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中华眼科医学杂志(电子版) ›› 2023, Vol. 13 ›› Issue (03) : 146 -151. doi: 10.3877/cma.j.issn.2095-2007.2023.03.004

论著

水通道蛋白1对人角膜内皮细胞增殖、迁移及凋亡影响的实验研究
樱峰, 王静, 刘雪清†(), 李潇   
  1. 830063　乌鲁木齐，新疆医科大学第二附属医院眼科
  • 收稿日期:2023-01-17 出版日期:2023-06-28
  • 通信作者: 刘雪清
  • 基金资助:
    新疆维吾尔自治区自然科学基金项目(2022D01C217)

The effects of aquaporin 1 on the proliferation, migration, and apoptosis of human corneal endothelial cells

Feng Ying, Jing Wang, Xueqing Liu†(), Xiao Li   

  1. Ophthalmology Department of the Second Affiliated Hospital of Xinjiang Medical University, Urumqi 830063, China
  • Received:2023-01-17 Published:2023-06-28
  • Corresponding author: Xueqing Liu
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樱峰, 王静, 刘雪清, 李潇. 水通道蛋白1对人角膜内皮细胞增殖、迁移及凋亡影响的实验研究[J]. 中华眼科医学杂志(电子版), 2023, 13(03): 146-151.

Feng Ying, Jing Wang, Xueqing Liu, Xiao Li. The effects of aquaporin 1 on the proliferation, migration, and apoptosis of human corneal endothelial cells[J]. Chinese Journal of Ophthalmologic Medicine(Electronic Edition), 2023, 13(03): 146-151.

目的

探讨水通道蛋白1(AQP1)对人角膜内皮细胞(HCE)增殖、迁移及凋亡影响的机制。

方法

以质粒互补脱氧核糖核酸3.1(pcDNA3.1)为载体，将AQP1过表达质粒(pcDNA3.1－AQP1)、空载对照质粒(pcDNA3.1－NC)、AQP1小干扰核糖核酸序列(siRNA－AQP1)及对照序列(siRNA－NC)分别转染于HCE细胞内。按其转染类别分为非转染对照组(Control组)、pcDNA3.1－AQP1组、pcDNA3.1－NC组、siRNA－AQP1组及siRNA－NC组。采用实时荧光定量聚合酶链反应法、5－溴－2－脱氧尿嘧啶实验、Transwell小室法及流式细胞术分别检测细胞的AQP1信使核糖核酸表达量、增殖能力、迁移能力及凋亡情况。为明确p38丝裂原活化蛋白激酶(p38 MAPK)在其机制中的作用，在pcDNA3.1－AQP1组添加p38 MAPK激活剂10 μmol/L茴香霉素，并将其命名为pcDNA3.1－AQP1+茴香霉素组，并采用蛋白印迹法检测紧密连接蛋白1(TJP1)、钠钾三磷酸腺苷酶、p38 MAPK及磷酸化p38 MAPK蛋白的表达含量。各组细胞AQP1信使核糖核酸相对表达量、细胞增殖活性、迁移率、凋亡率、TJP1、钠钾三磷酸腺苷酶蛋白表达量及磷酸化p38 MAPK/p38 MAPK比值均满足方差齐性和正态分布，以±s表示，组间比较采用独立样本t检验。

