All experimental procedures were performed according to the ARVO Declaration on the Use of Animals in Ophthalmic and Vision Research (17) and were approved by the Tianjin Medical University Animal Ethics Committee (Tianjin, China; approval no. TMUaMEC 2016009; June 6, 2016).

Adult C57BL/6J mice and Smad3-KO mice (18) were employed in the present study (age, 6-8 weeks; weight, 20±2 g; sex ratio, 76 male and 76 female; 76 KO and 76 WT mice). C57BL/6J mice were purchased from the Institute of Genetics and Developmental Biology at the Chinese Academy of Sciences (Beijing, China). The Smad3-KO mice were provided by Dr Xiao Yang from the Genetic Laboratory of Development and Disease (Institute of Biotechnology, Academy of Military Medical Sciences, Beijing, China). All mice were maintained under a 12 h light/dark cycle at 22-24°C with 45-50% humidity. Food and water was available ad libitum. The mice were anesthetized by intraperitoneal injections of pentobarbital sodium (70 mg/kg) (19). A 26-gauge hypodermic needle was applied to the right eye to puncture the corresponding central anterior capsule through the corneal limbus, following mydriasis and the administration of topical anesthesia. The depth of the puncture wound was ~25% of the length of the blade portion of the needle, as previously reported (17,19). The left eye served as an uninjured control. The mice were treated with erythromycin eye ointment and ofloxacin eye drops from 0 h to 4 weeks post-surgery. Finally, healing mice in the different groups were sacrificed by sevoflurane anesthesia and cervical dislocation to obtain the eyeballs for the subsequent analyses.

The HLE-B3 cell line was purchased from American Type Culture Collection (Manassas, VA, USA). The cells were cultured in Dulbecco's modified Eagle's medium/F-12 (HyClone; GE Healthcare Life Sciences, Logan, UT, USA) containing 20% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C with 5% CO₂. The cells were serum-starved overnight and then treated with TGF-β2 (10 ng/ml; R&D Systems, Inc., Minneapolis, MN, USA) for 24 h to measure the ECM component expression levels. In a separate group, the cells were pretreated with 3 µM SIS3 (p-Smad3 inhibitor; Sigma-Aldrich; EMD Millipore, Billerica, MA, USA) for 4 h prior to being treated with TGF-β2 (10 ng/ml). SIS3 was dissolved in 100% dimethyl sulfoxide (DMSO). DMSO was maintained at a final concentration of 0.1% in all experiments.

Following extraction from the mice, the eyeballs were fixed in 40 g/l paraformaldehyde solution for 24 h at 4°C. Subsequently, the fixed eyeballs were rinsed with PBS three times for 5 min each, and the eyeballs were dehydrated in 15% sucrose solution for 24 h, prior to being placed in 30% sucrose solution. Each eye was then embedded in optimum cutting temperature compound and sliced into 6-µm sagittal sections with a cryostat (Leica CM 1850; Leica Microsystems GmbH, Wetzlar, Germany). The sections were stored at −80°C until staining.

For the α-SMA, E-cadherin, lumican, OPN, fibronectin, HA and collagen type I immunofluorescence localization experiments, the frozen sections were removed from the −80°C freezer, warmed for 30 min at room temperature and rinsed with PBS three times for 5 min. The sections were then permeabilized with 0.1% Triton X-100 for 15 min; 5% bovine serum albumin (BSA; Roche Diagnostics, Basel, Switzerland) was added to the slides for non-specific antigen blocking, and then the slides were incubated for 60 min at room temperature. The sections were subsequently incubated with primary antibodies specific for α-SMA (1:500 dilution; cat. no. ab5694), OPN (1:200 dilution; cat. no. ab8448), lumican (1:200 dilution; cat. no. ab168348), fibronectin (1:500 dilution; cat. no. ab2413) E-cadherin (1:500 dilution; cat. no. ab11512), HA (1:100; cat. no. Sc-7392) and collagen type I (1:500 dilution; cat. no. ab34710) overnight at 4°C. With the exception of the HA antibody, which was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA), all of the primary antibodies were purchased from Abcam (Cambridge, MA, USA). The following day, the slides were washed with PBS three times, and they were then incubated with an Alexa Fluor 488-conjugated goat anti-rabbit (A11034), goat anti-rat (A21210), and goat anti-mouse (A11029) secondary antibodies (1:200 dilution; Thermo Fisher Scientific, Inc.) for 60 min at room temperature. The HLE-B3 cells were cultured in 24-well plates (2×10⁴ cells per well) and then treated with TGF-β2 (10 ng/ml) with or without SIS3 (3 µM) for 24 h. The cells were rinsed with PBS three times for 5 min each and then fixed with 4% paraformaldehyde for 30 min. Following washing with PBS, the cells were incubated with 5% BSA for 30 min and permeabilized with 0.1% Triton X-100 for 15 min. The cells were then incubated with primary antibodies specific for fibronectin (dilution 1:200) and E-cadherin (dilution 1:200) overnight at 4°C. The cells were washed with PBS three times, and then incubated with an Alexa Fluor 488-conjugated goat anti-rabbit and goat anti-mouse secondary antibody (1:200 dilution; Thermo Fisher Scientific, Inc.) for 60 min at room temperature. Following washing with PBS, the sections and cells were sealed with mounting medium with DAPI (Beijing Solarbio Science & Technology Co., Ltd., cat. no. S2110). Finally, LEC protein expression was observed with a laser scanning confocal microscope (FV1000; Olympus Corporation, Tokyo, Japan), and images were captured.

