Open Access
Cornea  |   June 2017
SERPINA3K Ameliorates the Corneal Oxidative Injury Induced by 4-Hydroxynonenal
Author Affiliations & Notes
  • Xiling Zheng
    Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China
  • Huixia Cui
    Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China
  • Yuanyuan Yin
    Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China
  • Yajing Zhang
    Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China
  • Rongrong Zong
    Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China
  • Xiaorui Bao
    Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China
  • Jian-xing Ma
    Department of Physiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States
  • Zuguo Liu
    Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China
  • Yueping Zhou
    Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China
  • Correspondence: Yueping Zhou, Eye Institute of Xiamen University, 168 Daxue Road, Xiamen, Fujian 361005, People's Republic of China; ypzhou@yahoo.com
  • Footnotes
     XZ and HC contributed equally to the work presented here and should therefore be regarded as equivalent authors.
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 2874-2883. doi:10.1167/iovs.17-21544
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      Xiling Zheng, Huixia Cui, Yuanyuan Yin, Yajing Zhang, Rongrong Zong, Xiaorui Bao, Jian-xing Ma, Zuguo Liu, Yueping Zhou; SERPINA3K Ameliorates the Corneal Oxidative Injury Induced by 4-Hydroxynonenal. Invest. Ophthalmol. Vis. Sci. 2017;58(7):2874-2883. doi: 10.1167/iovs.17-21544.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: We previously demonstrated that SERPINA3K has anti-inflammatory, antiangiogenic, and antioxidant effects in corneas. Here we further investigated the effects of SERPINA3K on the corneal oxidant injury setting recently developed and induced by 4-hydroxynonenal (4-HNE).

Methods: We applied the 4-HNE–induced corneal oxidant stress in cultured human corneal epithelial (HCE) cells in vitro and to the cornea of rats in vivo. The following experiments were conducted: cell counting kit 8 assay to detect cell viability; quantitative real-time PCR assay; Western blotting and immunofluorescent staining to measure gene expressions or protein levels of key reactive oxygen species (ROS)–associated factors (3-nitrotyrosine [3-NT]; nicotinamide adenine dinucleotide phosphate [NADPH]–oxidase 4 [NOX4]; superoxide dismutase [SOD]); catalase and nuclear factor [erythroid-derived 2]–like 2 [NRF2]); as well as main factors of the Wnt/β-catenin signaling pathway (p-LRP6, β-catenin and transcription factor 4 [TCF4]); histologic staining; and TUNEL staining to examine sections of rat corneas.

Results: We found that SERPINA3K concentration dependently protected cell viability, decreased levels of ROS marker 3-NT, suppressed NOX4, and upregulated SOD and catalase. Furthermore, SERPINA3K inhibited the activation of the ROS pathway NRF2 and its downstream factors, NAD(P)H dehydrogenase (quinone) 1 (NQO1) and heme oxygenase 1 (HO1), and also suppressed the activation of the Wnt signaling pathway p-LRP6, β-catenin, and TCF4 in HCE cells treated with 4-HNE. Meanwhile, SERPINA3K ameliorated the oxidant injury of rat corneas induced by 4-HNE and downregulated ROS systems and the Wnt/β-catenin pathway.

Conclusions: Our findings show that SERPINA3K protected the oxidant damage induced by 4-HNE in the cornea and its underlying mechanism was through suppression of the ROS system and inhibition of the activated Wnt/β-catenin signaling pathway.

It is believed that oxidative stress is associated with the pathogenesis of inflammation and angiogenesis of corneas. Some corneal diseases and ocular surface diseases are mainly caused by oxidant injury, such as pterygium.1 However, the role of oxidant stress in corneal diseases needs to be further elucidated, and new effective potential antioxidants to treat corneal diseases have to be explored with more effort. 
SERPINA3K (SA3K), also called kallikrein-binding protein, is a member of the serine proteinase inhibitor family.24 It was recently reported that SERPINA3K is an inhibitor of Wnt/β-catenin signaling pathway.5 We have demonstrated that SERPINA3K has beneficial effects of antineovascularization, anti-inflammation, and antioxidative stress in corneas,68 indicating that SERPINA3K has the potential to be served as a novel therapeutic agent to treat corneal diseases, while further investigations are necessary. 
