EPHX2基因分型与糖肾方治疗糖尿病肾病疗效相关性研究及其机制探讨

Recent studies have shown that the levels of EETs play an important role in the inflammatory response of diabetic nephropathy (DN). The sEH protein encoded by EPHX2 gene is a key enzyme in the metabolism of EETs. Previous studies have found that the change of EPHX2 rs751141 polymorphism led to decreased sEH enzyme activity and related to the occurrence and progression of DN. The research group developed an Chinese herbal medicine, Tangshen Formula, which can significantly reduce the level of DN expression in rat model of sEH and improve the renal micro-inflammation. Clinical studies found that TSF exhibited the renoprotective effect on DN patients with overt proteinuria, however, there is heterogeneity in clinical efficacy. We suggest that the EPHX2 rs751141 polymorphism may be associated with the treatment response to DN in patients. This study combined with the previous multicenter clinical trial of drug efficacy analysis of blood samples of patients with DN EPHX2 rs751141 polymorphism, and the monitoring of EETs and inflammatory factor levels before and after treatment; and at the cellular level, to explore the mechanism of Tangshen Formula on different genotypes by using plasmid transfection and the other methods. Therefore, we will clarify the relationship between the EPHX2 genotype and the effect of the treatment of diabetic nephropathy and the molecular mechanism of the difference between the different types of EPHX2, and the study will further provide the scientific basis for the individualized treatment of diabetic nephropathy by Chinese herbal medicine Tangshen Formula.

EETs水平在糖尿病肾病（DN）炎症反应中起着重要作用。EPHX2基因编码的sEH蛋白是代谢EETs的关键水解酶。申请人前期研究发现改变sEH酶活性的EPHX2 rs751141多态性与DN疾病的发生和进展有关。课题组研发的中药糖肾方可显著下调DN模型大鼠sEH表达水平，改善肾脏的微炎症状态。临床研究发现糖肾方对显性蛋白尿DN患者肾脏具有保护作用，然而临床疗效存在异质性。我们认为rs751141多态性可能与DN患者对糖肾方的治疗反应有关。本研究拟结合前期多中心临床试验DN患者血液标本进行EPHX2 rs751141多态性分析，并对治疗前后EETs及炎症因子水平进行监测；进而在细胞水平上，采用质粒转染等技术探讨糖肾方对不同基因型的作用机制。从而阐明EPHX2基因型与糖肾方疗效相关性以及糖肾方对EPHX2不同基因型疗效差异的分子机制，为糖肾方个体化治疗DN提供科学依据。

EETs水平在糖尿病肾病（Diabetic nephropathy, DN）炎症反应中起着重要作用。EPHX2基因编码的sEH蛋白是代谢EETs的关键水解酶。课题组研发的复方中药制剂糖肾方可显著下调DN模型大鼠sEH表达水平，改善肾脏的微炎症状态。临床研究发现糖肾方对显性蛋白尿DN患者肾脏具有保护作用，然而临床疗效存在异质性。我们认为EPHX2 rs751141多态性可能与DN患者对糖肾方的治疗反应有关。本研究拟结合前期多中心临床试验药物疗效不同的DN患者血液标本EPHX2 rs751141多态性进行分析，并对治疗前后EET水平进行监测；进而在细胞水平上，采用质粒转染、real-time PCR、Western blot等技术探讨糖肾方对不同基因型的作用机制。从而阐明EPHX2基因型与糖肾方疗效相关性以及糖肾方治疗EPHX2不同基因型的机制，为复方中药治疗DN提供科学依据。申请人前期研究工作发现改变sEH酶活性的EPHX2 rs751141多态性与DN疾病的发生和进展有关。在人体研究发现，糖肾方可以明显改善机体的某些代谢物水平，体内研究发现经过不同浓度的糖肾方干预后，与未刺激组相比萤光素酶活性未见明显变化，表明在正常条件下糖肾方不能对此人EPHX2启动子片段的活性产生影响。当前的实验表明中药糖肾方对糖尿病肾病的治疗作用可能不是通过直接上调sEH启动子片段实现的。糖肾方研究发现：DKD患者血清同型半胱氨酸（Hcy）水平明显升高，糖肾方用药前、用药3个月、6个月的血液样本中随着用药时间增加DN患者体内Hcy水平明显降低。同时我们初步发现：高Hcy水平可以上调HK2细胞sEH mRNA的表达。因此，糖肾方可能通过其所降低的中间介质Hcy 抑制sEH表达。在体外细胞实验中，突变型R287Q重组质粒相对于野生型WT具有较低的11,12-EET水解能力。突变型R287Q相对于野生型WT具有较低的炎症水平，外源性11,12-EET可以降低WT组炎症水平。A等位基因保护作用可能通过调控PI3K/AKT信号通路参与DKD的分子机制。本研究推动了DN 药物新靶标的发现及中药复方治疗DN生物学内涵的阐释。