MAML1基因和NLRP3炎症小体对临床剂量三价砷肾毒性中炎症信号的细胞内调控及川黄方对其干预的研究

With the increasing environmental pollution and trivalent arsenic clinical anti-tumor applications, arsenic induced acute kidney injury (AKI) is becoming a new serious medical problem. Mastermind-like1 (MAML1) gene could regulate multiple inflammatory signal pathways to determine cell fate, NLRP3 inflammasome might involve in AKI incidence. We first reported the clinical dose of trivalent arsenic might induce human renal tubular epithelial cell apoptosis and autophagy, which is accompanied by the activations of inflammatory signals, including COX2, beta-catenin, TNF-α, p38 MAPK and NF-κB, etc., but the intracellular regulatory mechanisms of regulating these inflammatory signals remain unclear. Chuan Huang Decoction has been confirmed to inhibit inflammation and renal tubular epithelial cell apoptosis, prevent AKI and arsenic nephrotoxicity. Speculate "MAML1 gene and NLRP3 is the key of the potential intracellular regulatory mechanisms in regulating the inflammatory mechanisms of trivalent arsenic-induced AKI with the clinical relevant dose, and also is the key mechanism of Chuan Huang Decoction efficiently preventing arsenic nephrotoxicity." Establish vivo and vitro models, siRNA method was used to knockdown MAML1 gene and NLRP3, then detect the renal tubular epithelial cell apoptosis and autophagy, AKI biomarkers, and the expression of inflammatory signals in protein and mRNA level, finally to test the hypothesis, reveal the mechanism of trivalent arsenic nephrotoxicity and Chuan Huang Decoction efficacy, and provide new evidence and targets of using “Detoxification and promoting blood circulation to remove blood stasis” method of Traditional Chinese Medicine to combat trivalent arsenic-induced kidney injury with clinical relevant dose.

随着环境污染加剧和三价砷临床抗肿瘤应用的增多，三价砷导致的急性肾损伤（AKI）成为新医学问题。MAML1基因调控多个炎症信号参与决定细胞命运，NLRP3炎症小体能参与AKI发病。我们在国际上率先报道临床剂量三价砷诱使人肾小管上皮细胞凋亡和自噬，并有COX2、beta-catenin、TNF-α、p38 MAPK及NF-κB等炎症信号参与，但炎症信号的胞内调控机制尚不清楚。证实川黄方能抑制炎症信号和肾小管上皮细胞凋亡、防治AKI及砷肾毒性。推测“MAML1、NLRP3是调控临床剂量三价砷致AKI的炎症机制的关键细胞内环节，也是川黄方疗效机制的关键途径”。建立体内、外模型，siRNA分别阻断MAML1和NLRP3，并通过对肾小管上皮细胞凋亡及自噬、AKI标志物、炎症信号表达等检测以验证假说，揭示三价砷肾毒性机制，并为川黄方“解毒化瘀”中医途径防治本病提供新证据和作用靶点。

随着环境污染加剧和三价砷临床抗肿瘤应用的增多，三价砷导致的急性肾损伤（AKI）成为新医学问题。MAML1基因调控多个炎症信号参与决定细胞命运，NLRP3炎症小体能参与AKI发病。我们在国际上率先报道临床剂量三价砷诱使人肾小管上皮细胞凋亡和自噬，并有COX2、beta-catenin、TNF-α、p38 MAPK等炎症信号参与，但炎症信号的胞内调控机制尚不清楚。本研究分别建立三价砷致肾损伤大鼠和细胞模型，证实该肾损伤存在MAML1和NLRP3激活；经siRNA分别阻断MAML1和NLRP3以后，细胞模型中COX2、beta-catenin、TNF-α、p38 MAPK等表达量下降，并伴随胞内ROS降低、肾小管上皮细胞凋亡和自噬的减少，从而佐证炎症机制是介导三价砷致肾损伤的发病机制之一、MAML1和NLRP3是调控炎症机制的重要环节。同时川黄方在大鼠模型显示出明显的肾保护效应，与模型组相比，川黄方组在肾功能、AKI标志物以及肾组织病理学检测方面均有显著改善；机制研究结果证实，川黄方显著抑制模型大鼠肾组织局部ROS生成，抑制MAML1、NLRP3介导的炎症信号（COX2、beta-catenin、TNF-α、p38 MAPK等）激活，并抑制肾小管上皮细胞凋亡和自噬。在对模型中显著上调的HO-1的转录调控研究中发现三价砷通是过活化Nrf2、Nf-κB、AP-1等多个转录因子来完成，川黄方及其有效成分单体川芎嗪能通过对该途径的进一步调控发挥肾保护效应。综合起来，本研究除了佐证炎症机制是三价砷致肾损伤的发病机制之一、MAML1和NLRP3是调控炎症机制的两个重要环节以外，进一步证明川黄方是防治三价砷致肾损伤的有效方剂，其肾保护机制与抑制肾组织氧化应激损伤、抑制炎症信号（如COX2、p38 MAPK）、调控Nrf2等转录因子介导的HO-1有关，同时该方还显示出对三价砷诱使的程序性细胞死亡方式（凋亡和自噬）有良好的抑制作用。上述发现非常值得进一步深入研究。