In previous studies, high-energy, short-wavelength blue light was always harmful to the eye and biological rhythms [9, 17]. Hence, blue light filtering intraocular lenses (IOLs) have been used after extracapsular cataract extraction surgery to protect macular health and reduce retinal photoxicity induced by blue light [18]. However, the sensitivity to the effects was much different for various types of cells. Our findings strongly supported that 458 nm blue light attenuated TGF-β2-induced EMT in LECs, which was consistent with findings in a previous study that blue light irradiation inhibited cell proliferation, migration and EMT process in colorectal cancer [15]. Therefore, in order to reduce the probability of PCO after cataract extraction surgery, we speculated that partial blue light exposure in the eye was necessary.
Dysregulation of autophagy is related to many lens dysfunctions, such as congenital cataract, age-related cataract, and PCO [19–22]. Previous findings have shown that sulforaphane reduced growth, migration, and viability in lens cells through ER stress and autophagy upon reactive oxygen species (ROS) production, and thus could serve as a putative therapeutic agent for PCO [23]. Compared with rapamycin, PP242 (a new-generation mTOR inhibitor) strongly inhibited the crucial cellular events in the formation of PCO through induction of autophagy and apoptosis, involving proliferation, attachment, and migration [24]. Additionally, it is worth mentioning that the production of large amounts of TGF-β2 in response to cataract surgical stimulation can induce EMT progression in residual LECs with enhanced migration capability, therefore indicating the crucial significance in deciphering the mechanism of TGF-β2-induced-EMT for the sake of therapeutic exploration in PCO [3]. As briefly introduced before, the effect of autophagy regulation in the EMT process was ambiguous and there is a complex link between these two processes. On the one side, cells required autophagy activation to survive during the EMT. On the other side, autophagy functions, as an onco-suppressive signal, could hinder the early phases of metastasization and activation of the EMT in cancers [25]. In eye research, autophagy blockage reduced TGF-β2-triggered EMT in LECs, suggesting that it would be a novel therapy method of autophagy inhibition for prevention and treatment of the fibrotic cataract [6]. Hence, in this study, we established a cellar model of TGF-β2-induced EMT in HLE-B3 cells and then investigated the role of autophagy during the inhibition of EMT under blue light exposure for the very first time.
The TGF-β contributes to EMT activation mainly by mediating Smad and non-Smad signal transduction pathways [26]. During the process of EMT, TGF-β can induce accumulation of autophagosomes and activate the autophagy flux upon stimulating the expression of several autophagy-related genes, such as Beclin-1, Atg5, Atg7, and death-associated protein kinase (Dapk) [27]. In this study, expression of Fibronectin, N-cadherin and Vimentin was examined as EMT markers. Fibronectin, a high molecular weight glycoprotein, can bind to integrins in the extracellular matrix [28]. N-cadherin, one of cell adhesion molecules, functions in the formation of adherens junctions to bind cells with each other [29]. Vimentin is abundantly expressed in mesenchymal cells as a type III intermediate filament protein [30]. In the present study, in addition to the rise of autophagosomes under TEM, the conversion of LC3-I to LC3-II and the degradation of p62 was elevated in a dose-dependent manner in HLE-B3 cells after TGF-β2 stimulation, implying that autophagy as a mediator participated in the process of TGF-β2-induced EMT of LECs [5]. The significantly reduced translation and transcription levels of EMT markers and the decreased migration ability after blue light exposure suggested that blue light inhibited cell migration and EMT process induced by TGF-β2. Meanwhile, blue light exposure further enhanced TGF-β2-induced LC3-II conversion while reversing p62 degradation, leading to p62 accumulation, indicating that impaired autophagy after blue light exposure during the inhibition of TGF-β2-induced EMT. Moreover, the accumulation of autophagolysosomes after blue light exposure might be attributed to reduced autophagolysosome degradation due to blocked autophagosome-lysosome fusion [31]. Overall, according to our data, blue light induced autophagy impairment and alleviated TGF-induced EMT, suggesting that administration of blue light exposure may potentially offer a novel therapeutic approach for the prevention of PCO.
Therefore, we further determined the effect of autophagy regulation in EMT attenuated by blue light exposure. Our data showed the increased autophagy flow and a greater transdifferentiation capability from LECs into myofibroblasts by assessing the mesenchymal markers after rapamycin treatment, with or without TGF-β2 combination or/and blue light. Rapamycin also significantly improved the cell migration property, reversing the migration slowdown induced by blue light. Furthermore, hloroquine, as an autophagy inhibitor, reduced the expression of TGF-β2-induced EMT markers and cell migration ability. These results confirmed our conjecture that blue light attenuated TGF-β2-induced EMT through autophagy inhibition. Further studies are needed to elucidate the effects of LED with different wavelengths which may have different effects, and to determine the underlying molecular mechanisms.
In summary, our study provided a direct evidence that blue LED exposure has inhibited effects on TGF-β2-induced EMT in LECs, which characterized by inhibited expression of EMT related markers and reduced migration, and it might be caused by impaired autophagy. Collectively, these findings provided a novel potential therapeutic strategy for PCO in the future.