目的：研究黄芪含药血清对氯化钴(CoCl₂)诱导人视网膜色素上皮(ARPE-19)细胞缺氧损伤的保护作用，从而探讨黄芪是否通过抗氧化应激来改善糖尿病视网膜病变(DR)。

方法：建立CoCl₂诱导ARPE-19细胞缺氧模型，并分为以下5组：正常组(细胞正常培养，不加任何处理)、缺氧模型组(200μmol/L CoCl₂)、空白血清组(200μmol/L CoCl₂+空白血清)、低剂量含药血清组(200μmol/L CoCl₂+10%含药血清)、高剂量含药血清组(200μmol/L CoCl₂+20%含药血清)。CCK-8检测细胞活性； 试剂盒检测细胞上清液中还原型谷胱甘肽(GSH)、丙二醛(MDA)水平； ELISA检测细胞培养基中缺氧诱导因子-1α(HIF-1α)、血管内皮生长因子(VEGF)的含量； 实时荧光定量PCR(qPCR)检测VEGF、HIF-1α和脯氨酰羟化酶-2(PHD-2)的mRNA水平； Western Blot法检测VEGF、HIF-1α和PHD-2的表达情况。

结果：采用200μmol/L的CoCl₂浓度可成功建立ARPE-19细胞缺氧模型。低剂量和高剂量黄芪含药血清可抑制缺氧诱导的ARPE-19细胞增殖(P<0.05)，升高ARPE-19细胞缺氧损伤中GSH水平，降低MDA含量(P<0.05)。低剂量和高剂量黄芪含药血清可抑制ARPE-19细胞缺氧损伤上清液中HIF-1α和VEGF的表达(P<0.05)，以及ARPE-19细胞中VEGF、HIF-1α、PHD-2的mRNA表达和蛋白表达(P<0.05)。

结论：低剂量和高剂量黄芪含药血清通过抗氧化作用减轻CoCl₂诱导ARPE-19细胞缺氧损伤。

AIM: To study the protective effect of astragalus-containing serum on cobalt chloride(CoCl₂)-induced hypoxia injury of human retinal pigment epithelial cells(ARPE-19), so as to explore whether astragalus can improve diabetic retinopathy(DR)by anti-oxidative stress.

METHODS: The ARPE-19 hypoxia model induced by CoCl₂ was established and divided into the following 5 groups: normal group(cells were cultured normally without any treatment), hypoxia model group(200μmol/L CoCl₂), blank serum group(200μmol/L CoCl₂+blank serum), low-dose drug-containing serum group(200μmol/L CoCl₂+10% medicated serum)and high-dose drug-containing serum group(200μmol/L CoCl₂+20% medicated serum); CCK-8 detects cell viability; Detect the levels of reduced glutathione(GSH)and malondialdehyde(MDA)in the cell supernatant with a kit; ELISA was used to detect the content of hypoxia-inducible factor-1α(HIF-1α)and vascular endothelial growth factor(VEGF)in cell culture medium; Real-time quantitative PCR(qPCR)to detect the mRNA levels of VEGF, HIF-1α and Prolyl hydroxylase-2(PHD-2); The expressions of VEGF, HIF-1α and PHD-2 were detected by Western Blot.

RESULTS: Hypoxia model of ARPE-19 can successfully establish by CoCl₂ at 200μmol/L. Low-dose and high-dose astragalus-containing serum could inhibit hypoxia-induced ARPE-19 proliferation(P<0.05), increase the GSH level and reduce the MDA content in ARPE-19 with hypoxic injury(P<0.05). Low-dose and high-dose astragalus-containing serum could inhibit the expression of HIF-1α and VEGF in ARPE-19 hypoxic injury supernatant(P<0.05), as well as the mRNA and protein expressions of VEGF, HIF-1α and PHD-2 in ARPE-19(P<0.05).

CONCLUSION: Low-dose and high-dose astragalus-containing serum alleviates the hypoxia injury of ARPE-19 induced by CoCl₂ through anti-oxidant effect.

国家自然科学基金资助项目(No.31502059); 湖北省教育厅科学研究计划指导性项目(No.B2015447)