Xu Weixing, Cao Liu, Liu Hua
Henan Eye Hospital, Henan Provincial People's Hospital, People's Hospital of Zhengzhou University, Zhengzhou City, China.
School of Graduate, Dalian Medical University, Dalian City, China.
Invest Ophthalmol Vis Sci. 2025 Jan 2;66(1):63. doi: 10.1167/iovs.66.1.63.
To investigate the effect of Ca2+/calmodulin-dependent protein kinase II (CAMKII) δ subtypes (CAMK2D) on sodium iodate (NaIO3)-induced retinal degeneration in mice.
Bioinformatics analysis and Western blot experiments were used to screen the significantly differentially expressed genes in age-related macular degeneration (AMD) disease. CAMK2D knockdown and overexpression models were constructed by lentivirus (LV) infection of adult retinal pigment epithelial cell line-19 (ARPE-19) cells in vitro. Flow cytometry was used to detect ARPE-19 cell apoptosis induced by NaIO3. In vivo, CAMK2D knockdown and overexpression mouse models were generated by infecting mouse retinal pigment epithelium (RPE) with adeno-associated virus (AAV). Retinography, optical coherence tomography (OCT), and histological analysis (hematoxylin and eosin staining) were used to detect NaIO3-induced retinal structural changes in mice. Electroretinography (ERG) was used to detect NaIO3-induced retinal function changes in mice. TdT-mediated dUTP nick-end labeling (TUNEL) staining was used to detect the apoptosis of retinal cells induced by NaIO3. RNA sequencing (RNA-Seq) and bioinformatics analysis were used to screen for target genes affected by CAMK2D in CAMK2D-overexpressing ARPE-19 cells. And flow cytometry, OCT, and ERG were used to evaluate the regulatory effect of CAMK2D on target genes.
Bioinformatics analysis found the expression of genes related to Ca2+ signal was significantly reduced in AMD patients. Western blot showed that in a mouse model of dry AMD induced by NaIO3, CAMK2D expression in RPE-Choroid tissue significantly lower than normal mice. In vitro, our results showed that overexpression of CAMK2D in ARPE-19 cells decreased apoptosis induced by NaIO3 and knockdown increased apoptosis. In vivo, CAMK2D overexpression in RPE cells can attenuate the retina degeneration induced by NaIO3 and CAMK2D knockdown aggravated degeneration. The bioinformatics analysis indicated that CAMK2D might affect AMD pathology through complement factor I (CFI). In vitro, knockdown of CFI in ARPE-19 cells increased apoptosis induced by NaIO3. In knockdown CFI ARPE-19 cells, overexpression of CAMK2D reduced the above apoptosis. In mice retina, CFI knockdown can aggravate the retina degeneration induced by NaIO3. In knockdown CFI mice, overexpression of CAMK2D in RPE can attenuate the above retina degeneration. Western blot confirmed that CAMK2D regulated the expression of CFI in mice.
CAMK2D can attenuate the retinal degeneration induced by NaIO3, which was achieved by regulating the CFI.
研究钙/钙调蛋白依赖性蛋白激酶II（CAMKII）δ亚型（CAMK2D）对碘酸钠（NaIO3）诱导的小鼠视网膜变性的影响。
采用生物信息学分析和蛋白质免疫印迹实验筛选年龄相关性黄斑变性（AMD）疾病中显著差异表达的基因。通过慢病毒（LV）体外感染成人视网膜色素上皮细胞系-19（ARPE-19）细胞构建CAMK2D基因敲低和过表达模型。采用流式细胞术检测NaIO3诱导的ARPE-19细胞凋亡。在体内，通过腺相关病毒（AAV）感染小鼠视网膜色素上皮（RPE）构建CAMK2D基因敲低和过表达小鼠模型。采用视网膜成像、光学相干断层扫描（OCT）和组织学分析（苏木精-伊红染色）检测NaIO3诱导的小鼠视网膜结构变化。采用视网膜电图（ERG）检测NaIO3诱导的小鼠视网膜功能变化。采用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）染色检测NaIO3诱导的视网膜细胞凋亡。采用RNA测序（RNA-Seq）和生物信息学分析筛选CAMK2D过表达的ARPE-19细胞中受CAMK2D影响的靶基因。并采用流式细胞术、OCT和ERG评估CAMK2D对靶基因的调控作用。
生物信息学分析发现AMD患者中与Ca2+信号相关的基因表达显著降低。蛋白质免疫印迹显示，在NaIO3诱导的干性AMD小鼠模型中，RPE-脉络膜组织中CAMK2D表达明显低于正常小鼠。在体外，我们的结果显示，ARPE-19细胞中CAMK2D过表达可减少NaIO3诱导的细胞凋亡，而基因敲低则增加细胞凋亡。在体内，RPE细胞中CAMK2D过表达可减轻NaIO3诱导的视网膜变性，而CAMK2D基因敲低则加重变性。生物信息学分析表明，CAMK2D可能通过补体因子I（CFI）影响AMD病理过程。在体外，ARPE-19细胞中CFI基因敲低可增加NaIO3诱导的细胞凋亡。在CFI基因敲低的ARPE-19细胞中，CAMK2D过表达可减少上述细胞凋亡。在小鼠视网膜中，CFI基因敲低可加重NaIO3诱导的视网膜变性。在CFI基因敲低的小鼠中，RPE中CAMK2D过表达可减轻上述视网膜变性。蛋白质免疫印迹证实CAMK2D在小鼠中调节CFI的表达。
CAMK2D可减轻NaIO3诱导的视网膜变性，这是通过调节CFI实现的。
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