基于目络瘀阻探讨活血通络利水法干预视网膜Müller细胞水液代谢的分子机制

The retinal edema associated with ischemic retinal diseases play an important role in severe visual impairment. Modern researchs find that the transport dysfunction of the water and potassium of müller cell is one of the important pathophysiological mechanisms for the formation of the retinal edema. Based on years of experience on clinical practice, the workgroup put forward a pathogenesis theory of ocular collateral stasis syndrome. Therefore in virtue of the guidance of theory, an effective Chinese herbal compound (i.e. Huoxuetongluolishui recipe) was developed. Based on excellent therapeutic effect of the herbal compositions, we conduct this study to investigate the formation mechanism of ischemic retinal edema through the malfunction of aquaporin 4 and Kir 4.1, and the role of their signal transduction pathways (i.e. PKC-ERK1/2 and p38 MAPKs pathway) involved in retinal edema. The study also analyzes the inter-relationships among the aquaporin 4, Na/K-ATPase, inflammatory factors, oxidative stress and excitatory amino acids in the ischemicl retinal tissues. Based on researches mentioned above, the analysis is carried on in multi-angles and multi-levels so as to characterize the micro syndrome of "ocular collateral stasis syndrome" theory, and explore setting up the auxiliary microcosmic diagnostic index system which reflects the complex interaction and transformation between deficiency, blood-stasis, toxin and fluid retention. Furthermore, we investigate the protective effects of Huoxuetongluolishui recipe on ischemic retinal disease and further explore its possible acting mechanism and underlying targets in order to develop new drugs for the treatment of ischemic eye disease.

缺血性视网膜病变伴发的视网膜水肿是造成患者视力损害的重要因素,而Müller细胞上水通道蛋白4(AQP4)与内向整流钾通道(Kir4.1)水钾转运功能失调是视网膜水肿形成的重要病理机制。本课题组经长期临床实践提出"目络瘀阻"病机理论假说，在此理论指导下研发出活血通络利水方，并在该复方治疗缺血性视网膜水肿有效的基础上，体内外实验相结合，从AQP4/Kir4.1失衡角度探讨缺血性视网膜水肿形成机理，研究对AQP4具有调控作用的PKC-MAPK信号通路中关键蛋白的变化；分析视网膜病变组织中Na+-K+-ATP酶、兴奋性氨基酸、炎性因子、氧化应激与AQP4之间的内在联系。从多角度、多层次阐释"目络瘀阻"病机理论的微观辨证特征，探索能够反映虚、瘀、毒、水等病理因素之间相互兼杂的微观证侯指标群，进一步探讨活血通络利水中药复方治疗缺血性视网膜疾病和视网膜水肿的作用靶点和可能作用机制，为新药开发奠定基础。

缺血性视网膜病变是常见的眼底疾病。课题组依据络病理论提出“目络瘀阻”病机假说，认为目络瘀阻，瘀毒水互结是缺血性视网膜病变进行性发展的主要中医病机，根据多年临床经验创制活血通络利水方并取得了良好临床疗效。为探讨“目络瘀阻”微观病理特征及中药复方解毒利水的药理作用机理，本项目分别采用大鼠视网膜分支静脉阻塞（BRVO）模型、Müller细胞缺氧模型、兔视网膜缺血再灌注（I/R）模型，体内外实验相结合，研究了对AQP4具有调控作用的PKC-MAPK信号通路的影响；分析Na-K-ATP酶、兴奋性氨基酸、炎性因子、氧化应激、AQP4与视网膜缺血水肿之间的内在联系。结果发现，大鼠BRVO时，视网膜电流图振幅下降，PKC-ERK1/2通路和p38通路被激活，进而诱导AQP4过量表达，视网膜水肿可能与AQP4/Kir4.1升高有关。而中药复方能通过抑制PKC-ERK1/2信号通路和p38通路而降低AQP4表达，减少Kir4.1和AQP1表达，从而减轻视网膜水肿。细胞实验也发现中药血清可通过抑制缺氧Müller细胞p38信号通路和ERK1/2信号通路降低Müller 细胞上AQP4的表达，同时增高Kir4.1的表达，从而恢复AQP4与Kir4.1的偶联关系，并具有显著的量效关系。兔I/R模型实验结果显示，造模后视网膜水肿，SOD活性逐渐降低，而Na-K-ATPase、MDA、NO、IL-6、IL-1β、TNF-α含量、Asp、Glu 水平及AQP4蛋白表达量均不同程度的升高，而中药复方能减轻水肿，其机制可能与提高视网膜抗氧化应激能力，减轻炎症反应，抑制兴奋氨基酸毒性损伤，降低AQP4表达，抑制BRB通透性升高有关。结论：缺血性视网膜病变“目络瘀阻”的微观病理特征包括视网膜水肿、毛细血管周围炎性浸润，细胞超微结构异常、线粒体等亚细胞器高度肿胀、核染色质浓缩、AQP4/Kir4.1失衡及毒性因子损伤等。活血通络利水方的解毒利水作用可能与抑制PKC- ERK1/2信号通路和p38通路，降低Müller细胞AQP4的过度表达，恢复AQP4/Kir4.1偶联关系，抑制AQP1表达增高，减少促炎因子、氧化应激因子和兴奋性氨基酸释放，降低血视网膜通透性有关。本研究突破了以往治疗缺血性视网膜病变多集中在活血化瘀方药的局限性，开拓了缺血性眼病的防治新思路。