP、Western-Blot、qRT-PCR等方法，探讨HIF/JMJD1A/MALAT1信号通路在RGEC损伤中的作用，以及肾康丸对该通路的影响，为研究中药治疗DN的机制和靶点提供科学证据。

糖尿病肾病是糖尿病患者最常见、最严重的微血管并发症之一。糖尿病微血管病变机制复杂，其中血管内皮损伤是其发生发展的重要机制，而缺氧是内皮损伤的重要环节。临床试验证明肾康丸能显著减少DN患者尿蛋白，有效的延缓DN进展，但具体作用机制尚不清楚，有待进一步研究。本课题组利用STZ诱导单侧肾切除DN小鼠和JMJD1A基因敲除小鼠作为体内实验模型，缺氧高糖条件下诱导的人脐静脉内皮细胞HUVECs作为体外模型，给予肾康丸干预，通过分析HIF-1a/JMJD1A通路基因和蛋白水平、氧化应激指标ROS、MDA及SOD水平等变化，染色质免疫共沉淀（Chromatin Immunoprecipitation Assey, ChIP）技术与荧光素酶报告探索HIF-1α/JMJD1A之间的作用方式及结合位点，揭示了肾康丸通过HIF-1a/JMJD1A途径改善DN的机制。以上研究结果显示，体内实验中肾康丸组可以减轻肾组织中肾小球肥大、系膜扩张以及炎症浸润和纤维化损伤的病理变化，下调HIF/JMJD1A信号通路及炎症因子TNF-α、IL-1β、IL-6、MCP-1、ICAM-1 mRNA的基因与蛋白表达水平。在体外缺氧高糖条件下培养HUVECs，HIF-1α表达增加而上调JMJD1A、IL-6、IL-8、ICAM-1、MCP-1基因与蛋白水平表达，增加ROS及MDA的产生，降低SOD活力（P<0.05）；下调HIF-1α水平能够降低缺氧高糖诱导的JMJD1A和炎症因子的分泌以及ROS及MDA水平，增加SOD活力，缓解炎性及氧化应激损伤。进一步实验表明沉默JMJD1A能够改善HUVECs炎性损伤及氧化应激水平，利用Ch-IP实验与荧光素酶报告证实HIF-1α能够直接结合JMJD1A的启动子区启动下游转录。以上结果表明肾康丸可以减缓缺氧高糖下HUVECs炎性损伤及氧化应激水平，其作用与HIF-1α/JMJD1A的表达水平下调相关。本研究阐明了肾康丸通过HIF/JMJD1A信号通路与DN发病的相关性以及作用机制，为肾康丸防治糖尿病肾病的临床应用提供了科学依据。