Total RNA was extracted from the lenses (n=4 samples per group) with the RNeasy Mini kit in accordance with the manufacturer's protocol (Qiagen GmbH, Hilden, Germany). When the HLE-B3 cells had been subjected to the above treatments, they were lysed in RLT buffer. The total RNA concentration was measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc.). A total of 2 µg of total RNA was used as a template to synthesize cDNA with M-MLV Reverse Transcriptase kit (Promega Corporation, Madison, WI, USA) according to the manufacturer's protocols. The cDNA was then stored at −20°C for later use. qPCR was performed with SYBR Premix Ex Taq™ (Takara Biotechnology Co., Ltd., Dalian, China), and the data was collected using an StepOnePlus™ Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The qPCR thermocycling conditions were as follows: 95°C for 3 min, followed by 40 cycles at 95°C for 12 sec and 62°C for 40 sec, a final extension step of 95°C for 15 sec, 60°C for 1 min, 95°C for 15 sec. The relative expression levels of genes were detected and GAPDH served as an internal control. The primer sequences are presented in Table I. The data were calculated by the 2^−ΔΔCq method (20).

The lenses (n=4 per group) from each group were collected and homogenized in lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) containing a protease and phosphatase inhibitor cocktail (Biotool, Shanghai, China). The precipitate was removed through centrifugation (12,000 × g for 5 min at 4°C), and the protein concentration of the supernatant was measured using a bicinchoninic acid assay kit (Thermo Fisher Scientific, Inc.). The protein sample from the supernatant was then mixed with 5X SDS-PAGE loading buffer and heated for 5 min at 98°C. Equal quantities of proteins (30 µg) were separated by 8-10% SDS-PAGE and then transferred onto a PVDF membrane for 2 h at 300 mA. The membrane was then incubated with 5% skim milk for 2 h at room temperature, and then incubated with primary antibodies specific for α-SMA (1:2,000 dilution), lumican (1:2,000 dilution), fibronectin (1:10,000 dilution), OPN (1:5,000 dilution) and GAPDH (1:10,000 dilution) in Blotto overnight at 4°C. Following three washes with PBST, the membrane was incubated with the appropriate secondary antibody (1:20,000; cat. no. ab6721; Abcam) for 2 h at room temperature. The membrane was then subjected to three washes with PBST, and protein expression was evaluated by chemiluminescence reagent (EMD Millipore) and exposure to chemiluminescent film. The grayscale images of the proteins were analyzed using ImageJ software (v.1.42q; National Institutes of Health, Bethesda, MD, USA).

Apoptosis was measured using a TUNEL detection kit (Roche Diagnostics). Briefly, the frozen sections were removed from storage and air-dried for 30 min at room temperature. Following washing with PBS, the sections were permeabilized with citrate buffer containing 0.1% Triton X-100 for 2 min on ice. DNase I was prepared to treat the positive control samples prior to TUNEL staining. The TUNEL reaction mixture was then added to the sections, which were incubated for 1 h at 37°C. Finally, the sections were mounted in medium with DAPI, and the resultant images were observed under a fluorescence microscope (DM4000 B LED; Leica Microsystems GmbH). The TUNEL-positive cell nuclei contained green fluorescent particles. For quantitative analysis, five fields in each of the sections from each group were randomly selected and observed at ×400 magnification, and the numbers of TUNEL-positive nuclei among 120 LECs were counted.

