基于AMPK/mTOR自噬调控通路探讨活血通络方早期干预糖尿病视网膜病变的分子机制

Diabetic retinopathy (DR) has high incidence and severe impairment. Recent studies show that oxidative stress plays important roles in the occurance of DR, which could be improved by regulating autophagy and reducing the apoptosis of retinal ganglion cells. AMPK/mTOR pathway autophagy may be a forceful breakthrough point, intervention on this target through autophagy mediation at early stages could protect retina and significantly reduce or delay the onset of DR development by early preventive medication, which can reduce the rate of blindness and medical costs.. Western medicine could not get a significant benefit of conventional hypoglycemic and solve the problem of preventing DR completely. DR falls the categories of “collateral disease”, the pathogenesis is related to eye collateral obstruction. Invigorating blood and unblocking the collaterals are important treated principle. Compound formula of invigorate Blood and unblock the Collaterals was accorded with this principle. Preliminary research shows that this compound formula could protect rat retina and significantly reduce or delay the onset of DR development, speculated this formula could protect the retina and alleviate the oxidative stress by up-regulating AMPK pathway and inhibiting mTOR pathway, which could provide new targets and ideas for the early treatment of DR in traditional Chinese medicine.

糖尿病视网膜病变（DR）发病率极高且危害严重。新近研究表明，氧化应激损伤在DR的发病中起重要作用，调控自噬可明显改善氧化应激损伤，减少视网膜神经节细胞的凋亡，从而延缓或减轻DR的发展。AMPK/mTOR通路为自噬调控的重要通路,针对该靶点调控自噬可能是早期干预DR的重要途径,有望逆转DR的发病进程，是降低致盲率及医疗费用的关键。. 西医强化降糖或改善视网膜微循环不能获得常规的显著获益，完全解决DR的早期治疗问题。DR归属中医“络病”范畴，目络瘀阻是其基本病机。治疗当以活血化瘀通络为主,并在此治则的指导下以丹参、三七和水蛭组成活血通络方。申请者前期研究表明，本方可明显延缓或减轻DR的发展，有望成为早期干预DR的有效手段。推测活血通络方可上调AMPK通路，抑制mTOR通路调控自噬，减少视网膜神经节细胞凋亡，从而起到早期治疗DR的作用，为中医药早期干预DR提供新的靶点和思路。

据IDF最新报道，2021年全球约5.37亿成年人（20-79岁）患有糖尿病，预计到到2045年将上升到7.83亿。糖尿病视网膜病变(diabetic retinopathy，DR)是一种糖尿病微血管并发症，严重威胁患者视力，最终可导致失明。1型糖尿病患者发展为DR的风险超过90%，且与2型糖尿病（T2DM）患者相比，1型糖尿病（T1DM）患者患DR的可能性更高。氧化应激和自噬在DR的发展中起着至关重要的作用，缓解机体氧化应激状态及靶向自噬通路蛋白是控制DR的一种新型潜在治疗策略。活血通络方含丹参、三七、水蛭、大黄、黄芪五种药物，具有化瘀通络、养血活血之功效。有研究证明中药复方可通过NF-κB途径有效改善DR。在临床应用中亦发现对于缓解DR具有显著疗效，本研究在明确活血通络方改善DR的基础上，从氧化应激介导下的自噬调控入手，围绕AMPK/mTOR等通路，揭示中药活血通络方对糖尿病大鼠视网膜病变的保护作用机制。本实验以雄性SD大鼠腹腔注射链脲佐菌素造DR模型，后随机分为模型组（DM）、羟苯磺酸钙治疗组（CD）、和活血通络方低剂量治疗组（HTF-L）、活血通络方中剂量治疗组（HTF-M）、活血通络方高剂量治疗组（HTF-H），健康SD大鼠作对照组。干预3月后，在运用苏木精-伊红、PAS染色和TUNEL法评估视网膜细胞凋亡情况的基础上，进一步运用Elisa、WB and RT-PCR等探索氧化应激及自噬调控相关靶点的表达情况。中药复方HTF干预后可缓解STZ导致的体重降低和血糖升高，抑制视网膜血管单位破坏，内皮细胞与周细胞凋亡以及神经节细胞损伤，通过抑制机体氧化应激状态（下调ROS水平，上调SOD、GSH水平），并激活AMPK/mTOR自噬通路(如增加ULK1、Beclin、LC3、AMPK的表达水平，抑制mTOR的表达水平)。