[1]陈晓娟,曹鑫,薛理丹,等.高迁移率族蛋白B1对高糖诱导的晶状体上皮细胞凋亡和自噬的影响[J].眼科新进展,2023,43(1):013-18.[doi:10.13389/j.cnki.rao.2023.0003]
　CHEN Xiaojuan,CAO Xin,XUE Lidan,et al.Effects of high mobility group box 1 protein on apoptosis and autophagy of lens epithelial cells induced by high glucose[J].Recent Advances in Ophthalmology,2023,43(1):013-18.[doi:10.13389/j.cnki.rao.2023.0003]
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高迁移率族蛋白B1对高糖诱导的晶状体上皮细胞凋亡和自噬的影响/HTML
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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
43卷
期数:
2023年1期
页码:
013-18
栏目:
实验研究
出版日期:
2023-01-05

文章信息/Info

Title:
Effects of high mobility group box 1 protein on apoptosis and autophagy of lens epithelial cells induced by high glucose
作者:
陈晓娟; 曹鑫; 薛理丹; 宋愈
226006　江苏省南通市，南通大学第二附属医院眼科
Author(s):
CHEN Xiaojuan; CAO Xin; XUE Lidan; SONG Yu
Department of Ophthalmology,the Second Affiliated Hospital of Nantong University,Nantong 226006,Jiangsu Province,China
关键词:
高迁移率族蛋白B1; 糖尿病性白内障; 晶状体上皮细胞; 凋亡; 自噬
Keywords:
high mobility group box 1;  diabetic cataract;  lens epithelial cells;  apoptosis;  autophagy
分类号:
R776.1
DOI:
10.13389/j.cnki.rao.2023.0003
文献标志码:
A
摘要:
目的　探讨高迁移率族蛋白B1（HMGB1）对高糖诱导的晶状体上皮细胞（LEC）凋亡和自噬的影响。
方法　本研究以糖尿病性白内障患者和年龄相关性白内障（ARC）患者晶状体前囊膜以及高糖和正常糖条件下培养的人晶状体上皮细胞株（SRA01/04）为研究对象。选取2021年10月至2022年3月在南通大学第二附属医院眼科确诊为白内障的患者20例，依据白内障类型分为糖尿病性白内障组（DC组）和ARC组，每组各10例，取患者晶状体前囊膜进行实验。将细胞分为正常糖组和高糖组，正常糖组培养基含5.5 mmol·L-1葡萄糖，高糖组培养基含25.0 mmol·L-1葡萄糖，处理24 h后进行实验。采用Western blot检测各组HMGB1蛋白的表达水平。利用siRNA技术对HMGB1进行敲降，将细胞分为正常对照组、scr-siRNA组和si-HMGB1组。正常对照组：不作任何处理；scr-siRNA组：加入等量的Lipofectamine 3000和scr-siRNA；si-HMGB1组：加入等量的Lipofectamine 3000和si-HMGB1。三组细胞均用含5.5 mmol·L-1葡萄糖的培养基培养48 h。采用实时荧光定量PCR实验检测HMGB1 mRNA的表达情况以验证敲降效率。随后将细胞分为正常糖组、高糖组、高糖+scr-siRNA组、高糖+si-HMGB1组。高糖+scr-siRNA组、高糖+si-HMGB1组细胞先进行相应转染，待收集细胞前24 h，正常糖组细胞用含5.5 mmol·L-1葡萄糖培养基培养，高糖组、高糖+scr-siRNA组、高糖+si-HMGB1组细胞用含25.0 mmol·L-1葡萄糖的培养基培养。采用Western blot检测各组细胞凋亡相关蛋白（Bax和Bcl-2）和自噬相关蛋白（LC3B和p62）的表达水平，采用TUNEL法检测细胞凋亡情况。
结果　DC组患者晶状体前囊膜HMGB1蛋白相对表达量为1.18±0.02，高于ARC组（1.00±0.02），差异有统计学意义（P<0.001）；高糖组SRA01/04细胞中HMGB1蛋白相对表达量同样高于正常糖组，差异有统计学意义（P<0.001）。si-HMGB1组SRA01/04细胞HMGB1 mRNA相对表达量显著低于scr-siRNA组，差异有统计学意义（P<0.001）。高糖组SRA01/04细胞中促凋亡蛋白Bax的表达水平高于正常糖组，而抗凋亡蛋白Bcl-2表达水平低于正常糖组，差异均有统计学意义（均为P<0.05）。敲降HMGB1后，高糖+si-HMGB1组SRA01/04细胞中Bax的表达水平低于高糖+scr-siRNA组，而Bcl-2表达水平高于高糖+scr-siRNA组，差异均有统计学意义（均为P<0.05）。高糖组SRA01/04细胞LC3II的表达水平高于正常糖组，而p62的表达水平低于正常糖组，差异均有统计学意义（均为P<0.05）；HMGB1敲降后，高糖+si-HMGB1组SRA01/04细胞LC3II的表达水平低于高糖+scr-siRNA组，而p62的表达水平高于高糖+scr-siRNA组，差异均有统计学意义（均为P<0.05）。
结论　HMGB1可能通过调控LEC凋亡和自噬过程参与糖尿病性白内障的发生发展。
Abstract:
Objective　To investigate the effects of high mobility group box 1 (HMGB1) protein on the high glucose-induced apoptosis and autophagy of lens epithelial cells (LEC).
Methods　The anterior lens capsules from patients with diabetic cataract (DC) and age-related cataract (ARC) and human lens epithelial cell line (SRA01/04) cultured under high glucose (HG) and normal glucose (NG) conditions were chosen for the study. Twenty patients diagnosed with cataract in the Department of Ophthalmology of the Second Affiliated Hospital of Nantong University from October 2021 to March 2022 were selected. According to the types of cataracts, the patients were divided into DC group and ARC group, with 