目的 研究白芷颗粒对早期糖尿病视网膜病变（diabetic retinopathy，DR）的保护作用。方法 采用ip链脲佐菌素（streptozotocin，STZ）法制备糖尿病模型，设置对照组、模型组、白芷（1.25 g/kg）组及羟苯磺酸钙（135 mg/kg）组。连续给药12周后采用苏木素-伊红（HE）和高碘酸-席夫（PAS）染色观察大鼠视网膜病理学改变；采用TUNEL染色法检测视网膜细胞凋亡情况；采用Western blotting检测视网膜组织紧密连接蛋白及磷脂酰肌醇3-激酶（phosphatidylinositol 3-kinase，PI3K）/蛋白激酶B（protein kinase B，Akt）通路相关蛋白表达。人视网膜上皮细胞（adult retinal pigment epithelial cell line-19，ARPE-19）分为对照组、高渗组、高糖组、白芷（150 μg/mL）组和羟苯磺酸钙（20 μmol/L）组，对照组在含5.5 mmol/L葡萄糖的培养基中培养；高渗组在含5.5 mmol/L葡萄糖和25 mmol/L甘露醇的培养基中培养；高糖组和各给药组在含30 mmol/L葡萄糖的培养基中培养，并给予相应药物。采用TUNEL染色、免疫荧光、Western blotting法观察细胞凋亡、紧密连接蛋白及PI3K/Akt通路相关蛋白表达。使用PI3K抑制剂LY294002后，观察其下游凋亡相关蛋白的变化。结果 HE染色结果显示，模型组大鼠视网膜组织排列紊乱，视网膜变薄，白芷干预后较模型组有所缓解。TUNEL染色显示视网膜组织中细胞凋亡率增加。Western blotting结果显示，与模型组比较，白芷组咬合蛋白（Occludin）、闭锁小带蛋白1（Zonula occluden-1，ZO-1）、p-PI3K/PI3K、p-Akt/Akt蛋白表达水平明显升高（P＜0.05），Bcl-2相关X蛋白（Bcl-2 associated X protein，Bax）/ B淋巴细胞瘤-2（B-cell lymphoma-2，Bcl-2）、剪切型半胱氨酸天冬氨酸蛋白酶-3（cleaved cystein-asparate protease-3，cleaved Caspase-3）蛋白表达水平明显降低（P＜0.05）。体外实验结果显示，白芷干预后细胞凋亡率降低（P＜0.05），Bax/Bcl-2、cleaved Caspase-3蛋白表达水平明显降低（P＜0.05），Occludin、ZO-1蛋白表达水平明显升高（P＜0.05）；免疫荧光结果显示白芷干预后Occludin、ZO-1表达升高；此外，白芷能够促进高糖环境下PI3K/Akt的磷酸化，在使用PI3K抑制剂干预后抑制了Akt的磷酸化，Bax/Bcl-2、cleaved Caspase-3蛋白表达水平明显升高（P＜0.05）。结论 白芷颗粒能够通过抑制细胞凋亡保护血-视网膜屏障损伤，从而延缓早期DR的发展，其作用机制可能与调节PI3K/Akt信号通路有关。

Objective To study the protective effect of Angelica dahurica granules on diabetic retinopathy (DR). Methods Diabetes mellitus model was established by intraperitoneal injection of streptozotocin, rats were randomly divided into control group, model group, A. dahurica (1.25 g/kg) group and calcium dobesilate (135 mg/kg) group. After 12 weeks of continuous administration, the pathological changes in retina of rats were observed by HE and PAS staining; TUNEL staining was used to detect cell apoptosis in retina; Western blotting was used to detect the expressions of tight junction proteins and phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway related protein. Adult retinal pigment epithelial cell line-19 (ARPE-19) was divided into control group, hyperosmotic group, high glucose group, A. dahurica (150 μg/mL) group and calcium dobesilate (20 μmol/L) group. The control group was cultured in medium containing 5.5 mmol/L glucose; The hypertonic group was cultured in medium containing 5.5 mmol/L glucose and 25 mmol/L mannitol; The high glucose group and each administration group were cultured in a culture medium containing 30 mmol/L glucose and given corresponding drugs. TUNEL staining, immunofluorescence and Western blotting were used to observe cell apoptosis, expressions of tight junction protein, and PI3K/Akt pathway related protein. After using the PI3K inhibitor LY294002, the changes in downstream apoptosis related proteins were observed. Results HE staining results showed that the retinal tissue arrangement of model group rats was disordered, and the retina became thinner. After intervention with A. dahurica, there was some relief compared to the model group. TUNEL staining showed an increase in cell apoptosis rate in retinal tissue. Western blotting results showed that compared with model group, the expression levels of occludin, Zonula occlude-1 (ZO-1), p-PI3K/PI3K and p-Akt/Akt proteins in A. dahurica group were significantly increased (P < 0.05). Bcl-2 associated X protein (Bax)/B-cell lymphoma-2 (Bcl-2), cleaved cystein-asparate protease-3 (cleaved Caspase-3) protein expression levels were significantly reduced (P < 0.05). In vitro experiments results showed after intervention with A. dahurica, the apoptosis rate of cells was decreased (P < 0.05), and the expression levels of Bax/Bcl-2 and cleaved Caspase-3 proteins were significantly decreased (P < 0.05), while the expression levels of Occludin and ZO-1 proteins were significantly increased (P < 0.05); The immunofluorescence results showed that the expressions of Occludin and ZO-1 were increased after intervention with A. dahurica; In addition, A. dahurica could promote the phosphorylation of PI3K/Akt in high glucose environments. After intervention with PI3K inhibitors, Akt phosphorylation was inhibited, and the expression levels of Bax/Bcl-2 and cleaved Caspase-3 proteins were significantly increased (P < 0.05). Conclusion A. dahurica granules can protect against damage to the blood retinal barrier by inhibiting cell apoptosis, thereby delaying the development of early DR. Its mechanism may be related to the regulation of the PI3K/Akt signaling pathway.

R285.5

天津市教委科研计划项目（2020KJ190）；天津市卫计委中西医结合重点项目（2021034）