结果

Control组、pcDNA3.1－NC组、siRNA－NC组、pcDNA3.1－AQP1组及siRNA－AQP1组HCE细胞AQP1信使核糖核酸相对表达量分别为(1.00±0.08)、(1.05±0.07)、(0.97±0.07)、(2.14±0.12)及(0.33±0.02)。与Control组比较，pcDNA3.1－NC组和siRNA－NC组比较的差异均无统计学意义(t＝0.653，0.684；P>0.05)，说明pcDNA3.1－NC组和siRNA－NC组未影响细胞，故后续指标检测不再检测该两组。pcDNA3.1－AQP1组HCE细胞增殖活性、细胞迁移率、细胞凋亡率、TJP1、钠钾三磷酸腺苷酶蛋白表达量及磷酸化p38 MAPK/p38 MAPK比值分别为(63.31±7.35)%、(74.28±7.04)%、(3.64±1.48)%、(1.73±0.13)、(2.04±0.15)及(0.18±0.02)；siRNA－AQP1组分别为(22.15±3.26)%、(35.73±3.86)%、(20.35±2.83)%、(0.23±0.02)、(0.21±0.02)及(0.75±0.09)；pcDNA3.1－AQP1+茴香霉素组分别为(52.35±4.38)%、(58.74±5.42)、(6.73±0.53)%、(1.38±0.21)、(1.18±0.19)及(0.33±0.03)。pcDNA3.1－AQP1组与Control组各指标比较的差异均有统计学意义(t＝3.844，3.534，3.253，4.253，4.753，4.321；P<0.05)；siRNA－AQP1组与Control组各指标比较的差异均有统计学意义(t＝4.136，3.741，3.621，3.426，4.122，4.894，3.795；P<0.05)；pcDNA3.1－AQP1+茴香霉素组与pcDNA3.1－AQP1组各指标比较的差异均有统计学意义(t＝3.251，3.363，3.053，3.242，3.674，4.264；P<0.05)。

结论

AQP1过表达能够促进HCE细胞增殖和迁移，抑制细胞凋亡，并参与维持HCE细胞功能，其作用机制可能与抑制p38 MAPK信号通路激活有关。

关键词: 水通道蛋白1, 角膜内皮细胞, 增殖, 迁移, 凋亡, p38丝裂原活化蛋白激酶信号通路
Objective

The aim of this study is to investigate the effects of aquaporin 1 (AQP1) on proliferation, migration and function maintenance of human corneal endothelial (HCE) cells and its related mechanisms.

Methods

pcDNA3.1 as a carrier, AQP1 overexpression plasmid (pcDNA3.1-AQP1), empty control plasmid (pcDNA3.1-NC), AQP1 small interfering ribonucleic acid sequence (siRNA-AQP1), and control sequence (siRNA-NC) were transfected into HCE cells, respectively. According to transfection types, cells were divided into non transfection control group (Control group), pcDNA3.1-AQP1 group, pcDNA3.1-NC group, siRNA-AQP1 group, and siRNA-NC group. The effect of AQP1 on the proliferation, migration, and apoptosis of HCE cells were evaluated.The expression of AQP1 mRNA in cells was detected by qRT-PCR. The ability of cell proliferation was detected by EdU assay. The cell migration ability was detected by Transwell chamber method. The apoptosis was detected by flow cytometry.To clarify the role of p38 mitogen activated protein kinase (p38 MAPK) signaling pathways in its mechanisms, p38 MAPK activator 10 μmol/L anisomycin was added to the pcDNA3.1-AQP1 group and called the pcDNA3.1-AQP1+ anisomycin group. The protein expression levels of human tight junction protein 1 (TJP1), sodium potassium triphosphate adenylase (Na+ /K+ -ATPase), p38 MAPK, and phosphorylated p38 MAPK in HCE cells were detected by Western blot.The relative expression level of AQP1 mRNA, cell proliferation activity, migration rate, apoptosis rate, the expression level of TJP1 and sodium potassium triphosphatase protein, and phosphorylation p38 MAPK/p38 MAPK ratio in each group of cells conformed to the homogeneity of variance and normal distribution, represented by ±s, and independent sample t-test was used for intergroup comparison.

