高糖对体外培养视网膜Müller细胞自噬及凋亡的影响

doi:10.3760/cma.j.cn115909-20210408-00143

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摘要目的：探究高糖诱导体外培养的视网膜Müller细胞自噬及凋亡的变化。方法：实验研究。将体外培养的SD大鼠视网膜Müller细胞根据不同糖浓度分为5.5 mmol/L葡萄糖组（正常组）及20、30、40、 50 mmol/L高糖组，并对40 mmol/L高糖处理Müller细胞组采用不同时间3、6、12、24、36 h进行培养。使用蛋白免疫印迹法及细胞免疫荧光检测自噬指标LC3Ⅰ/LC3Ⅱ、自噬信号通路P62、Beclin-1 蛋白表达及细胞定位；使用透射电镜及GFP-LC3慢病毒转染观察自噬小体的形成情况；使用TUNEL 实验检测细胞凋亡状态。不同组别间数据比较采用单因素方差分析。结果：正常组及20、30、40、 50 mmol/L高糖组中Müller细胞质的Beclin-1、P62及LC3Ⅰ/LC3Ⅱ蛋白相对表达量总体比较，差异均有统计学意义（F=131.21，P=0.029；F=197.25，P=0.012；F=100.02，P=0.045），其中与正常组比较各浓度高糖组Müller细胞质内Beclin-1、LC3Ⅰ/LC3Ⅱ蛋白均明显降低（P<0.05），P62蛋白相对表达量明显增加（P<0.001）。正常组以及6、12、24、36 h高糖组中Müller细胞质的Beclin-1、P62及LC3Ⅰ/LC3Ⅱ 蛋白相对表达量总体比较，差异均有统计学意义（F=98.46，P=0.015；F=109.48，P=0.004；F=99.27， P=0.032），其中6 h高糖组LC3Ⅰ/LC3Ⅱ、Beclin-1蛋白明显增加（P=0.002、0.015），12、24、36 h高糖组与正常组相比较LC3Ⅰ/LC3Ⅱ、Beclin-1蛋白均明显下降（P<0.05），P62蛋白明显增加（P<0.001）。正常组及6、24 h高糖组视网膜Müller细胞中GFP-LC3转染阳性颗粒、自噬小体相对数量组间总体差异有统计学意义（F=240.49，P=0.014；F=198.98，P=0.001），其中与正常组比较，6 h高糖组GFP-LC3 转染阳性颗粒、自噬小体相对数量增加（P=0.002、0.029），24 h高糖组明显降低（P=0.019、0.036）。正常组及6、24 h高糖组及3MA组视网膜Müller细胞凋亡指数分别为3.14±1.01、15.34±3.87、 25.82±4.96、24.79±4.01，总体比较差异有统计学意义（F=234.12，P=0.003），与正常组比较，6、24 h 高糖组TUNEL阳性细胞增多（P=0.022、0.039），24 h高糖组与6 h高糖组比较TUNEL 阳性细胞进一步增加（P=0.017）。结论：高糖诱导体外培养的视网膜Müller细胞早期自噬增加，细胞凋亡抑制，随着时间不断延长，高糖抑制自噬形成，增加细胞凋亡。高糖抑制自噬可能是促进Müller细胞凋亡的重要原因。

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关键词 ：糖尿病视网膜病变, 高糖, 自噬, 凋亡, Müller细胞

