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ENGLISH ABSTRACT
腐皮镰孢菌对角膜上皮细胞AMPK磷酸化及IL-6表达的影响
魏静静
谢艳亭
岳娟
董军璐
司玮
王春梅
张红敏
王丽娅
作者及单位信息
·
DOI： 10.3760/cma.j.cn115989-20200730-00544
Effects of Fusarium solani on AMPK phosphorylation and IL-6 expression in corneal epithelial cells
Wei Jingjing
Xie Yanting
Yue Juan
Dong Junlu
Si Wei
Wang Chunmei
Zhang Hongmin
Wang Liya
Authors Info & Affiliations
Wei Jingjing
People's Hospital of Zhengzhou University, Department of Ophthalmology, Henan Provincial People's Hospital, Henan Eye Hospital, Henan Eye Institute, Henan Key Laboratory of Ophthalmology and Visual Science, Zhengzhou 450003, China
Xie Yanting
People's Hospital of Zhengzhou University, Department of Ophthalmology, Henan Provincial People's Hospital, Henan Eye Hospital, Henan Eye Institute, Henan Key Laboratory of Ophthalmology and Visual Science, Zhengzhou 450003, China
Yue Juan
People's Hospital of Zhengzhou University, Department of Ophthalmology, Henan Provincial People's Hospital, Henan Eye Hospital, Henan Eye Institute, Henan Key Laboratory of Ophthalmology and Visual Science, Zhengzhou 450003, China
Dong Junlu
People's Hospital of Zhengzhou University, Department of Ophthalmology, Henan Provincial People's Hospital, Henan Eye Hospital, Henan Eye Institute, Henan Key Laboratory of Ophthalmology and Visual Science, Zhengzhou 450003, China
Si Wei
People's Hospital of Zhengzhou University, Department of Ophthalmology, Henan Provincial People's Hospital, Henan Eye Hospital, Henan Eye Institute, Henan Key Laboratory of Ophthalmology and Visual Science, Zhengzhou 450003, China
Wang Chunmei
People's Hospital of Zhengzhou University, Department of Ophthalmology, Henan Provincial People's Hospital, Henan Eye Hospital, Henan Eye Institute, Henan Key Laboratory of Ophthalmology and Visual Science, Zhengzhou 450003, China
Zhang Hongmin
People's Hospital of Zhengzhou University, Department of Ophthalmology, Henan Provincial People's Hospital, Henan Eye Hospital, Henan Eye Institute, Henan Key Laboratory of Ophthalmology and Visual Science, Zhengzhou 450003, China
Wang Liya
People's Hospital of Zhengzhou University, Department of Ophthalmology, Henan Provincial People's Hospital, Henan Eye Hospital, Henan Eye Institute, Henan Key Laboratory of Ophthalmology and Visual Science, Zhengzhou 450003, China
·
DOI： 10.3760/cma.j.cn115989-20200730-00544
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摘要

目的研究角膜上皮细胞是否存在腺嘌呤核糖核苷酸活化的蛋白激酶(AMPK)磷酸化及真菌对角膜上皮细胞AMPK磷酸化和白细胞介素6(IL-6)表达的影响。

方法取人永生化角膜上皮细胞系，采用实时无标记细胞多功能分析仪筛选AMPK激动剂5-氨基-4-甲酰胺咪唑核糖核苷酸(AICAR)(100、300、500和1 000 μmol/L)和抑制剂化合物C(10.0、12.5、15.0、17.5和20.0 μmol/L)对角膜上皮细胞作用的安全浓度范围。以单纯角膜上皮细胞作为正常对照组，角膜上皮细胞与孢子共培养作为孢子对照组，在孢子对照组基础上分别加入AICAR和化合物C作为AICAR组和化合物C组，分别培养4 h。采用Western blot法检测角膜上皮细胞磷酸化AMPK(p-AMPK)和AMPK的表达，采用酶联免疫吸附测定(ELISA)法测定角膜上皮细胞培养上清液中IL-6质量浓度。

结果不同浓度AICAR作用于角膜上皮细胞不同时间后，细胞指数总体比较差异均无统计学意义(均 P>0.05)。10.0 μmol/L和12.5 μmol/L化合物C作用于角膜上皮细胞后细胞指数较未处理细胞升高。正常对照组、孢子对照组、AICAR组和化合物C组p-AMPK水平分别为0.67±0.15、2.57±0.12、3.67±0.58和1.50±0.50，各组间总体比较差异有统计学意义( F＝32.820， P<0.001)。孢子对照组p-AMPK水平较正常对照组升高，差异有统计学意义( P<0.001)；AICAR组较孢子对照组p-AMPK水平升高，化合物C组较孢子对照组p-AMPK水平降低，差异均有统计学意义(均 P＝0.010)。正常对照组、孢子对照组、AICAR组和化合物C组AMPK相对表达量总体比较差异无统计学意义( F＝0.120， P＝0.950)。正常对照组、孢子对照组、AICAR组和化合物C组IL-6质量浓度分别为(107.81±17.15)、(156.32±9.94)、(167.96±14.16)和(127.42±19.75)pg/ml，各组间总体比较差异有统计学意义( F＝15.210， P<0.001)，其中孢子对照组IL-6质量浓度较正常对照组升高，差异有统计学意义( P<0.001)；AICAR组IL-6质量浓度较孢子对照组略升高，差异无统计学意义( P＝0.260)，化合物C组IL-6质量浓度较孢子对照组降低，差异有统计学意义( P＝0.010)。

结论角膜上皮细胞有AMPK磷酸化表达，且真菌孢子刺激角膜上皮细胞后AMPK磷酸化增强，IL-6分泌增多。

真菌;角膜炎;上皮细胞;白细胞介素6;腺嘌呤核糖核苷酸活化的蛋白激酶;磷酸化
ABSTRACT

ObjectiveTo investigate the expression of adenosine 5'-monophosphate-activated protein kinase (AMPK) phosphorylation in corneal epithelial cells and the effects of fungus on AMPK phosphorylation and interleukin-6 (IL-6) production in corneal epithelial cells.

