Protective Effects of One 2,4-Dihydro-3H-Pyrazol-3-one Derivative against Posterior Capsular Opacification by Regulation of TGF-β2/SMADs and Non-SMAD Signaling, Collagen I, and Fibronectin Proteins

Many elderly individuals frequently experience cataracts that interfere with vision. After cataract surgery, the left lens epithelial cell (LEC) exhibited fibrosis and posterior capsule opacification (PCO). Sometimes, there is a need for a second surgery; nevertheless, people try other methods, such as a good pharmacological agent, to treat PCO to reduce transforming growth factor-β2 (TGF-β2) amounts to avoid secondary surgery. The aim of the present study was to explore the potential anti-PCO activity of five 2,4-dihydro-3H-pyrazol-3-one (DHPO) derivatives in a TGF-β2-induced fibrogenesis SRA01/04 cell model. The 2-phenyl-5-propyl-DHPO (TSE; no. 2: TSE-2) compound showed the best activity of reduced expression levels of TGF-β2 among five derivatives and therefore was chosen to evaluate the anti-PCO activity and molecular mechanisms on the Sma and mad protein (SMAD) signaling pathway (including TGF-β2, SMADs, and the inhibition of nuclear translocation of SMADs), non-SMAD pathway proteins, including p-extracellular, regulated protein kinases (ERK) 1/2, or p-c-Jun N-terminal kinase (JUN) by Western blotting, PCR, or confocal immunofluorescence analyses. Following treatment with 10 μg/mL of the five compounds, the cells displayed great viability by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTT) assay. In this study, the result of lactate dehydrogenase (LDH) activity measurement did not affect the cytotoxicity of the five compounds. In TGF-β2-induced fibrogenesis in SRA01/04 cells, treatment with the TSE compound decreased the TGF-β2/SMAD signaling genes, including reduced mRNA or expression levels of TGF-β2, SMAD3, and SMAD4, leading to inhibition of TGF-β2-induced fibrogenesis. Our confocal immunofluorescence analyses demonstrated that TSE treatment displays a suppressive effect on SMAD2/3 or SMAD4 translocation to the nucleus. Furthermore, TSE treatment exhibits a reduction in the non-SMAD target gene expression levels of p- c-Jun N-terminal kinase (JUN), p- extracellular, regulated protein kinases (ERK)1/2, p- p38 mitogen-activated protein kinase (p38), p-phosphatidylinositol 3-kinase (PI3K), p-mammalian target of rapamycin complex (mTORC), p-Akt (Ser473), and p-Akt (Thr308). The overall effect of TSE is to reduce the expression levels of collagen I and fibrinogen (FN), thus contributing to antifibrotic effects in cell models mimicking PCO. Our findings reveal the benefits of TSE by regulating TGF-β/SMAD signaling and non-SMAD signaling-related gene proteins to display antifibrotic activity in cells for the possibility of preventing PCO after cataract surgery.


Introduction
Cataracts frequently occur in elderly individuals. Posterior capsular opacification (PCO) is an ocular complication disease after cataract surgery, and the principal reasons are fibrosis [1] of the entire posterior and left anterior capsule with lens epithelial cells (LECs) adhered [2,3]. These quickly develop and finally interfere with the visual axis for the next visual deprivation [2][3][4] after cataract surgery. Finally, once there is worse enough inhibition of visual quality, another surgery must be performed [2,3].
There are various means in the management and prevention of PCO, including appropriate anti-inflammatory drugs [5], anti-metabolic agents (such as 5-fluorouracil, mitomycin) [6,7], improved intraocular lens (IOL) designs (such as different materials, construction and designs to create a barrier to retard cell growth on the posterior capsule) [8,9], good surgical advances, drug delivery systems (such as a closed bag drug delivery system), and biological targets [1]. The design of IOLs and improving surgical skills and materials result in the amelioration of PCO [2,3] but have not eradicated the problem [10], and trauma and toxic side effects (such as releasing drugs toward other tissues that are harmful to the corneal endothelium) exist [11]. Numerous growth factors function in certain signaling pathways; thus, therapeutic intervention is thought to be via amelioration or regulation of crucial signaling targets [2,3].
Transforming growth factor-β2 (TGF-β2) signaling has been shown to play an important role in PCO growth following cataract surgery [1] Accumulating evidence reveals that TGF-β2 plays a core role in the regulation of LEC behavior during lens repair after injury [1][2][3]12] and cataract surgery [3,13]. Therefore, previous evidence has shown that strategies attenuating TGF-β2 signaling could be efficacious in the inhibition of capsular fibrosis [10] or PCO after cataract surgery.
Furthermore, TGF-β2 has been identified to activate not only its SMAD signaling pathway downregulation but also the constitution of PCO [14]. Saika et al. and other researchers demonstrated that SMADs are targets associated with intracellular signal transduction from cell exterior TGF-β receptors to nuclear gene promoters [3,13,15], and once ligands bind with the TGF-β receptor, translocation of a complex of phosphorylated SMAD2 and 3 occurs to the nuclei in a complex with SMAD4 and sequela [13]. The TGF-β family is a representative epithelial-mesenchymal transition (EMT) inducer in both development and disease through SMAD-and non-SMAD-regulated pathways [15,16]. Non-SMAD signaling pathways induce epithelial-mesenchymal transition (EMT) with translation via the PI3K/AKT/mTOR pathway and promote cytoskeletal remodeling [17].