The reactive oxygen species (ROS) system is composed of ROS generation and ROS degradation. The research of the role of ROS, the ROS system and pathway, and the exploration of new antioxidants has recently drawn a lot of attention. 4-Hydroxynonenal, also known as 4-HNE or HNE, is a main product generated during lipid peroxidation. Many reports documented the role of 4-HNE in the oxidative stress.911 4-Hydroxynonenal has been applied as an important marker of lipid peroxidation/oxidative stress and it has been used to induce cell oxidative stress in experimental research. We recently reported that 4-HNE plays an oxidant role in the corneal epithelium through the regulation of the ROS system and suggested that 4-HNE can be applied as an oxidant injury setting in experimental corneal research.12 
In this present study, we continued to further investigate the antioxidant activities of SERPINA3K in cornea, using the novel corneal oxidant injury setting induced by 4-HNE; we also focused on the underlying mechanism associated with the ROS system and Wnt/β-catenin signaling pathway. 
Materials and Methods
We purchased 4-HNE from Enzo Life Sciences (Farmingdale, NY, USA). The cell counting kit (CCK)-8 was purchased from Dojindo (Kyushu, Japan). The antibodies of anti–3-nitrotyrosine (3-NT), anti–nicotinamide adenine dinucleotide phosphate [NADPH]–oxidase 4 (NOX4), anti-SOD2, anti-catalase, anti–nuclear factor (erythroid-derived 2)-like 2 (NRF2), anti-NQO1, and anti–HO-1 were purchased from Abcam (Cambridge, UK). The antibodies of anti–phospho-LRP6, anti–β-catenin, and anti-transcription factor 4 (TCF4) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The antibody specific for β-actin was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG, HRP-conjugated goat anti-mouse IgG, and HRP-conjugated rabbit were purchased from Sigma-Aldrich Corp. AlexaFluor488 donkey anti-rabbit IgG, AlexaFluor488 donkey anti-mouse IgG and AlexaFluor594 donkey anti-mouse IgG were purchased from Invitrogen (Carlsbad, CA, USA). Hematoxylin and eosin (H&E)-methylene blue were purchased from Solabio Life Science, Inc. (Beijing, China). We purchased TUNEL kits from Beyotime Biotechnology (Haimen, China). 
Purification of SERPINA3K
The SERPINA3K/pET28 construct was introduced into Escherichia coli strain BL21. The purification procedure of SERPINA3K has been previously reported.7 The purity of recombinant SERPINA3K was examined by SDS-PAGE. Endotoxin concentration was monitored by using a limulus amebocyte kit. Activity was checked by MTT assay with HUVEC cells. 
Cell Culture and Procedures
Human corneal epithelial (HCE) cells, simian virus 40 transformed, were obtained from RIKEN BioResource Center (Tokyo, Japan), and were passaged in Dulbecco's modified Eagle medium: Nutrient mixture F-12 (DMEM-F12) media (Invitrogen, Carlsbad, CA, USA) supplemented with 6% heat-inactivated fetal bovine serum, bovine insulin (7 μg/mL), recombinant human epidermal growth factor (10 ng/mL), and 1% penicillin and streptomycin. 
To establish experimental settings for oxidative stress, 4-HNE at a concentration of 30 μM was selected and applied or the same amount of vehicle (n-hexane) was added as conditioned medium and cultured for 24 hours; simultaneously, SERPINA3K at different concentrations of 80 nM and 160 nM were given in the treated groups. 
For cell viability assay, the HCE cells were plated at a density of 8000 cells per well in 96-well culture plates. When the HCE cells were cultured to about 70% confluency, the media were removed and changed to DMEM-F12 basic media (serum free) in the presence of 4-HNE or SERPINA3K at a specific concentration and in the absence of 4-HNE or SERPINA3K. For all other experiments, the HCE cells were plated at a density of 30 × 104 cells per well in 6-well or 10 × 104 cells per well in 12-well culture plates, cultured in the presence of 4-HNE or SERPINA3K at a specific concentration and in the absence of 4-HNE or SERPINA3K. After treatment for a specified time period, the cells were harvested or prepared for CCK-8 assay, immunofluorescent staining, Western blot, and quantitative real-time PCR (qRT-PCR) analysis following the methods and procedures described below. 
In Vivo Experimental Procedures
Wistar rats (male, 180–220 g, aged 4–6 weeks) were purchased from Shanghai Shilaike Laboratory Animal Co., Ltd., (Shanghai, China). The animal experiments were carefully performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the animal experimental protocol was approved by the Animal Ethics Committee of Medical College of Xiamen University (approval ID: XMUMC: 2014-10-20). The animals were kept in an air-conditioned facility, with food and water ad libitum. The rats were randomly divided into four groups (n = 6): (1) control group without any treatment (saline containing same amount of vehicle); (2) positive control group, 4-HNE plus PBS only group; (3) another positive control group, 4-HNE plus bovine serum albumin (BSA) group; and (4) SERPINA3K treatment group, 4-HNE plus SERPINA3K group. 
