三七粉防治有色素大鼠早期糖尿病视网膜微血管渗漏和新生血管形成的机制研究

According to epidemiological studies, diabetic retinopathy (DR) is the No. 1 cause of blindness among all blinding retinal vascular diseases. Clinical laser photocoagulation treatment and vitrectomy can partially improve the effective vision of patients, but they have little influence over the entire course of retinopathy. Drug treatments, including anti-VEGF drugs, Ang-1 factor and stem cell therapy, have not made gratifying progress until now. Diabetic retinopathy pathologic basis is a new blood vessel formation and leakage of microcirculation, HIF/VEGF signal pathways play an important role in the process. We've learned from the past research, DR is closely relative to the course of diabetes mellitus, which drug can prevent diabetes develop into diabetic retinopathy ?From our prior work we found that Panax notoginseng can reduce platelet adhesion rate and aggregation rate of the rats’ blood, to clarify blood stasis ,and have certain therapeutic effect for DR . So we put forward the hypothesis that early application of Panax notoginsengn can improved the retinal ischemia and hypoxia state and delay the happening of the diabetic retinopathy through HIF/VEGF signaling pathway .This experiment is designed to make diabetic rats model with STZ. After 12 weeks we will carry out the FFA fundus angiography, find microvascular lesion rats to form the DR group, and set up a negative control group. Through 12 weeks’ intervention with Panax notoginseng and equivalent physiological saline respectively. Then we will investigate the mechanism of Panax notoginseng to prevent DR by the detection of rat serum platelet adhesion rate and accumulation rate, Evans blue (EB) leakage test; we also investigate HIF-1α、VEGF and CD31、MMP-9 by western-blot and immunohistochemistry methods in rat retina. We look forward to provide a theoretic basis for the theory "preventive disease " and “righteousness existing, evil not invading” in traditional Chinese medicine through our studies.

糖尿病视网膜病变（DR）位居致盲性视网膜血管疾病之首。目前应用药物治疗、激光光凝及玻璃体切除术仅部分延长患者视力,对DR病程无重要影响。DR病理基础是视网膜新生血管形成和微循环渗漏，HIF/VEGF信号通路在此过程中起重要作用。研究已知,DR与糖尿病病程正相关，在糖尿病期即应用药物治疗是否对DR有预防作用？前期工作中我们发现，三七粉能降低DR大鼠血小板黏附率和聚集率，改善视网膜血流高粘滞状态,对DR有一定治疗作用。据此提出三七粉早期应用改善了视网膜缺血、缺氧状态，继而通过HIF/VEGF信号通路延缓DR发生的假说。本课题采用BN有色素大鼠,沿用经典STZ造糖尿病大鼠模型，三七粉预防和早期治疗给药，模拟人FFA荧光眼底血管造影活体诊断DR、伊文思蓝渗漏实验、免疫组化和western-blot等探讨三七粉通过HIF/VEGF信号通路发挥对DR的防治作用，并确定三七的最佳给药时间窗和量效关系。

背景：糖尿病视网膜病变的发病率逐年升高，其中的抗-VEGF为PDR治疗的有效药物，但因为昂贵的医疗费用和反复球内给药的患者接受度及并发症,使它的应用受到限制。因此,寻找一种有效、经济的治疗和预防早期DR的方法迫在眉睫。内容：重点研究三七粉中可能对DR有治疗作用的三种单体成分：三七总皂苷、槲皮素和阿拉伯糖早期干预后对DR的HIF-1a-VEGF信号通路以及炎症介导的血管内皮损伤、BRB的破坏和血管渗漏的机制影响，明确三七粉中具有抗炎、抗VEGF功效的有效成分，以及这些单体成分对早期糖尿病视网膜病变可能的治疗作用及分子机制。结果：分别在给药4周、8周和12周成功应用活体荧光血管造影技术观察到糖尿病12周的BN鼠出现了视网膜血管扩张迂曲、无灌注区、微血管渗漏等如人背景期糖尿病视网膜病变的眼底改变，而给药组三七总皂苷组BN鼠眼底情况良好。分别在给药4周、8周和12周进行Western Blot检测结果显示：在第4周每组VEGF因子表达无差别，HIF-1-α和ICAM-1在空白对照组和皂苷组分子量表达低；第8周时VEGF、HIF-1-α和ICAM-1在空白对照组和皂苷组分子量表达低；第12周VEGF和HIF-1-α在空白对照组和皂苷组分子量表达低，ICAM-1因子在空白组表达量低，在其余各组表达量无明显差异。此外，我们分别在给药4周、8周和12周进行了视网膜免疫组化及在给药8周和12周行血脂（包括甘油三酯、总胆固醇和高密度脂蛋白）检测，无统计学差异。此外，我们分别在给药4周、8周和12周通过使用三诺血糖仪分别监测四组BN鼠血糖。结果显示：皂苷组大鼠血糖显著低于对照大鼠组(P<0.01)，提示三七总皂苷具有强的降糖作用，槲皮素组大鼠和阿拉伯糖组大鼠血糖低于对照大鼠组(P<0.05)，提示槲皮素和阿拉伯糖也具有降糖作用(P<0.05)。意义：三七中的三七总皂苷具有一定的抗VEGF的作用，指导我们进一步开发祖国医学。