Cell Lines
SRA01/04 cells (RCB1591), an SV40 T-antigen-transformed human LEC line, SV40 T-antigen-transformed human lens epithelial cell line [20], were purchased from Cell Bank Riken BioResource Research Center, Japan. The cells were cultured in media specific to the cell line, and this process was performed as described in a previous study [21].

Lactate Dehydrogenase (LDH) Activity Assay
After treatment with these five compounds, this part was performed as described in a previous study [23].

Immunoblotting Analysis
SRA01/04 cell lysates were lysed in radioimmunoprecipitation assay (RIPA) buffer, and the immunoblotting analysis was performed as described in previous studies [21,24] with the following specific antibodies: anti-TGFβ2 and anti-SMADs (Cell Signaling, Beverly, MA, USA).

Confocal Immunofluorescence Microscopy
Cells were seeded at a density of 2 × 10 5 cells/well in 6-well plates as described in a previous study [25].

Statistical Analyses
Data are the mean and standard error. All results were analyzed with analysis of variance with Dunnett's multiple range test by SPSS software. p < 0.05 was considered statistically significant.

LDH Assay
Following treatment with five compounds 24 h later, the cell-mediated lysis percentages were 4.47 ± 0.9% for the control and 10 µg/mL TSE groups and 7.36 ± 1.1% for the control and 10 µg/mL TSE groups 48 h later. TSE did not display significant cytotoxicity action ( Figure 3B).

Effects of TSE on the mRNA Levels of Targeted Genes in Cells
The mRNA levels of TGF-β2, SMAD3, and SMAD4 were markedly enhanced in SRA01/04 cells compared to the CON group (p < 0.001, p < 0.001, p < 0.001, respectively). A reduction in the mRNA levels of TGF-β2 was observed in the 5, 10, and 20 mg/mL TSE-treated groups compared to the vehicle-treated TGF-β2 group (p < 0.05, p < 0.001, p < 0.001, respectively) ( Figure 4A,B). A reduction in the mRNA levels of SMAD3 was observed in the 5, 10, and 20 mg/mL TSE-treated groups compared to the vehicle-treated TGF-β2 group (p < 0.05, p < 0.001, p < 0.001, respectively) ( Figure 4A,B). A reduction in the mRNA level of SMAD4 was observed in the 5, 10, and 20 mg/mL TSE-treated groups compared to the vehicle-treated TGF-β2 group (p < 0.01, p < 0.001, p < 0.001, respectively) ( Figure 4A,B).

LDH Assay
Following treatment with five compounds 24 h later, the cell-mediated lysis percentages were 4.47 ± 0.9% for the control and 10 μg/mL TSE groups and 7.36 ± 1.1% for the control and 10 μg/mL TSE groups 48 h later. TSE did not display significant cytotoxicity

TSE Inhibits the Nuclear Translocation of SMADs
By immunofluorescence assay, the expression levels of SMAD2/3 and SMAD4 protein in SRA01/04 cells were scanned and examined. SMAD2/3 and SMAD4 may be expressed within the cells and nearly in the cytoplasm. As shown in Figures 5 and 6, the nuclear SMAD2/3 or SMAD4 staining in the control group (in the absence of TGF-β2) in SRA01/04 cells was very weak. These results were evaluated under confocal microscopy. Following treatment with 5, 10, or 20 mg/mL TSE, nuclear SMAD2/3 and nuclear SMAD4

TSE Inhibits the Nuclear Translocation of SMADs
By immunofluorescence assay, the expression levels of SMAD2/3 and SMAD4 protein in SRA01/04 cells were scanned and examined. SMAD2/3 and SMAD4 may be expressed within the cells and nearly in the cytoplasm. As shown in Figures 5 and 6, the nuclear SMAD2/3 or SMAD4 staining in the control group (in the absence of TGF-β2) in SRA01/04 cells was very weak. These results were evaluated under confocal microscopy. Following treatment with 5, 10, or 20 mg/mL TSE, nuclear SMAD2/3 and nuclear SMAD4 expression was decreased in the SRA01/04 cell line compared with the control under microscopy at 200× magnification, and the suppressive activity of 20 mg/mL TSE was the most effective  Figure 5. Effects of TSE on epithelial-mesenchymal translocation of SMAD4 using confocal mic copy. The mesenchymal phenotypic marker SMAD4 (red). The nuclei were stained with D (blue). Magnification, ×200.