Abstract：Objective: To investigate whether high glucose (HG) can regulate the autophagy and apoptosis in Müller cells. Methods: In this experimental research, primary SD rat retinal Müller cells were exposed to normal glucose (NG) or 3 h, 6 h, 12 h, 24 h, 36 h of high glucose. And Müller cells were exposed to 5.5, 20, 30, 40, 50 mmol/L for 24 h. LC3Ⅰ/LC3Ⅱ, P62, and Beclin-1 protein expression was examined by Western blot analysis in the various experimental groups. And in fluorescence microscopy experiments, autophagy was evaluated by the autophagy markers LC3Ⅰ/LC3Ⅱ and P62. The formation of autophagosomes and autolysosomes were examined by electron microscopy. TUNEL assay was used to detect apoptosis in high glucose Müller cells. One-way analysis of variance was used to compare data between different groups. Results: The relative expression levels of Beclin-1, P62 and LC3Ⅰ/LC3Ⅱ in Müller cytoplasm of the normal glucose group, and 20 mmol/L, 30 mmol/L, 40 mmol/L and 50 mmol/L high glucose groups were compared. There were statistically significant differences (F=131.21, P=0.029; F=197.25, P=0.012; F=100.02, P=0.045). Compared to the normal group, Beclin-1 and LC3Ⅰ/LC3Ⅱ protein in Müller cytoplasm of the high glucose groups significantly decreased (P<0.05). The relative expression of P62 protein significantly increased (P<0.001). The relative expression levels of autophagy related protein Beclin-1, P62 and LC3Ⅰ/LC3 in Müller cytoplasm of the normal group and high glucose groups at 6 h, 12 h, 24 h and 36 h were statistically significant (F=98.46, P=0.015; F=109.48, P=0.004; F=99.27, P=0.032). LC3Ⅰ/LC3 and Beclin-1 protein significantly increased in the 6 h high glucose group (P=0.002, 0.015); LC3Ⅰ/LC3Ⅱ in and Beclin-1 protein significantly decreased in 12 h, 24 h and 36 h compared to the normal group (P<0.05), but the P62 protein significantly increased (P<0.001). There were statistically significant differences in the relative number of GFP-LC3-transfected positive particles and autophagosomes in the normal group, and high glucose 6 h and 24 h retinal Müller cells (F=240.49, P=0.014; F=198.98, P=0.001). Compared to the normal group, the relative number of GFP-LC3-transfected positive particles and autophagosomes in the 6h group increased. There were statistically significant difference (P=0.002, 0.029) and there were significant decreases in 24 h (P=0.019, 0.036). The apoptosis indexes of retinal Müller cells were 3.14±1.01, 15.34±3.87, 25.82±4.96 and 24.79±4.01 in the normal group, 6 h and 24 h groups and 3MA group, respectively, and the overall difference was statistically significant (F=234.12, P=0.003). Compared to the normal group, the TUNEL positive cells increased in the 6 h and 24 h groups (P=0.022, 0.039), and the TUNEL positive cells further increased in the 6 h and 24 h groups (P=0.017). Conclusions: HG can induce early autophagy and inhibit apoptosis in cultured retinal Müller cells, and HG can inhibit autophagy and inrease apoptosis over time. This study suggests that high glucose inhibition of autophagy may be an important factor in promoting the apoptosis of Müller cells.

Key words： diabetes retinopathy high glucose autophagy apoptosis Müller cells

收稿日期: 2021-04-08

PACS:

基金资助:苏州市民生科技项目（SYS2018004，SS201758）；江苏省自然科学基金（BK20191177）

通讯作者: 宋鄂（ORCID：0000-0003-4259-9739），Email：songe@suda.edu.cn

引用本文:

王立, 颜世传, 牛兰俊, 等. . 高糖对体外培养视网膜Müller细胞自噬及凋亡的影响[J]. 中华眼视光学与视觉科学杂志, 2022, 24(1): 17-26. Li Wang,Shichuan Yan,Lanjun Niu,et al. Autophagy and Apoptosis of Retinal Müller Cells Induced by High Glucose. Chinese Journal of Optometry Ophthalmology and Visual science, 2022, 24(1): 17-26. DOI: 10.3760/cma.j.cn115909-20210408-00143

链接本文:

https://www.cjoovs.com/CN/10.3760/cma.j.cn115909-20210408-00143 或 https://www.cjoovs.com/CN/Y2022/V24/I1/17

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