10 patients in each group. The anterior lens capsules of the patients were extracted for the experiments. The cells were divided into the NG group (cultured in the medium with 5.5 mmol·L-1 glucose) and HG group (cultured in the medium with 25.0 mmol·L-1 glucose), which were studied after treatment for 24 h, respectively. The expression level of HMGB1 protein was detected by Western Blot. HMGB1 knockdown was performed by small interfering ribonucleic acid (siRNA) technology, and the cells were divided into normal control group (no treatment was made), scrambled small interfering ribonucleic acid (scr-siRNA) group (same amount of Lipofectamine 3000 and scr-siRNA were added) and small interfering HMGB1 (si-HMGB1) group (same amount of Lipofectamine 3000 and si-HMGB1 were added). The cells in the three groups were cultured in the medium containing 5.5 mmol·L-1 glucose for 48 h. Real-time quantitative PCR was used to detect the expression of HMGB1 messenger ribonucleic acid (mRNA) to verify the knockdown efficiency. Then the cells were divided into the NG group, HG group, HG + scr-siRNA group and HG + si-HMGB1 group. The transfection was carried out on cells in the HG + scr-siRNA group and HG + si-HMGB1 group until 24 h before cell collection. The cells in the NG group were cultured in the medium containing 5.5 mmol·L-1 glucose, while the cells in the HG group, the HG + scr-siRNA group and the HG + si-HMGB1 group were cultured in the medium with 25.0 mmol·L-1 glucose. The expression levels of apoptosis-related proteins (Bax and Bcl-2) and autophagy-related proteins (LC3B and p62) were detected by Western Blot. The apoptosis rate was examined by TUNEL assay.
Results　The relative expression level of HMGB1 protein from the anterior lens capsules of patients in the DC group was 1.18±0.02, which was higher than that in the ARC group (1.00±0.02), and the difference was statistically significant (P<0.001). The relative expression level of HMGB1 protein from the SRA01/04 cells in the HG group was also significantly higher than that in the NG group (P<0.001). The relative expression level of HMGB1 mRNA from SRA01/04 cells in the si-HMGB1 group was significantly lower than that in the scr-siRNA group (P<0.001). The expression level of pro-apoptotic protein Bax from SRA01/04 cells in the HG group was higher than that in the NG group, while the expression level of anti-apoptotic protein Bcl-2 was lower than that in the NG group (both P<0.05). After HMGB1 knockdown, the expression level of Bax from SRA01/04 cells in the HG + si-HMGB1 group was lower than that in the HG + scr-siRNA group, while the expression level of Bcl-2 was higher than that in the HG + scr-siRNA group, showing significant differences (both P<0.05). The expression level of LC3II from SRA01/04 cells in the HG group was higher than that in the NG group, while the expression level of p62 was lower than that in the NG group, and the differences were statistically significant (both P<0.05).
Conclusion　HMGB1 may be involved in the occurrence and development of DC via regulating the apoptosis and autophagy of LEC.

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备注/Memo

备注/Memo:
南通市科学技术局项目（编号：JC2021188，MSZ21084）
更新日期/Last Update: 2023-01-05