The experimental procedure was as follows: rats were anesthetized with pentobarbital (50 mg/kg intraperitoneally), then 10 μL 4-HNE (250 μM) was topically administered. After 30 minutes, 10 μL PBS only, 10 μL BSA (10 μg in PBS), or 10 μL SERPINA3K (10 μg in PBS) was topically given. After 12 hours, the administrations were given at both eyes of the rats again. After 24 hours, the rats were killed, followed by removal and dissection of the eyeballs or cornea. The dissected eyeballs were stored in a −80°C freezer for the use of hematoxylin and eosin (H&E) staining, TUNEL staining, and immunofluorescent staining. The method of dissection of corneal tissue for Western blot and quantitative real-time PCR was previously reported.8,12 The whole corneal tissue was carefully dissected immediately under a surgical microscope by an experienced person. 
Cell Viability Assay
Cell viability was measured using a CCK-8 assay and conducted following the protocol of the manufacturer. After HCE cells were cultured for 24 hours in the conditional media, then the conditional media were replaced by CCK-8 constituted in culture media, followed by incubation for 4 hours at 37°C in the dark. The solution was detected directly after incubation. The absorbance was measured spectrophotometrically at 450 nm with a microplate reader (Bio Tek ELX800; Bio Tek Instruments, Winooski, VT, USA). 
Immunofluorescent Staining
For the immunofluorescent staining with anti-3-NT, anti-NOX4, and anti-NRF2 antibodies, the HCE cells were fixed in 4% paraformaldehyde for 1 hour and then kept in 70% ethanol for use, the corneal sections were fixed in cold acetone for 10 minutes. We incubated the HCE cells or the corneal sections with anti–3-NT antibody (1:150); anti-NOX4 antibody (1:150); and anti-NRF2 antibody (1:150) at 4°C overnight, respectively. After three washes with PBS, The HCE cells or the corneal sections were further incubated in AlexaFluor488-conjugated IgG (1:300) or AlexaFluor594-conjugated IgG (1:300) for 1 hour. After three washes with PBS (10 minutes each time), the HCE cells or the corneal sections were mounted with mounting medium with 4,6-diamidino-2-phenylindole (VECTASHIELD; Vector Laboratories, Burlingame, CA, USA), and photographed with a microscope (Leica DM2500; Leica microsystems, Wetzlar, Germany). 
Western Blot Analysis
The treated HCE cells or dissected corneal tissues were lysed and total cellular protein concentrations were measured by a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein were resolved by electrophoresis through 10% tris-glycine SDS polyacrylamide gel and electro-transferred onto a PVDF membrane. The membrane was blocked with 1% (wt/vol) BSA in tris-buffered saline with 0.1% TWEEN-20 (TBST) for 1 hour and subsequently incubated overnight at 4°C with primary antibodies of anti–3-NT (1:300); NOX4 (1:500); anti-NRF2 (1:500); anti-SOD2 (1:5000); anti-catalase (1:2000); anti-NQO1 (1:1000); anti-HO1 (1:250); anti–phospho-LRP6 (1:1000); anti–β-catenin (1:1000) and anti-TCF4 (1:1000) at 4°C overnight. After three washes with TBST, the membrane was incubated for 1 hour with a 1:10,000 dilutions of an HRP-conjugated IgG antibody in TBST containing 1% BSA. After three washes with TBST, the bands were detected using a commercial imaging system (Molecular Imager ChemiDoc XRS; Bio-Rad Laboratories, Hercules, CA, USA). As needed, the membrane was stripped in stripping buffer (CWBIO, Beijing, China) and reblotted with an antibody specific for β-actin for loading control. The band intensities were semiquantified by densitometry using analytical software (Quantity-One; Bio-Rad Laboratories). 
