PCO is a common fibrotic disease and the most common complication after cataract surgery. EMT is the common pathological basis of many fibrotic diseases such as PCO. Understanding the pathological mechanism of EMT will not only help the prevention and treatment of PCO, but may also provide a new understanding of the occurrence and development of fibrotic diseases.
When trauma or surgical damage leads to the destruction of the lens structure, the remaining lens epithelial cells proliferate excessively and migrate to the posterior capsule, resulting in EMT, which changes the color of the lens or reduces the transparency, and ultimately leads to the occurrence of cataracts. The typical characteristics of EMT include: E-cadherin, a key molecule that maintains cell polarity and intercellular adhesion, is degraded or lost function, and the abundance of tight junction proteins such as occludin and zonules occlusion 1 (ZO1) is decreased. The expression of keratin decreases. At the same time, the characteristic proteins of mesenchymal cells such as Fibronectin, α-SMA, Vimentin, N-Cadherin (N-Cadherin), and Col Ⅰ increase[24, 25]. The connections between cells are dissolved, the cytoskeleton is reconstructed, and the cell morphology is transformed into a mesenchymal-like multidendrite spindle shape, and cell migration and invasion capabilities are enhanced. TGF-β2 is currently known as a potent inducer of EMT. TGF-β2 is not only involved in EMT during growth and development, but is also closely related to EMT during wound healing, fibrosis and cancer[28, 29]. In this study, the real-time fluorescent quantitative PCR test results showed that the transparent lens capsule and the fibrotic lens capsule were compared between healthy donors and fibrotic cataract patients, the expression levels of fibrotic lens epithelial mesenchymal marker α-SMA, ECM markers FN, Col I, and Acan were significantly increased. TGF-β2 was used to stimulate human lens in vitro implants. In the LEC cell line SRA01/04 treated with TGF-β2, α-SMA, FN, Col I and Acan increased, and the closed zone tissue (ZO1) and E-cadherin (The expression of E-cad) decreased, and the expression of N-cadherin and vimentin (Vim) increased, confirming that lens fibrosis involves hyperproliferation and EMT, and TGF-β2 can significantly promote the occurrence of EMT in lens epithelial cells.
The human TLR3 gene is located on chromosome 4, and its expression product TLR3 receptor protein can recognize double-stranded RNA associated with viral infection. Studies have shown that TLR3, as an innate immune receptor, plays a role in activating anti-inflammatory signaling pathways during injury and infection. Chronic liver alcohol accumulation can inhibit TLR3-dependent signaling pathways in NK cells during the late stage of liver fibrosis and alcoholic liver disease. The lack of TLR3-mediated killing of NK cells played an important role in accelerating the progression of the disease. However, the expression and function of TLR3 in the lens have not been reported yet. In this study, we detected the expression of TLR3 in human lens epithelial cells. More importantly, we found that TLR3 was significantly increased in the mouse lens anterior capsule injury model, and in the EMT model of lens epithelial cells induced by TGF-β, 2 TLR3 also increased significantly, suggesting that TLR3 may be involved in the lens EMT process, but its role in the lens epithelial cell EMT process is still unclear.
In order to further study the role of TLR3 in EMT of lens epithelial cells, we constructed siRNA and TLR3 overexpression lentiviral vectors that specifically interfere with TLR3, aiming to observe differences in TLR3 expression on the proliferation, migration, invasion and EMT of lens epithelial cells cultured in vitro. To explore the regulatory effect of TLR3 on the EMT of lens epithelial cells. We found that under the stimulation of TGF-β2, the expression levels of α-SMA, Vimentin, and Col Ⅰ of cells transfected with si-TLR3 increased significantly, while the expression level of E-Cadherin decreased significantly, indicating that both groups of cells had EMT, but the expression of FN, α-SMA, Col I and Acan in the si-TLR3 group decreased, the proliferation rate decreased, and migration and invasion were significantly decreased. The results of WB experiments showed that the epithelial markers ZO-1 and E-cad were retained, and the expression of N-cad and Vim protein was significantly reduced, indicating that TLR3 has a functional effect on the proliferation, migration, invasion and EMT of human lens epithelial cells. The Notch signaling pathway is highly conserved in evolution and is involved in physiological processes such as cell proliferation, differentiation, apoptosis, and maintenance of cell stemness. Notch ligands in mammals are also called DSL proteins. There are five types, namely Jagged1, Jagged2, DLL1, DLL3, and DLL4, all of which are type I transmembrane proteins. TGF-β2 can induce the expression of Notch ligands. In patients with diabetic nephropathy, the expression of Jagged1 and Hey1 induced by TGF-β2 plays a particularly important role, which may be related to the pathological process of the disease. It has been confirmed that Jagged is the target gene of TGF-β2 in a variety of mammalian cells. TGF-β2 relies on Smad3 to regulate the expression of Jagged1 and Hey1 in the process of inducing EMT in cells. In this study, Western blot results showed that Jagged-1, Notch-1, and Notch-2 proteins were significantly increased in human lens epithelial cells treated with TGF-β2. After transfection of si-TLR3 and overexpression of TLR3, Jagged-1, Notch-1, and Notch-2 proteins in the overexpression TLR3 group remained elevated, and the expression of si-TLR3 histone decreased significantly, indicating that TGF-β2 effectively activated Notch signal path. In order to study whether the Jagged-1/Notch signaling pathway mediates the biological function of TLR3 in the process of human lens fibrosis, transfection of si-TLR3 and the use of TLR3 activator valproic acid (VA) were used at the same time. The results showed that compared with the TGF-β2 group, the FN, α-SMA, Col I and E-cad genes in the si-TLR3 group were significantly decreased, the proliferation rate was significantly decreased, and the migration and invasion capabilities were significantly decreased; Compared with the si-TLR3 group, the FN, α-SMA, Col I, and E-cad genes were relatively higher in the VA group, and the proliferation rate increased significantly, and the migration and invasion capabilities increased significantly. The results of WB experiments showed that compared with the TGF-β2 group, the ZO-1 and E-cad protein expressions in the si-TLR3 group increased, and the N-cad and Vim proteins decreased. The VA group and the si-TLR3 group had the opposite results. Compared with the TGF-cβ2 group, Jagged-1/Notch signaling pathway related proteins decreased significantly in the si-TLR3 group, and improved in the VA group compared with the si-TLR3 group. It shows that in the process of human lens epithelial cell fibrosis, TLR3 promotes the proliferation, migration, invasion and EMT of human lens epithelial cells by directly acting on the Jagged-1/Notch signaling pathway.
Our results provide the first evidence that TLR3 and Jagged-1/Notch pathway play an important role in lens fibrosis. Our data indicate that the blockade of TLR3 and Jagged-1/Notch pathways may be a promising strategy for the prevention and treatment of organ fibrosis. These understandings of the regulatory relationship between TLR3 and Jagged-1/Notch signaling pathway are helpful to understand the pathogenesis of fibrotic diseases.