从缺氧-VEGF途径探讨止血化瘀利水方防治视网膜静脉阻塞黄斑水肿的作用机理

Retinal Vein Occasion（RVO）is the second most common retinal vascular disease..Macular edema (ME) and other new vessels related complications caused by retinal ischemia and hypoxia after RVO are the major reason of blindness.Prevention and treatment of ME and other complications has been hot since there is no specific clinical therapy for promotion of retinal vascular occlusion reperfusion.Intravitreal injection of anti-VEGF agents is the most effective way for the complications mentioned about, but with high cost and of high risk of operation and recrudescence.RVO belongs to the category "collateral stasis sudden blindness" in traditional Chinese medicine.The principle“Lixuezhishui” we suggested based on Blood Syndrome Theory，an ancient Chinese book，matches well with the idea of anti-VEGF in treatment of ME in western medicine.Our previous research shows that Liangxue Zhixue method,Huoxue Tongluo method could modulate the coagulant mechanisms and improve microcirculation,which is beneficial to change the ischemia and hypoxia condition in retina，and then reduce the retinal hemorrhage and edema .For further exploration of the target of Chinese medicine based on “Lixuezhishui” principle, we will detect retinal(both in vitro and in vivo) VEGF and its upstream regulatory factors HIF-1α、NF-kB and the expression of the vascular permeability related factor PKC、ICAM-1 by western blot,PCR technique in this project.We expect to investigate the action mechanisms of Zhixue Quyu Lishui Decoction in ME related to RVO of experimental models.

RVO是第二位视网膜血管病，RVO后视网膜缺血缺氧所致ME等新生血管性合并症是致盲的主要原因。因临床对RVO血管疏通尚无特效疗法，针对ME等合并症的防治成为热点，眼内注射抗VEGF制剂是目前国际上最有效的方法，但其费用昂贵，复发性高，手术风险大。中医学认为RVO属络瘀暴盲范畴，ME乃瘀血化水、血病致水病。我们在《血证论》治血四法基础上提出“理血治水”治则，与西医抗VEGF思路吻合。项目组前期国家自然科学基金课题提示，凉血止血、活血通络法可调节RVO凝血机制、改善微循环，有利于改善视网膜缺血缺氧，减少出血、水肿。为进一步明确理血治水法治疗ME的作用靶点，本项目拟通过免疫组化、western blot、PCR技术检测兔眼视网膜组织及体外培养人RPE细胞的VEGF及其上游缺氧调控因子HIF-1α、NF-kB，血管通透性因子PKC、ICAM-1的表达，探讨止血化瘀利水方干预RVO后ME的作用机制。

RVO是第二位视网膜血管病，RVO后视网膜缺血缺氧所致黄斑水肿等新生血管性合并症是致盲的主要原因。中医学认为RVO属“络瘀暴盲”范畴，黄斑水肿乃瘀血化水、血病致水病。课题组在《血证论》治血四法基础上提出“理血治水”治则，通过体外细胞实验及体内动物实验针对缺氧-VEGF高表达是激活相关信号通路导致RVO及其并发症的关键环节，建立细胞和动物缺氧模型，应用病理和分子生物学实验技术，探讨止血化瘀利水方在防治RVO黄斑水肿的可能作用环节及靶点，为该中药的临床应用提供实验依据，为探索RVO治疗方法提供新的策略与方向。研究结果表明：1.CoCl2可成功诱导ARPE-19细胞缺氧模型，以更好的模拟体内缺氧环境。2.缺氧诱导ARPE-19细胞中VEGF、HIF-1α表达升高，提示缺氧环境下细胞趋化两因子的高表达。3.止血化瘀利水方含药血清对缺氧损伤后ARPE-19细胞的增殖和VEGF、HIF-1α表达均起抑制作用，因此推测改善缺氧环境及抑制VEGF是中药在治疗RVO中发挥作用的关键机制。4.光化学法可成功诱导RVO兔模型，具备动物死亡率低及成功率高的优点。5.RVO兔模型视网膜中VEGF、HIF-1α、NF-κB p65、ICAM-1、GFAP表达升高，在14d时尤为明显，提示以上因子参与了RVO及其并发症的病理过程，14d为相关因子维持的时间转折点。6.止血化瘀利水方对RVO兔模型视网膜中VEGF、HIF-1α、NF-κB p65、ICAM-1、GFAP起到较好的抑制调节作用，表明该中药可能是通过改善视网膜缺血缺氧途径，减少VEGF等新生血管生长相关因子产生，并有助于修复血管内皮损伤，降低RVO造成的Müller细胞对损伤的过度反应，保护了正常的视网膜细胞，从而达到防治RVO引起的黄斑水肿及新生血管性并发症的目的。7.结合体外实验与体内实验的结果表明，止血化瘀利水方可能通过缺氧-VEGF途径对RVO及黄斑水肿、新生血管性并发症具有较好的防治作用。