RNA Extraction and Quantitative Real-Time PCR Assay
Total RNA was extracted from the treated HCE cells by using TRIzol reagent (Invitrogen). Reverse transcription was performed with Oligo18T primers and reverse transcription reagents according to the manufacturer's protocol (TaKaRa Bio, Inc., Shiga, Japan). Quantitative real-time PCR was performed with mRNA special primers. The following primers were used for the PCR: for SOD2, 5′-GAGAAGTACCAGGAGGCGTTG-3′ (forward) and 5′-GAGCCTTGGACACCAACAGAT-3′ (reverse); for catalase, 5′-ACTGAGGTCCACCCTGACTAC-3′ (forward) and 5′-TCGCATTCTTAGGCTTCTCA-3′ (reverse); for NRF2, 5′-AAACCAGTGGATCTGCCAAC-3′ (forward) and 5′-GACCGGGAATATCAGGAACA-3′ (reverse); for NQO1, 5′-ATGTATGACAAAGGACCCTTCC-3′ (forward) and 5′-TCCCTTGCAGAGAGTACATGG-3′ (reverse); for HO1, 5′-AAGATTGCCCAGAAAGCCCTGGAC-3′ (forward) and 5′-AACTGTCGCCACCAGAAAGCTGAG-3′ (reverse). We performed PCR reactions on a commercial PCR system (CFX-96 Touch Real-Time PCR Detection System; Bio-Rad Laboratories) with a master mix (SYBR Premix Ex Taq; TaKaRa Bio, Inc.) at 95°C for 10 minutes, followed by 45 cycles of 95°C for 10 seconds, 57°C for 30 seconds, and 75°C for 10 seconds, after which melt curve analysis was performed at once from 65°C to 95°C. All reactions were performed in triplicate and the average cycle threshold (Ct) values greater than 38 were treated as negative. 
Statistical Analysis
We conducted a 1-way ANOVA test to analyze the data from CCK-8 assay, Western blot, and quantitative real-time PCR, followed by a post hoc analysis Tukey test to compare the differences between the groups or a Student's t-test. A value of P < 0.05 was considered statistically significant. 
Results
SERPINA3K Protected HCE Cells Induced by 4-HNE
We previously demonstrated that SA3K has protective effects on the oxidant injury induced by hydrogen peroxide (H2O2) in HCE cells.8 Here we applied a recently developed oxidant damage setting induced by 4-HNE12 and investigated the antioxidant effects of SERPINA3K on the 4-HNE–induced oxidant injury. We treated HCE cells with various concentrations of 4-HNE (5, 10, 20, 30, 40, and 80 μM) for 24 hours and it was shown that 4-HNE decreased the cell viability of HCE cells in a concentration-dependent manner (Fig. 1A). We finally selected a concentration of 30 μM 4-HNE to induce the oxidant damage in HCE cells and applied the same concentration of 4-HNE in the following experiments. It was demonstrated that SERPINA3K significantly protected the cellular toxicity induced by 4-HNE, particularly at concentrations of 80 and 160 nM of SERPINA3K (Fig. 1B). 
Figure 1
 
SA3K protected HCE cells induced by 4-HNE. (A) Cell viability of HCE cells after treatment of various concentrations of 4-HNE (0, 5, 10, 20, 30, 40, and 80 μM) for 24 hours. Data are presented as mean ± SEM, n = 5 in each group. ***P < 0.001. A concentration of 30 μM HNE was selected and applied to induce oxidative stress in the following experiments in HCE cells. (B) SERPINA3K at 80 and 160 nM significantly ameliorated the cellular injury induced by 4-HNE. Data are presented as mean ± SEM, n = 3 in each group. **P < 0.01, ***P < 0.001. (C) Representative images of Western blotting of 3-NT, a marker of ROS. (D) Statistical analysis of Western blotting data of 3-NT. Data are presented as mean ± SEM, n = 3 in each group. *P < 0.05. ** P < 0.01. (E) Representative images of immunocytochemical staining of 3-NT in HCE cells. Scale bars: 10 μm.
Figure 1
 
SA3K protected HCE cells induced by 4-HNE. (A) Cell viability of HCE cells after treatment of various concentrations of 4-HNE (0, 5, 10, 20, 30, 40, and 80 μM) for 24 hours. Data are presented as mean ± SEM, n = 5 in each group. ***P < 0.001. A concentration of 30 μM HNE was selected and applied to induce oxidative stress in the following experiments in HCE cells. (B) SERPINA3K at 80 and 160 nM significantly ameliorated the cellular injury induced by 4-HNE. Data are presented as mean ± SEM, n = 3 in each group. **P < 0.01, ***P < 0.001. (C) Representative images of Western blotting of 3-NT, a marker of ROS. (D) Statistical analysis of Western blotting data of 3-NT. Data are presented as mean ± SEM, n = 3 in each group. *P < 0.05. ** P < 0.01. (E) Representative images of immunocytochemical staining of 3-NT in HCE cells. Scale bars: 10 μm.