Discussion
Cataract is one of the major elderly topics worldwide. Posterior capsu tion (PCO) is a familiar complicative issue following cataract surgery. Curre various means of management and prevention of PCO, including surgical a anti-metabolic agents, and biological targets. Targeting TGF-β signaling method for developing a novel therapeutic agent [27]. Therefore, we hypo there are therapeutic interventions with no toxic side effects and low cost TGFβ-2 and SMAD pathways, leading to antifibrotic effects and contributi vention of PCO. Consistently, we found that TSE decreased the mRNA an pression levels of TGF-β2, SMAD3, and SMAD4; moreover, TSE inhibite translocation of SMAD2/3 and SMAD4 in TGF-β2-induced fibrotic cells, and was associated with the management of PCO. Therefore, it is possible that a novel and prospective agent against PCO.

Discussion
Cataract is one of the major elderly topics worldwide. Posterior capsular opacification (PCO) is a familiar complicative issue following cataract surgery. Currently, there are various means of management and prevention of PCO, including surgical advances, IOL, anti-metabolic agents, and biological targets. Targeting TGF-β signaling is a modern method for developing a novel therapeutic agent [27]. Therefore, we hypothesized that there are therapeutic interventions with no toxic side effects and low cost that block the TGFβ-2 and SMAD pathways, leading to antifibrotic effects and contributing to the prevention of PCO. Consistently, we found that TSE decreased the mRNA and protein expression levels of TGF-β2, SMAD3, and SMAD4; moreover, TSE inhibited the nuclear translocation of SMAD2/3 and SMAD4 in TGF-β2-induced fibrotic cells, and this vital cell was associated with the management of PCO. Therefore, it is possible that TSE might be a novel and prospective agent against PCO.
Pirfenidone (PFD) is an anti-inflammatory, antioxidant, and antifibrotic agent used in animal studies [9,18,28]. Pirfenidone has been demonstrated to inhibit the growth of orbital fibroblasts at concentrations of 1.9 mg/mL in patients with thyroid-associated ophthalmopathy [10,29].
These five compounds (2,4-dihydro-3H-pyrazol-3-one (DHPO) derivatives) were synthesized by our team with a core structure similar to that of PFD. Our findings show that the activity of these five compounds displays almost similar activities at concentrations of 10 µg/mL but more efficacy than PFD (at concentrations of 0.25 mg/mL) on the expression levels of TGF-β2, SMAD3, or SMAD4, implying that these five compounds display antifibrotic activity approximately 25 times that of PFD in SRA01/04 cells. Based on the results of protein expression of TGF-β2, we selected the most efficient compound and examined the protective activity and its underlying molecular mechanism of TSE.
In this study, we investigated whether TSE displayed targeting TGFβ signaling to provide new insights for developing a novel therapeutic intervention. Our data showed that in the MTT test, 10 µg/mL TSE exhibited no toxic effect in SRA01/04 cells ( Figure 3A). Moreover, TSE displayed no LDH activity discharging from the injured cells without harming these cells ( Figure 3B). However, this study demonstrates for the first time the beneficial effects of anti-PCO in TGF-β2-induced SRA01/04 cells, thereby producing evidence of the role of TSE's anti-fibrotic effects by inhibition of TGF-β2-SMAD and non-SMAD signaling pathways.
Previous works from laboratories showed that the SRA01/04 model has been widely used [10,30] and is a key measure of the possible reasons for cataract development. Therefore, for further analysis, we used this cell line to investigate the potential anti-PCO activity of TSE.
Pirfenidone exerted its antifibrotic effect in the following cases. A recent study reported that pirfenidone decreases the levels of mRNA and TGF-β expression in animal models of lung fibrosis [30]. In addition, pirfenidone was found to downregulate the mRNA levels of TGF-β1 in a rodent hepatic fibrosis study [31]. Increasing evidence shows that pirfenidone not only inhibits collagen but also decreases TGF-β expression in a renal fibrosis model [32][33][34].
A recent publication confirmed many of our findings with a reduction in the nuclear accumulation and/or translocation of SMAD2/3 and SMAD4 following treatment with pirfenidone [10]. A recent study found that pirfenidone inhibits TGF-β signaling with suppression of active SMAD2/3 complexes in the nuclei accumulation but does not complete SMAD2/3 phosphorylation [35]. Therefore, there is a possibility that TSE acts as PFD and reduces the nuclear accumulation and/or translocation of SMAD2/3 and SMAD4. In addition to the abovementioned findings, all of these findings point to at least one pathway that results in TSE inhibiting TGF-β2-induced fibrogenic LECs with a decrease in the expression levels of collagen I and fibronectin and reducing the mRNA and expression levels of TGF-β2, SMAD3, or SMAD4, as well as a decrease in the expression levels of p-ERK p42/44 and p-JUN, resulting in the prevention of PCO occurrence.
In addition to SMAD2/3/4 nuclear translocation, there are many signaling pathways involved in the TGF-β receptor-induced response. TGF-β can induce EMT by direct phosphorylation of SMAD2/3 or activation of non-SMAD pathways, such as mitogenactivated protein kinase (MAP) kinase (MAPK; p-38, ERK1/2, JUN), mTOR, or PI3K/Akt, thus contributing to the inhibition of epithelial target genes and mesenchymal marker activation [36][37][38]. The additional exact molecular mechanisms involved in the suppression of the TGF-β2-SMAD signaling pathway by TSE remain to be elucidated. Further study will be needed to investigate other potential mechanisms, including activation of the non-SMAD signaling pathway (such as Hippo/YAP, β-catenin/Wnt, proKin, or Rho GTPase) of TSE for suppressive activity in TGF-β2-induced fibrogenic effects in LECs. Because SRA01/04 cells were used in this study, we speculate that TSE might be another candidate for protection against PCO.