Results

The relative expression of AQP1 mRNA of Control group, pcDNA3.1-NC group, siRNA-NC group, pcDNA3.1-AQP1 group and siRNA-AQP1 group were (1.00±0.08), (1.05±0.07), (0.97±0.07), (2.14±0.12) and (0.33±0.02). Compared with the Control group, there were no both signifcant differences between pcDNA3.1-NC group and siRNA-NC group (t=0.653, 0.684; P>0.05), indicating that cells with pcDNA3.1-NC and siRNA-NC were not affected, thus other indices of these cells were not detected again. The cell proliferation activity, the cell migration ratio, the apoptosis rate, the expression TJP1, Na+ /K+ -ATPase protein, and the ratio of phosphorylated p38 MAPK/p38 MAPK of pcDNA3.1-AQP1 group were (63.31±7.35)%, (74.28±7.04)%, (3.64±1.48)%, (1.73±0.13), (2.04±0.15) and (0.18±0.02), respectively; those of siRNA-AQP1 group were(22.15±3.26)%, (35.73±3.86)%, (20.35±2.83)%, (0.23±0.02), (0.21±0.02) and (0.75±0.09), respectively; those of pcDNA3.1-AQP1+ anisomycin group were (52.35±4.38)%, (58.74±5.42), (6.73±0.53)%, (1.38±0.21), (1.18±0.19) and (0.33±0.03), respectively. There were signifcant differences in all indices between pcDNA3.1-AQP1 group and Control group (t=3.844, 3.534, 3.253, 4.253, 4.753, 4.321; P<0.05); between siRNA-AQP1 group and Control group (t=4.136, 3.741, 3.621, 3.426, 4.122, 4.894, 3.795; P<0.05); between pcDNA3.1-AQP1 group and pcDNA3.1-AQP1+ anisomycin group(t=3.251, 3.363, 3.053, 3.242, 3.674, 4.264; P<0.05).

Conclusions

AQP1 overexpression can promote HCE cell proliferation and migration, inhibit apoptosis, and participate in the maintenance of HCE cell function, which may be relevant with the inhibition of p38 MAPK signaling pathway activation.