All experimental results are presented as the mean ± standard deviation of at least three independent experiments. All statistical analyses were performed using SPSS (v.19.0; IBM SPSS, Armonk, NY, USA). Student's t-test was used to assess the differences between two groups. P<0.05 was considered to indicate a statistically significant difference.

To determine whether the absence of Smad3 inhibited the EMT of the lens epithelium following injury, the present study first examined the expression levels of α-SMA, a commonly used EMT marker, using immunofluorescence staining. α-SMA was expressed in Smad3^−/− mice, however, the intensity of immunofluorescence staining intensity in the Smad3^−/− mice was reduced, compared with that in the WT mice on days 7, 14 and 28 post-injury (Fig. 1A). Additionally, the expression of α-SMA was detected by western blot analysis, the results of which confirmed those of the immunofluorescence experiments. Specially, western blot analysis revealed that the expression levels of α-SMA in the Smad3^−/− mice were reduced by 87.5, 92.7 and 90.6%, compared with those in the Smad3^+/+ mice on days 7, 14 and 28 post-injury, respectively (P<0.05; Fig. 1B). The expression of the epithelial marker, E-cadherin, was also detected, the downregulation of which is the most important molecular event in EMT development (21). The Smad3^−/− mice expressed higher levels of E-cadherin, compared with the WT mice at all time points during EMT. Immunofluorescence staining confirmed that the expression levels of E-cadherin were increased in Smad3^−/− mice, compared with those in WT mice (Fig. 1C). Additionally, to further confirm the EMT process, the EMT-related transcription factors Snail1, Slug, SIP1 and Twist1 were examined by RT-qPCR analysis. The results showed that the expression of these four transcription factors increased in trauma-induced EMT of LECs and decreased significantly in Smad3-KO mice, compared with WT mice (Fig. 1D-G). As shown in Fig. 1H, the RT-qPCR results revealed that the expression of α-SMA in the Smad3-KO mice had decreased to levels that were 35.5, 25.3 and 20% of those in the WT mice on days 7, 14 and 28, respectively (P<0.001). The mRNA expression levels of E-cadherin in the Smad3-KO mice were 4-, 6-, and 2.8-fold higher than those in the WT mice on days 7, 14 and 28 post-surgery, respectively (Fig. 1I).

For the in vitro experiments SIS3 was added to the culture to inhibit the phosphorylation of Smad3. As shown in Fig. 2A, the mRNA expression levels of α-SMA and Snail were decreased by 3.6- and 3.7-fold, respectively, and that of E-cadherin was increased by 5.2-fold in the TGF-β2+SIS3 group, compared with the TGF-β2 group. The immunofluorescence staining confirmed these results (Fig. 2B).

To examine ECM accumulation during lens epithelium wound healing, the present study detected the expression levels of lumican, OPN, fibronectin, collagen type I and HA (Fig. 3A-a-g). Lumican is an ECM protein belonging to the small leucine-rich proteoglycan family, which binds to cytokines, regulates ECM secretion by mesenchymal cells, and is involved in epithelial cell migration and tissue repair (22). The expression of lumican was examined using RT-qPCR and western blot analyses. As shown in Fig. 3A-a, the mRNA expression of lumican in the Smad3^−/− mice was decreased to levels that were 21.3, 11.5 and 13.2% of those in the WT mice on days 7, 14 and 28 post-surgery, respectively (P<0.001). The western blot analysis revealed that the expression of lumican in the Smad3-KO mice decreased to levels that were 52.6, 7.9 and 8.8% of those in the WT mice on days 7, 14 and 28 post-injury, respectively (P<0.05), which was consistent with the RT-qPCR results (Fig. 3B). Immunofluorescence staining confirmed that the expression of lumican was decreased in the absence of Smad3 (Fig. 3C).

