血红素氧合酶1对高糖诱导人晶状体上皮细胞的氧化应激和凋亡的影响

张奇; 马天驹; 黄杨; 李朝辉

doi:10.3969/j.issn.2095-5227.2021.04.011

摘要:

背景白内障是全世界最常见的致盲原因，在我国致盲性眼病中占比最高。近年来医疗水平虽有进步，但手术仍是现阶段治疗白内障最有效的方法。

目的研究血红素氧合酶1(heme oxygenase-1，HO-1)对高糖诱导人晶状体上皮细胞的氧化应激和凋亡的影响，为研发治疗糖尿病性白内障的药物奠定理论基础。

方法将晶状体上皮细胞(SRA01/04)暴露于不同浓度(5 mmol/L，15 mmol/L，30 mmol/L，50 mmol/L，100 mmol/L)的葡萄糖溶液中24 h，用CCK-8检测各组细胞的增殖活力，选择最佳葡萄糖浓度。晶状体上皮细胞(SRA01/04)暴露于5 mmol/L (Control)和100 mmol/L (D)浓度的葡萄糖、HO-1的诱导剂(钴原卟啉；cobalt protoporphyrin，CoPP)和HO-1的抑制剂(锌原卟啉；zinc protoporphyrin，ZnPP)下，将实验分为6组：Control 5 mmol/L组，Control 5 mmol/L + CoPP 10 μmol/L组，Control 5 mmol/L + ZnPP 10 μmol/L组，D 100 mmol/L组，D 100 mmol/L + CoPP 10 μmol/L组，D 100 mmol/L + ZnPP 10 μmol/L组。流式细胞术测定各组细胞内活性氧(reactive oxygen species，ROS)相对含量。Annexin V/PI对各组细胞凋亡染色，流式细胞仪器检测细胞凋亡情况。

结果晶状体上皮细胞(SRA01/04)暴露于100 mmol/L浓度的葡萄糖中，细胞增殖活力显著下降(P＜0.05)，此为本实验的最佳浓度。流式细胞仪测得各组ROS相对含量：Control 5 mmol/L组4.70% ± 1.27%，Control 5 mmol/L + CoPP 10 μmol/L组2.37% ± 1.37%，Control 5 mmol/L + ZnPP 10 μmol/L组34.4% ± 1.72%，D 100 mmol/L组51.63% ± 1.74%，D 100 mmol/L + CoPP 10 μmol/L组43.37% ± 2.08%，D 100 mmol/L + ZnPP 10 μmol/L组60.10% ± 1.54%，表明高糖会导致细胞内ROS相对含量升高，HO-1的表达上调会导致细胞内的ROS相对含量下降；反之，HO-1表达下调时，细胞内的ROS相对含量增多。用流式细胞仪器检测上述各组细胞凋亡比例分别为3.05% ± 0.35%、1.69% ± 0.53%、7.46% ± 1.85%、39.56% ± 0.50%、33.72% ± 2.05%、44.53% ± 0.48%，表明高糖会导致细胞凋亡比例升高，HO-1的表达上调会导致细胞凋亡比例下降；反之，HO-1表达下调时细胞凋亡比例升高。

结论上述研究结果表明通过对HO-1表达水平的上调，降低了细胞内ROS相对含量，从而减少了细胞的凋亡，对高糖诱导的人晶状体上皮细胞损伤起保护作用。因此HO-1是一种潜在治疗糖尿病性白内障的干预措施。

Abstract:

Background Cataract is the most common cause of blindness in the world, and ranks the first in the causes of blindness in China. Despite medical advances, surgery is still the most effective therapy for cataract at present.

Objective To investigate the effect of heme oxygenase-1 (HO-1) on oxidative response and apoptosis of human lens epithelial cells induced by high glucose, so as to provide evidences for the development of drugs for the treatment of diabetic cataract.

Methods Lens epithelial cells (SRA01/04) were exposed to different concentrations of glucose (5 mmol/L, 15 mmol/L, 30 mmol/L, 50 mmol/L, 100 mmol/L) for 24 h. The proliferation activity of cells in each group was determined by CCK-8, and the optimal experimental concentration of glucose was selected. Lens epithelial cells (SRA01/04) were exposed to high concentrations of glucose (D 100 mmol/L), HO-1 inducer (cobalt protoporphyrin, CoPP) and HO-1 inhibitor (zinc protoporphyrin, ZnPP); they were divided into control 5 mmol/L group, control 5 mmol/L + COPP 10 μmol/L group, control 5 mmol/L + ZnPP10 μmol/L group, D 100 mmol/L group, D 100 mmol/L + COPP 10 μmol/L group, and D 100 mmol/L + ZnPP 10 μmol/L group. Then the content of intracellular ROS in each group was determined by Rhydride cell assay. Finally, Annexin V/PI staining was used to detect the apoptosis of cells in each group, and flow cytometry was used to detect the apoptosis.

Results Lens epithelial cells (SRA01/04) exposed to 100mmol/L glucose showed the lowest cell proliferation activity (P < 0.05), which was selected as the optimal concentration of this experiment. The content of ROS in each group was 4.70% ± 1.27% in the control group, 2.37% ± 1.37% in the control + COPP group, 34.4% ± 1.72% in the control + ZnPP group, 51.63% ± 1.74% in the D group, 43.37% ± 2.08% in the D + COPP group, 60.10% ± 1.54% in the D + ZnPP group, respectively; High glucose led to the increase of intracellular ROS content, and the up-regulation of HO-1 expression led to the decrease of intracellular ROS content. On the contrary, when the HO-1 expression was down-regulated, intracellular ROS content was produced in large quantities. The apoptosis ratio detected by flow cytometry was 3.05% ± 0.35% in the control group, 1.69% ± 0.53% in the control + COPP group, 7.46% ± 1.85% in the control + ZnPP group, 39.56% ± 0.50% in the D group, 33.72% ± 2.05% in the D + COPP group, and 44.53% ± 0.48% in the D + ZnPP group, respectively. High glucose led to the increase of cell apoptosis ratio, and the up-regulation of HO-1 expression caused a decline in apoptosis ratio; on the other hand, the down regulation of HO-1 expression increased cell apoptosis.

Conclusion These results indicate that the up-regulation of HO-1 expression level can reduce the intracellular ROS content, thereby reducing cell apoptosis, which suggests that it plays a protective role in the high glucose induced injury of human lens epithelial cells. Therefore, HO-1 is a potential intervention for the treatment of diabetic cataract.

血红素氧合酶1对高糖诱导人晶状体上皮细胞的氧化应激和凋亡的影响

Effect of heme oxygenase 1 (HO-1) on oxidative stress and apoptosis induced by high glucose in human lens epithelial cells