小檗碱调控NLRP3炎症体活性的机制在对比剂急性肾损伤发病中作用的研究

With the wide application of iodinated contrast media in disease diagnosis and treatment, contrast induced acute kidney (CI-AKI) has became the third cause of hospital-acquired acute kidney injury. Our previous study has demonstrated that contrast induced kidney injury through NLRP3 inflammasome pathway. Recent studies have identified that berberine modulate NLRP3 inflammasome through blocking P2X7 receptor or ROS-TXNIP pathway, which indicate that berberine has a potential to prevent or treat CI-AKI. .Nucleotide-binding oligomerization domain receptor-like protein 3 (NLRP3) is a newly discovered pattern recognition receptors expressed in a variety of intrinsic renal cells such as renal tubular epithelial cells, podocytes, mesangial cells. After activated, it formed NLRP3 inflammasome with apoptosis-associated speck-like protein (apoptosis-associated speck-like protein containing CARD, ASC), then activating caspases 1 (caspase -1), causing interleukin 18 (IL-18) and interleukin-1β (IL-1β) mature and release. Four NLRP3 inflammasome activation measures had been found: 1, several microbial toxins; 2, extracellular ATP activate P2X7 receptor, resulting in the activation of K + outflow; 3, lysosomal rupture and release cathepsin B; 4, local oxidative stress. Berberine is involved in at least two pathways of NLRP3 inflammasome activation..Our current study aims to investigate the protective role of berberine in CI-AKI in vitro and in vivo. In the first part, we will find out whether berberine alleviate CI-AKI induced kidney injury. The second part of the study was designed to suppress NLRP3 inflammasome directly as to investigate whether berberine modulate CI-AKI through NLRP3 inflammasome. The third part of the study was designed to block P2X7 receptor or THNIP protein to investigate the role of berberine modulating NLRP3 inflammasome in CI-AKI.

随着含碘对比剂的广泛运用，对比剂急性肾损伤（contrast-induced acute kidney injury CI-AKI）已成为院内获得性急性肾损伤的第三大原因。本课题组前期通过体内及体外实验发现NLRP3炎症体通路活化是对比剂急性肾损伤的重要致病原因，阻断这一通路可显著缓解对比剂急性肾损伤。近期研究发现，小檗碱可分别通过抑制P2X7受体活性或抑制ROS-TXNIP通路活性调控NLRP3炎症体，具有潜在的防治CI-AKI的作用，值得进一步研究。.本课题组拟通过已有的体内外两部分模型，探讨小檗碱对CI-AKI发病与进展的防治作用;通过直接阻断NLRP3炎症体，分析小檗碱调控NLRP3炎症体通路的机制在CI-AKI发病中的作用；进一步分别阻断小檗碱调控NLRP3炎症体的两条上游通路，分析CI-AKI中小檗碱调控NLRP3炎症体活性的具体途径。本研究可为防治CI-AKI提供新的靶点与理论。

项目背景. 对比剂所致急性肾损伤（contrast-induced acute kidney injury，CI-AKI）已成为院内获得性急性肾损伤的第三大原因。多中心临床研究发现：CI-AKI发病率约为3%-15%，在高危患者中发病率可达50%以上。CI-AKI可导致多种不良事件：1、延长患者住院时间；2、增加并发症；3、增加远期不良事件；4、在接受心血管造影和心脏介入手术的患者中，发生CI-AKI的患者远期死亡率、中风与心脏病发作风险增加。最严重的是，CI-AKI一旦发生尚无有效措施逆转其病程。CI-AKI的发病机制尚未完全明确。.主要研究内容. 本研究拟通过体外人近端肾小管上皮细胞培养和体内CI-AKI小鼠模型实验研究：.（1）小檗碱是否可以预防或减轻对比剂所致的急性肾损伤.（2）探讨小檗碱是否通过调控NLRP3炎症体活性达成上述目标。.（3）探讨小檗碱是否通过调节NLRP3炎症体上游P2X7受体或TXNIP蛋白活性达到调控NLRP3炎症体活性的目的。.本研究结果可为CI-AKI的防治方法提供新的依据，有可能成为预防与治疗CI-AKI的有效药物。.重要结果. 小檗碱可有效防治对比剂急性肾损伤的作用. 小檗碱通过调控NLRP3炎症体活性发挥对对比剂急性肾损伤的保护作用. 小檗碱通过抑制P2X7受体活性发挥调节NLRP3炎症体活性的作用.关键数据. 小檗碱有效抑制对比剂急性肾损伤所致的HK-2细胞中NLRP3、ASC蛋白升高，及cleaved Caspase-3、BAX蛋白升高、BCL-2蛋白下降。. 小檗碱有效抑制对比剂诱导的HK-2细胞中P2X7受体活化。. 小檗碱有效降低对比剂诱导的小鼠肾脏中肌酐、及尿素氮升高，肾小管上皮脱水，空泡变性等损伤。. 小檗碱有效抑制对比剂急性肾损伤所致的小鼠肾脏组织中NLRP3、ASC蛋白升高，及cleaved Caspase-3、BAX蛋白升高、BCL-2蛋白下降。.科学意义. 本研究发现：小檗碱通过抑制P2X7受体活性阻断NLRP3炎症体通路，进而减轻碘海醇诱导的肾脏小管上皮细胞凋亡、肾脏损伤。结论：小檗碱通过调节NLRP3炎症体减轻对比剂急性肾损伤，具防治CI-AKI的潜力。