晚期糖基化终末产物通过上调NOX4促进胰腺纤维化的机制研究

Pancreatic fibrosis is a characteristic pathological change in chronic pancreatitis (CP), and activation of pancreatic stellate cell (PSC) is a key event in CP fibrosis. Previous studies have found that reactive oxygen species play an important role in PSC activation. NADPH oxidase (NOX) is an important source of intracellular reactive oxygen species, while NOX4 is involved in cardiac remodeling and liver and kidney fibrosis. Recent studies have found that NOX expression could be regulated by advanced glycation end products (AGEs) through their receptors (RAGE) to participate in diseases such as diabetic complications and liver fibrosis. Our previous study found that serum AGEs were increased in CP patients. Transcriptome sequencing indicated that NOX4 expression was increased and AGEs-RAGE pathway was enriched in activated PSCs. After stimulated with AGEs, primary PSCs were activated and NOX4 increased. It is speculated that AGEs bind to the RAGE receptor on the surface of PSC and induce oxidative stress by promoting NOX4 expression, which activates PSCs. In this study, we intends to use NOX4 inhibitors, siRNA interference technology and NOX4 knockout mice to explicit the molecular mechanism of AGEs promoting pancreatic fibrosis in vitro and in vivo, and evaluating the potential value of NOX4 as a therapeutic target for of CP.

胰腺纤维化是慢性胰腺炎（CP）特征性病理改变，胰腺星状细胞（PSC）活化是CP纤维化关键事件。既往研究发现活性氧在PSC活化中扮演重要角色，NADPH氧化酶（NOX）是细胞内活性氧的重要来源，其中NOX4可参与心脏重塑及肝、肾纤维化。近期研究发现晚期糖基化终末产物（AGEs）可通过其受体（RAGE）调节NOX表达参与糖尿病并发症、肝纤维化等疾病。申请人前期研究发现CP患者血清AGEs增多；转录组测序提示小鼠活化PSC中NOX4表达升高，AGEs-RAGE通路富集；进一步用AGEs刺激原代PSC后细胞活化且NOX4表达增加。据此推测，AGEs与PSC表面RAGE受体结合后促进NOX4表达诱发氧化应激，导致PSC活化。本研究拟利用NOX4抑制剂、siRNA干扰技术以及NOX4基因敲除小鼠在体外及体内水平阐述AGEs促进胰腺纤维化的分子机制，探讨NOX4作为靶点治疗CP的潜在价值。

胰腺纤维化是慢性胰腺炎（CP）特征性病理改变，胰腺星状细胞（PSC）活化是CP纤维化关键事件。活性氧在PSC活化中发挥重要作用，NADPH氧化酶（NOX）作为活性氧的重要来源参与了氧化应激，目前已有研究证实NOX4在心脏重塑及肝、肾纤维化方面发挥重要作用。我们使用免疫荧光检测CP小鼠胰腺组织氧化应激增强，转录组测序提示小鼠活化PSC中NOX4表达升高，且存在AGEs-RAGE通路富集；进一步刺激人源PSC活化后，NOX4表达升高，而NOX选择性抑制剂GKT137831作用后PSC活化指标表达降低。近期研究发现晚期糖基化终末产物（AGEs）可通过其受体（RAGE）调节NOX表达参与糖尿病并发症、肝纤维化等疾病。AGEs是过量的糖和蛋白质结合的产物，可通过促进自由基产生而加速人体的衰老并导致慢性退化型疾病。我们前期研究发现CP患者血清AGEs含量显著高于健康志愿者AGEs增多，用AGEs刺激原代PSC后细胞活化且NOX4表达增加，进一步使用siRNA 敲减NOX4等实验发现AGEs通过与PSC表面RAGE受体结合后促进NOX4表达导致PSC活化。为探究NOX抑制剂抑制PSC活化的机制，使用NOX广谱抑制剂DPI处理活化的人源PSC进行转录组测序分析，发现864个差异表达基因，其中463个上调，401个下调；GO功能富集分析发现固醇类合成相关通路有显著变化。最后在体内水平探讨以NOX4为靶点治疗胰腺纤维化的潜在价值，使用雨蛙素造模NOX4基因敲除小鼠，与正常雨蛙素造模小鼠进行对比，NOX4基因敲除小鼠纤维化程度较轻，星状细胞激活减少，细胞增殖活性减弱，巨噬细胞浸润减少。本研究首次探讨了AGEs对胰腺纤维化的作用，并阐述AGEs通过上调NOX4促进CP发生发展的分子机制。通过CP模型小鼠及NOX4敲除转基因小鼠来探究NOX4作为治疗靶点的可行性，为改善或逆转胰腺纤维化提供新的思路。