AOPP-RAGE轴诱导EMT发生的机制及复方黄甘干预靶点的研究

Advanced oxidation protein products (AOPPs) as a new kind of uremic toxin, is rising in the chronic kidney disease (CKD) patients along with the deterioration of renal function. Studies have shown that AOPPs can induce the epithelial-mesenchymal transition (EMT), and develop into renal fibrosis, which are the common pathological features and the ending of CKDs caused by various pathogenesis. Therefore, how to reverse, suppress, or slow down the process of renal fibrosis are the focus and difficulty for many scholars research. Our previous studies showed that in different CKD models, AOPPs were significantly increased, and the expression of advanced glycosylation end product-specific receptor (RAGE) and EMT marker protein α-SMA were abnormally increased at the same time, along with the activation of Wnt/β-catenin signaling pathway. And our patent medicine Huang Gan Formula particles could inhibit this process, but the specific mechanism was unclear. Based on these, our study adopts to using CRISPR-Cas9 gene knockout technology to explore whether AOPPs can induce EMT through RAGE- Wnt/β-catenin signaling pathway, and to discuss the function of AGER1 in this process, and to find the target for Compound Huang Gan through the intervention of Compound Huang Gan drug-containing serum, so as to provide new theoretical basis for the treatment of CKD.

晚期氧化蛋白产物(AOPP)是一种新型尿毒毒素，在慢性肾脏病（CKD）患者体内随着肾功能的恶化而持续升高。研究表明，AOPPs可诱导上皮间质转化（EMT），促使肾纤维化，而肾纤维化是各种病因所致CKD的共同病理特征和最终结局。因此，如何延缓、抑制或是逆转肾纤维化过程，是广大学者研究的焦点与难点。我们前期研究表明：在不同CKD模型动物体内，AOPPs均显著升高，且同时伴随晚期糖基化终产物受体（RAGE）与EMT标志蛋白α-SMA表达异常增加，以及Wnt/β-catenin信号通路激活，而我们的专利药复方黄甘颗粒可抑制这一过程，但具体机制不明。基于此，本研究预采用CRISPR-Cas9基因敲除技术来探究AOPP是否通过RAGE-Wnt/β-catenin信号轴诱导EMT发生，以及AGER1在此过程中的调节作用，并通过复方黄甘含药血清的干预寻求其作用靶点，以期为CKD的治疗提供新的理论依据。

晚期氧化蛋白产物(AOPP)是一种新型尿毒毒素，可诱导上皮间质转化（EMT），促使肾纤维化，而肾纤维化是各种病因所致CKD的共同病理特征和最终结局。我们前期研究表明：在不同CKD模型动物体内，AOPPs均显著升高，且同时伴随晚期糖基化终产物受体（RAGE）与EMT标志蛋白α-SMA表达异常增加，以及Wnt/β-catenin信号通路激活，而我们的专利药复方黄甘颗粒可抑制这一过程，但具体机制不明。基于此，我们采用western blot和RT-PCR技术，证明AOPP处理HKC-8细胞后，可显着诱导EMT标志蛋白α-SMA表达和抑制E-cadherin表达，并上调Wnt1，β-catenin，Tcf4和Gsk-3β的表达。而通过CRISPR-Cas9基因敲除技术构建RAGE敲除的HKC-8细胞系后，这一过程被显著抑制。然而，我们继续通过siRNA沉默AGER1基因表达，又显著增强了这些作用。这些结果表明，AOPP可通过激活RAGE-Wnt /β-catenin信号传导途径诱导HKC-8细胞EMT，而AGER1可能促进了这一过程的发生。当复方黄甘不同剂量作用于HKC-8的EMT模型后，我们通过western blot和RT-PCR技术研究发现，复方黄甘可显著抑制Wnt/β-catenin通路相关mRNA和蛋白的表达，与Wnt/β-catenin通路特异性性抑制剂ICG-001组相比，无显著性差异。在共聚焦下观察细胞免疫荧光染色，可直观发现复方黄甘可抑制EMT标志性蛋白α-SMA和wnt通路β-catenin蛋白的表达。这些结果可能为CKD发生发展中的EMT提供新的理论基础，并为复方黄甘的临床应用提供了新的理论依据。