加味桃红四物汤通过HIF-1α-STAT3调节VEGF/PEDF平衡对视网膜静脉阻塞的作用机制研究

Macular edema of retinal vein occlusion (RVO) is a severe reason of visual impairment, and the treatment is limited. It is known that vascular endothelial growth factor (VEGF) is the key mechanism, while the HIF-1α-STAT3 pathway is an important pathway involved in VEGF formation of Müller cells. Also, HIF-1α is involved in the regulation of pigment epithelium-derived factor (PEDF) which is the most important inhibitors of VEGF, and it might be associated with autophagy. Our previous studies revealed that modified Tao-Hong-Si-Wu decoction (THSW) had a satisfying improvement on RVO microcirculation, and inhibited VEGF secretion significantly. So we propose a hypothesis: as follows: THSW protects RVO by regulating the expression of VEGF/PEDF through HIF-1α-STAT3 pathway. We will evaluate it both in vivo and in vitro. firstly, RVO rat models will be established, and histological structure of retina and Müller cells, as well as expressions of HIF-1α、STAT3、VEGF、PEDF will be observed to explore effect of THSW. Then we will culture human retinal Müller cells with hypoxia, make STAT3 signaling silence by siRNA transfection to study the regulation of THSW on HIF-1α-STAT3 signaling pathway. Hereby, this project aims to explore the underlying pathway between HIF-1α-STAT3 and VEGF/PEDF, as well as clarify molecular mechanism of protective effects of THSW on RVO.

视网膜静脉阻塞（RVO）黄斑水肿是危害视力的祸首，也是治疗难点。已知血管内皮生长因子（VEGF）是其关键机制，且HIF1α- STAT3通路是Müller细胞分泌VEGF重要途径；同时HIF1α参与VEGF抑制剂色素上皮细胞衍生因子（PEDF）眼内调控，这可能与细胞自噬有关。前期研究发现，加味桃红四物汤（THSW）明显改善RVO微循环并减少VEGF分泌，效果良好。基于以上研究基础我们提出假说：THSW通过HIF1α -STAT3通路调控Müller细胞对 VEGF/PEDF表达平衡治疗RVO。拟采用SD大鼠RVO模型，观察THSW对视网膜结构、Müller细胞和HIF1α、STAT3、VEGF、PEDF表达；缺氧培养Müller细胞，siRNA干扰技术沉默STAT3，研究THSW对Müller细胞HIF1α -STAT3通路影响。本研究旨在明确中药对RVO调控机制，为THSW临床提供依据。

视网膜静脉阻塞（RVO）黄斑水肿是危害视力的祸首，也是治疗难点。已知血管内皮生长因子（VEGF）是其关键机制，且HIF1α-STAT3通路是Müller细胞分泌VEGF重要途径；同时HIF1α参与VEGF抑制剂色素上皮细胞衍生因子（PEDF）眼内调控。前期研究发现，加味桃红四物汤（THSW）明显改善RVO微循环并减少VEGF分泌，效果良好。基于以上研究基础我们提出假说：THSW通过HIF1α-STAT3通路调控Müller细胞对VEGF/PEDF表达平衡治疗RVO。本次研究我们在RVO动物模型以及缺氧状态下培养视网膜Müller细胞分别在细胞水平和体内水平研究加味桃红四物汤干预RVO后VEGF/PEDF的变化，结果发现：1）加味桃红四物汤灌胃SD大鼠RVO模型改善了视网膜微循环，FFA显示视网膜血管渗透性降低，从而促进视网膜出血、渗出吸收，缓解静脉血管的迂曲扩张。在RVO模型组中，VEGF的表达随时间增加，在7天时表达最高。加味桃红四物汤灌胃之后，VEGF的含量比同时间点的模型组表达下降，且三组中药组均和模型组有差异。高浓度组更有意义，中、低浓度组间区别不明显。加味桃红四物汤可能通过抑制STAT3通路的活化从而下调VEGF的表达。2）加味桃红四物汤可改善缺氧诱导的视网膜Müller细胞rMC-1增殖下降，抑制其缺氧诱导的VEGF分泌，下调VEGF的基因表达，上调PEDF基因表达，维护了两者的平衡，从而减轻视网膜缺氧，发挥治疗RVO的作用，其作用机制与抑制STAT3/HIF-1α通路有关。另外，HIF-1α基因被沉默后MTSD对缺氧下VEGF的分泌仍具有显著的下调作用，说明MTSD对VEGF的作用不仅仅通过HIF-1α通路，还可能对VEGF依赖的其它通路也具有调控作用，这也是中药多靶点调控的可能。3）其有效单体-芍药苷可促进视网膜Müller细胞rMC-1增殖，抑制缺氧诱导的VEGF和IL-1β分泌，其机制可能与抑制NF-κB/NLRP3通路有关。本研究旨在明确中药对RVO调控机制，为THSW临床提供了依据。