Up-regulation of p21 activated kinase 4 expression in the retina of diabetes mice and its effects on the behavior and mitochondrial function in retinal vascular endothelial cells
10.3760/cma.j.cn511434-20220418-00224
- VernacularTitle:糖尿病小鼠视网膜p21活化激酶4表达上调及其对视网膜血管内皮细胞行为和线粒体功能的影响
- Author:
Mingfei JIAO
1
;
Hui LI
;
Jingjing CAO
;
Zhenyu KOU
;
Guijia WU
;
Aihua LIU
;
Lijie DONG
Author Information
1. 天津医科大学眼科医院、眼视光学院、眼科研究所 国家眼耳鼻喉疾病临床医学研究中心天津市分中心 天津市视网膜功能与疾病重点实验室, 天津 300384
- Keywords:
Diabetic retinopathy;
P21 activated kinase 4;
Retinal vascular endothelial cells;
Mitochondrial function;
Cell migration;
Leukocyte adhesion;
Animal experim
- From:
Chinese Journal of Ocular Fundus Diseases
2023;39(5):401-407
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To observe the effects of p21 activated kinase 4 (PAK4) on the mitochondrial function and biological behavior in retinal vascular endothelial cells.Methods:The experimental study was divided into two parts: in vivo animal experiment and in vitro cell experiment. In vivo animal experiments: 12 healthy C57BL/6J male mice were randomly divided into normal control group and diabetes group, with 6 mice in each group. Diabetes mice were induced by streptozotocin to establish diabetes model. Eight weeks after modeling, quantitative real-time polymerase chain reaction and Western blots were performed to detect the expression of PAK4 in diabetic retinas. In vitro cell experiments: the human retinal microvascular endothelial cells (hRMEC) were divided into three groups: conventional cultured cells group (N group), empty vector transfected (Vector group); pcDNA-PAK4 eukaryotic expression plasmid transfected group (PAK4 group). WB and qPCR were used to detect transfection efficiency, while scratching assay, cell scratch test was used to detect cell migration in hRMEC of each group. In vitro white blood cell adhesion experiment combined with 4 ', 6-diamino-2-phenylindole staining was used to detect the number of white blood cells adhering to hRMEC in each group. The Seahorse XFe96 cell energy metabolism analyzer measures intracellular mitochondrial basal respiration, adenosine triphosphate (ATP) production, maximum respiration, and reserve respiration capacity. The t-test was used for comparison between the two groups. Single factor analysis of variance was used for comparison among the three groups. Results:In vivo animal experiments: compared with normal control group, the relative expression levels of PAK4 mRNA and protein in retina of diabetic mice were significantly increased, with statistical significance ( t=25.372, 22.419, 25.372; P<0.05). In vitro cell experiment: compared with the N group and Vector group, the PAK4 protein, mRNA relative expression and cell mobility in the hRMEC of PAK4 group were significantly increased, with statistical significance ( F=36.821, 38.692, 29.421; P<0.05). Flow cytometry showed that the adhesion number of leukocytes on hRMEC in PAK4 group was significantly increased, and the difference was statistically significant ( F=39.649, P<0.01). Mitochondrial pressure measurement results showed that the capacity of mitochondrial basic respiration, ATP production, maximum respiration and reserve respiration in hRMEC in PAK4 group was significantly decreased, with statistical significance ( F=27.472, 22.315, 31.147, 27.472; P<0.05). Conclusion:Over-expression of PAK4 impairs mitochondrial function and significantly promotes leukocyte adhesion and migration in retinal vascular endothelial cells.