Conclusions
In summary (Figure 9), the present study demonstrated that TSE has therapeutic potential in treating PCO associated with its antifibrotic activity in a TGF-β2-induced fibrogenesis SRA01/04 cell model. The major target of TSE is the decrease in the expression levels of numerous target genes involved in the TGF-β2/SMAD and non-SMAD signaling pathways and the inhibition of nuclear translocation of SMADs. TSE treatment decreased the mRNA or expression levels of TGF-β2, SMAD3, or SMAD4, with consequent reduced accumulation of SMADs in the nuclei and translocation, as well as decreased non-SMAD pathway gene expression levels of p-JUN/ JUN, p-ERK1/2/ ERK1/2, p-p38/ p38, p-PI3K/ PI3K, p-mTOR/ mTOR, p-Akt (Ser 473 )/ t-Akt, and p-Akt (Thr 308 )/ t-Akt in TGF-β2-induced fibrogenic cells. Furthermore, TSE decreased the expression levels of collagen I and fibronectin. The overall effect of TSE is to decrease TGF-β2-induced fibrogenesis in cells, thus resulting in antifibrotic activity with the potential to prevent PCO occurrence.
TGF-β2-SMAD signaling pathway by TSE remain to be elucidated. Further study needed to investigate other potential mechanisms, including activation of the non signaling pathway (such as Hippo/YAP, β-catenin/Wnt, proKin, or Rho GTPase) for suppressive activity in TGF-β2-induced fibrogenic effects in LECs. Because SR cells were used in this study, we speculate that TSE might be another candidate f tection against PCO.

Conclusions
In summary (Figure 8), the present study demonstrated that TSE has therape tential in treating PCO associated with its antifibrotic activity in a TGF-β2-induce genesis SRA01/04 cell model. The major target of TSE is the decrease in the exp levels of numerous target genes involved in the TGF-β2/SMAD and non-SMAD si pathways and the inhibition of nuclear translocation of SMADs. TSE treatment de the mRNA or expression levels of TGF-β2, SMAD3, or SMAD4, with consequent r accumulation of SMADs in the nuclei and translocation, as well as decreased non pathway gene expression levels of p-JUN/ JUN, p-ERK1/2/ ERK1/2, p-p38/ p38, p-PI3K/ mTOR/ mTOR, p-Akt (Ser 473 )/ t-Akt, and p-Akt (Thr 308 )/ t-Akt in TGF-β2-induced fibrogen Furthermore, TSE decreased the expression levels of collagen I and fibronectin. Th all effect of TSE is to decrease TGF-β2-induced fibrogenesis in cells, thus resulting fibrotic activity with the potential to prevent PCO occurrence.   Informed Consent Statement: Not applicable.

Data Availability Statement:
All data used to support the findings of this study are available from the corresponding author, Chun-Ching Shih, upon reasonable request. Corresponding author's email: ccshih@ctust.edu.tw.

Conflicts of Interest:
The authors have declared no conflict of interest.