MethodsThe human immortalized corneal epithelial cell line was selected.The safe concentration range of AMPK agonist 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) (100, 300, 500, 1 000 μmol/L) and inhibitor Compound C (10.0, 12.5, 15.0, 17.5, 20.0 μmol/L) on corneal epithelial cells was screened by multi-function real-time unlabeled cell analyzer.Corneal epithelial cells without any treatment were used as the normal control group, and those co-cultured with spores were used as the spore control group.Corneal epithelial cells co-cultured with spores were treated with AICAR and Compound C for 4 hours in the AICAR group and Compound C group, respectively.The expression of phosphorylated AMPK (p-AMPK) and AMPK in corneal epithelial cells was detected by Western blot, and the concentration of IL-6 in the culture supernatant was determined by enzyme-linked immunosorbent assay (ELISA).

ResultsAfter treatment with different concentrations of AICAR for different periods, there was no statistical significance in the cell index of corneal epithelial cells (all at P>0.05). The cell index of corneal epithelial cells was increased with 10.0 μmol/L and 12.5 μmol/L Compound C treatment compared with that of the normal control group.The expression levels of p-AMPK were 0.67±0.15, 2.57±0.12, 3.67±0.58 and 1.50±0.50, respectively, in the normal control group, spore control group, AICAR group and Compound C group, showing a statistically significant difference among them ( F=32.820, P<0.001). The expression level of p-AMPK was significantly higher in the spore control group compared with the normal control group ( P<0.001). The expression level of p-AMPK in the AICAR group was higher than that in the spore control group, and the expression level of p-AMPK in the Compound C group was lower than that in the spore control group, and the differences were statistically significant (both at P=0.010). There was no significant difference in the relative expression level of AMPK among the four groups ( F=0.120, P=0.950). The expression levels of IL-6 concentration in the normal control group, spore control group, AICAR group and Compound C group were (107.81±17.15), (156.32±9.94), (167.96±14.16) and (127.42±19.75)pg/ml, respectively, showing a statistically significant difference among them ( F=15.210, P<0.001). The IL-6 concentration of the spore control group was higher than that of the normal control group, and the difference was statistically significant ( P<0.001). The IL-6 concentration of the AICAR group was higher than that of the spore control group, but the difference was not statistically significant ( P=0.260). The IL-6 concentration of the Compound C group was lower than that of the spore control group, and the difference was statistically significant ( P=0.010).

ConclusionsIn corneal epithelial cells, AMPK phosphorylation is found, which is enhanced after fungal spores stimulation, and the secretion of IL-6 increases.

Fungi;Keratitis;Epithelial cells;Interleukin-6;Adenine ribonucleotide activated protein kinase;Phosphorylation
Zhang Hongmin, Email: mocdef.3ab616090mhz
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引用本文

魏静静,谢艳亭,岳娟,等. 腐皮镰孢菌对角膜上皮细胞AMPK磷酸化及IL-6表达的影响[J]. 中华实验眼科杂志,2022,40(02)：133-138.

DOI:10.3760/cma.j.cn115989-20200730-00544

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腺嘌呤核糖核苷酸活化的蛋白激酶(adenosine 5’-monophosphate-activated protein kinase，AMPK)是一种由α、β和γ亚基组成的异源三聚体蛋白激酶，参与调节葡萄糖、脂质以及蛋白质的代谢和维持能量稳态，被称为细胞能量调节器 [ 1 , 2 ]。近年来研究发现，AMPK信号通路除在能量代谢调节方面起关键作用外，在感染和宿主防御方面也起到重要作用 [ 3 , 4 ]。真菌性角膜炎是由致病真菌引起的严重感染性、致盲眼病，临床抗真菌类滴眼制剂种类有限，治疗棘手 [ 5 , 6 ]。位于角膜组织最外层的上皮细胞是角膜防御致病微生物侵袭的第一道屏障，是防御真菌感染的关键，目前角膜上皮细胞是否存在AMPK磷酸化尚不清楚。在真菌性角膜炎炎症初期及进展期，大量多形核中性粒细胞浸润 [ 7 ]，白细胞介素6(interleukin-6，IL-6)是一种由184个氨基酸组成的单链糖蛋白 [ 8 ]，其能促进多形核中性粒细胞的激活 [ 9 ]。IL-6以自分泌-旁分泌的方式起作用，诱导驻留的角膜细胞产生巨噬细胞炎性蛋白(macrophage inflammatory protein，MIP)-2和MIP-1α [ 10 ]，MIP能促进角膜局部炎症免疫细胞聚集，从而发挥杀菌作用。本实验拟研究角膜上皮细胞是否存在AMPK磷酸化以及真菌对角膜上皮细胞AMPK磷酸化及IL-6表达的影响，探讨AMPK磷酸化和IL-6在真菌性角膜炎中的作用。
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备注信息
A
张红敏，Email： mocdef.3ab616090mhz
B

魏静静：设计实验、实验操作、论文撰写、数据整理、统计分析；谢艳亭、岳娟、董军璐、司玮、王春梅：实验操作、数据分析；张红敏：设计实验、研究指导、论文修改、经费支持；王丽娅：论文修改，研究指导

C
所有作者均声明不存在任何利益冲突
D
国家自然科学基金面上项目 (81770902)
国家自然科学基金河南联合基金重点支持项目 (U1704283)
河南省立眼科医院基础研究重点专项项目 (21JCZD003)
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