Key words: Aquaporin 1, Corneal endothelial cells, Proliferation, Migration, Apoptosis, p38 mitogen activated protein kinase signaling pathway
图6 AQP1对细胞p38丝分裂原活化蛋白激酶信号通路相关蛋白表达影响的比较　图5A和图5B分别示各组细胞p38丝分裂原活化蛋白激酶信号通路相关蛋白印迹图和比较柱状图　注：AQP1，水通道蛋白1；pcDNA3.1，质粒互补脱氧核糖核酸3.1；NC，空载对照；siRNA，小干扰核糖核酸；Control，对照；*，与对照组比较P<0.05
[1]
Zhu Q, Zhu Y, Tighe S, et al.Engineering of human corneal endothelial cells in vitro[J]. Int J Med Sci, 2019, 16(4): 507－512.
[2]
Liu Y, Sun H, Hu M, et al. Human corneal endothelial cells expanded in vitro are a powerful resource for tissue engineering[J]. Int J Med Sci, 2017, 14(2): 128－135.
[3]
Price MO, Mehta JS, Jurkunas UV, et al. Corneal endothelial dysfunction: evolving understanding and treatment options[J]. Prog Retin Eye Res, 2021, 82: 100904.
[4]
Okumura N, Kagami T, Watanabe K, et al. Feasibility of a cryopreservation of cultured human corneal endothelial cells[J]. PLoS One, 2019, 14(6): e0218431.
[5]
Joko T, Shiraishi A, Kobayashi T, et al. Mechanism of proliferation of cultured human corneal endothelial cells[J]. Cornea, 2017, 36(S1): S41－S45.
[6]
ZBogner B, Schroedl F, Trost A, et al. Aquaporin expression and localization in the rabbit eye[J]. Exp Eye Res, 2016, 147: 20－30.
[7]
Patil R, Wang H, Sharif NA, et al. Aquaporins: novel targets for age－related ocular disorders[J]. J Ocul Pharmacol Ther, 2018, 34(1－2): 177－187.
[8]
Nuzzi R, Buono L, Scalabrin S, et al. Effect of stem cell－derived extracellular vesicles on damaged human corneal endothelial cells[J]. Stem Cells Int, 2021, PMID: 33531909.
[9]
Tran TL, Hamann S, Heegaard S. Aquaporins in the eye[J]. Adv Exp Med Biol, 2017, 969: 193－198.
[10]
Hua Y, Ying X, Qian Y, et al. Physiological and pathological impact of AQP1 knockout in mice[J]. Biosci Rep, 2019, 39(5): BSR20182303.
[11]
Jiang Y, Ma R, Zhao Y, et al. MEF2C/miR－133a－3p.1 circuit－stabilized AQP1 expression maintains endothelial water homeostasis[J]. FEBS Lett, 2019, 593(18): 2566－2573.
[12]
Maltaneri RE, Schiappacasse A, Chamorro ME, et al. Aquaporin－1 plays a key role in erythropoietin－induced endothelial cell migration[J]. Biochim Biophys Acta Mol Cell Res, 2020, 1867(1): 118569.
[13]
Arras W, Vercammen H, NíDhubhghaill S, et al. Proliferation increasing genetic engineering in human corneal endothelial cells: aliterature review[J]. Front Med, 2021, PMID: 34268324.
[14]
Zhao C, Li W, Duan H, et al. NAD+ precursors protect corneal endothelial cells from UVB－induced apoptosis[J]. Am J Physiol Cell Physiol, 2020, 318(4): C796－C805.
[15]
Schlötzer－Schrehardt U, Zenkel M, Strunz M, et al. Potential functional restoration of corneal endothelial cells in fuchs endothelial corneal dystrophy by ROCK inhibitor (ripasudil) [J]. Am J Ophthalmol, 2021, 224: 185－199.
[16]
Duan S, Zhang Y, Yuan F, et al. Corneal endothelial expansion using human umbilical cord mesenchymal stem cell－derived conditioned medium[J]. J Cell Physiol, 2021, 236(4): 2606－2615.
[17]
Kwang－Hua CW. Temperature－dependent viscosity dominated transport control through AQP1 water channel[J]. J Theor Biol, 2019, 480: 92－98.
[18]
Kong B, Zhao S, Kang X, et al. MicroRNA－133a－3p inhibits cell proliferation, migration and invasion in colorectal cancer by targeting AQP1[J]. Oncol Lett, 2021, 22(3): 649.
[19]
Osorio G, Zulueta－Dorado T, González－Rodríguez P, et al. Expression pattern of aquaporin 1 and aquaporin 3 in melanocytic and nonmelanocytic skin tumors[J]. Am J Clin Pathol, 2019, 152(4): 446－457.
[20]
Grönroos P, Ilmarinen T, Skottman H. Directed differentiation of human pluripotent stem cells towards corneal endothelial－like cells under defined conditions[J]. Cells, 2021, 10(2): 331.
[21]
Thériault M, Gendron SP, Brunette I, et al. Function－related protein expression in fuchs endothelial corneal dystrophy cells and tissue models[J]. Am J Pathol, 2018, 188(7): 1703－1712.
[22]
Thiagarajah JR, Verkman AS. Aquaporin deletion in mice reduces corneal water permeability and delays restoration of transparency after swelling[J]. J Biol Chem, 2012, 277(21): 19139－19144.
[23]
Hongo A, Okumura N, Nakahara M, et al. The effect of a p38 mitogen－activated protein kinase inhibitor on cellular senescence of cultivated human corneal endothelial cells[J]. Invest Ophthalmol Vis Sci, 2017, 58(9): 3325－3334.
[24]
Nakahara M, Okumura N, Nakano S, et al. Effect of a p38 mitogen－activated protein kinase inhibitor on corneal endothelial cell proliferation[J]. Invest Ophthalmol Vis Sci, 2018, 59(10): 4218－4227.
[25]
Li B, Liu C, Tang K, et al. Aquaporin－1 attenuates macrophage－mediated inflammatory responses by inhibiting p38 mitogen－activated protein kinase activation in lipopolysaccharide－induced acute kidney injury[J]. Inflamm Res, 2019, 68(12): 1035－1047.
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