OPN is a glycoprotein, which is secreted into the ECM by several cells and is expressed in PCO and anterior subcapsular cataract (ASC) tissues (23). To obtain additional insight into the role of OPN in the EMT of the lens epithelium and to identify the steps of the EMT that are dependent on Smad3, the present study examined OPN expression patterns in WT and KO lenses following injury. The WT LECs exhibited strong immunoreactivity to OPN protein, compared with the Smad3-KO LECs at all time points following injury (Fig. 3D). As shown in Fig. 3A-b, the mRNA expression of OPN in the Smad3-KO mice was decreased to levels that were 4.4, 2.2 and 5.6% of those in the WT mice on days 7, 14 and 28 post-surgery, respectively (P<0.001). The protein expression levels of OPN were measured by western blot analysis on day 14, and the protein expression of OPN in the Smad3-KO mice was 79.2% lower than that in the WT mice (P<0.001; Fig. 3E).

Fibronectin is a fibrotic marker and is a major ECM molecule, which is absent in the clear adult lens; however, fibronectin accumulates around LECs in PCO (24). Therefore, the present examined the expression of fibronectin in Smad3-KO and WT mice following puncturing the central anterior lens capsule. The absence of Smad3 decreased the mRNA expression of fibronectin in the Smad3-KO mice to levels that were 22.2, 25 and 29.4% of those in the WT mice on days 7,14 and 28 post-injury, respectively, as shown in Fig. 3A-c. Additionally, the western blot analysis revealed that the expression of fibronectin in the Smad3-KO was decreased by 85.5%, compared with that in the WT mice on day 14 post-injury (Fig. 3F). Immunoreactivity to fibronectin was compared between the two groups. Fibronectin immunofluorescence intensity in the Smad3-KO group was lower than that in the WT group (Fig. 3G). In the in vitro experiments described above, the mRNA expression levels of fibronectin were 3-fold lower in the TGF-β2+SIS3 group, compared those in the TGF-β2 group (Fig. 2A and B), and the immunofluorescence staining confirmed these results.

In the transparent lens, collagen expression is limited to the capsule of the lens; however, abnormal collagen deposition has been identified in ASC plaques and PCO (25). Analysis of the expression of collagen type I was performed using RT-qPCR and immunofluorescence. The mRNA expression of collagen type I in the Smad3-KO mice was decreased to levels that were 65.5, 73 and 71.4% of those in the WT mice on days 7, 14 and 28 following anterior capsular injury, respectively (Fig. 3A-d). Immunofluorescence staining confirmed that the expression of collagen type I was decreased in the absence of Smad3 (Fig. 3H). In the in vitro experiments, the mRNA expression levels of collagen type I were 2.2-fold lower in the TGF-β2+SIS3 group than in the TGF-β2 group (Fig. 2A).

HA is found throughout the body, regulates cell proliferation, migration and differentiation, and is key in inflammation and tumorigenesis (26). Hyaluronan synthase (HAS) is an enzyme, which is vital in HA synthesis and exists as one of the following three subtypes: HAS1, HAS2 and HAS3. In the in vivo mouse model, the expression of HA was increased following injury; however, Smad3-KO decreased the expression of HA (Fig. 3I). The expression levels of HAS1, HAS2 and HAS3 were further examined (Fig. 3A-e-g). As shown in Fig. 3A-f, the expression of HAS2 in the Smad3-KO mice was decreased to levels that were 58, 23.4 and 27.6% of those in the WT mice on days 7, 14 and 28 post-injury, respectively (P<0.05), and the expression of HAS3 in the Smad3-KO mice was decreased to levels that were 20, 48.9 and 63% of those in the WT mice on days 7, 14 and 28 post-injury, respectively (P<0.05) (Fig. 3A-g). However, the expression of HAS1 in the Smad3-KO mice was decreased to levels that were 42.6, 43.3 and 42.1% of those in the WT mice on days 7, 14 and 28, respectively (Fig. 3A-e).

Taken together, the above data indicated that Smad3 signaling is important in trauma-induced EMT in LECs in vivo and in vitro. The deletion of the Smad3 gene reduced cell transdifferentiation and ECM accumulation, and suppressed fibrosis in trauma-induced EMT in LECs.

TUNEL labeling was applied to compare apoptosis levels between the two experimental groups in the present study. LECs around the capsular puncture undergo apoptosis relatively early in the healing process (27). Therefore, lens epithelial cells apoptosis was detected on day 7 using TUNEL staining. The Smad3^−/− lenses exhibited a significantly higher percentage of TUNEL-positive cells (39.82±4.4%), compared with the Smad3^+/+ lenses (1.1±0.44%) (Fig. 4).