We then investigated the changes of 3-NT, a marker of ROS,13 after treatment of SERPINA3K in 4-HNE–induced oxidant injury in HCE cells. As shown by the Western blotting analysis in Figures 1C and 1D, SERPINA3K significantly suppressed the increased levels of 3-NT induced by 4-HNE (Figs. 1C, 1D). Meanwhile, it also revealed by immunofluorescent staining images that SERPINA3K inhibited the cellular intensity signals of 3-NT after induction of 4-HNE (Fig. 1E). 
Taken together, the novel experimental data suggest that SERPINA3K ameliorates the oxidant damages induced by 4-HNE, recently developed in human corneal epithelial cells. 
SERPINA3K Suppressed the ROS System and Pathway
It is well known that the ROS system can be divided into ROS generation and ROS degradation.14,15 A key enzyme of ROS generation is NOX4,1619 whereas SOD and catalase are two classic main enzymes of ROS degradation.20,21 We first determined if SERPINA3K targets on NOX4 by Western blot and immunofluorescent staining. It demonstrated that SERPINA3K significantly inhibited the upregulated NOX4 protein level (Figs. 2A, 2B) as well as the immunofluorescent signals of NOX4 (Fig. 2C). Furthermore, we also detected the gene expressions and protein levels of SOD and catalase. It showed that SERPINA3K significantly increased the downregulated gene expressions and protein levels of SOD and catalase (Figs. 2D–I). 
Figure 2
 
SA3K suppressed ROS system induced by 4-HNE in HCE cells. The level of ROS generation enzyme NOX4 was measured by Western blotting and immunocytochemical staining. (A) Representative images of Western blotting of NOX4. (B) Statistical analysis of Western blotting data of NOX4. Data are presented as mean + SEM, n = 4 in each group. *P < 0.05, **P < 0.01. (C) Representative images of immunocytochemical staining of NOX4 in HCE cells. Scale bars: 10 μm. The gene expressions and protein levels of ROS degradation factors SOD and catalase were detected by qRT-PCR assay and Western blotting. (D) Quantitative RT-PCR of SOD gene expression in HCE cells. Data are presented as mean ± SEM, n = 4 in each group. *P < 0.05, ***P < 0.001. (E) Quantitative RT-PCR of catalase gene expression in HCE cells. Data are presented as mean ± SEM, n = 4 in each group. *P < 0.05, ***P < 0.001. (F) Representative images of Western blotting of SOD. (G) Statistical analysis of Western blotting data of SOD. Data are presented as mean ± SEM, n = 6 in each group. *P < 0.05, ***P < 0.001. (H) Representative images of Western blotting of catalase. (I) Statistical analysis of Western blotting data of catalase. Data are presented as mean ± SEM, n = 7 in each group. *P < 0.05, **P < 0.01.
Figure 2
 
SA3K suppressed ROS system induced by 4-HNE in HCE cells. The level of ROS generation enzyme NOX4 was measured by Western blotting and immunocytochemical staining. (A) Representative images of Western blotting of NOX4. (B) Statistical analysis of Western blotting data of NOX4. Data are presented as mean + SEM, n = 4 in each group. *P < 0.05, **P < 0.01. (C) Representative images of immunocytochemical staining of NOX4 in HCE cells. Scale bars: 10 μm. The gene expressions and protein levels of ROS degradation factors SOD and catalase were detected by qRT-PCR assay and Western blotting. (D) Quantitative RT-PCR of SOD gene expression in HCE cells. Data are presented as mean ± SEM, n = 4 in each group. *P < 0.05, ***P < 0.001. (E) Quantitative RT-PCR of catalase gene expression in HCE cells. Data are presented as mean ± SEM, n = 4 in each group. *P < 0.05, ***P < 0.001. (F) Representative images of Western blotting of SOD. (G) Statistical analysis of Western blotting data of SOD. Data are presented as mean ± SEM, n = 6 in each group. *P < 0.05, ***P < 0.001. (H) Representative images of Western blotting of catalase. (I) Statistical analysis of Western blotting data of catalase. Data are presented as mean ± SEM, n = 7 in each group. *P < 0.05, **P < 0.01.
Nuclear factor (erythroid-derived 2)–like 2 is an important factor of the ROS signaling pathway.2225 Our previous data indicated that 4-HNE can activate the NRF2 signaling pathway.12 We then investigated if SERPINA3K acts through NRF2 signaling pathway and its downstream factors NQO1 and HO1. As shown in Figure 3, SERPINA3K decreased the upregulated protein level of NRF2 (Figs. 3A, 3B) and the protein levels of its downstream factors: NQO1 (Figs. 3C, 3D) and HO1 (Figs. 3E, 3F) in the HCE cells after exposure to 4-HNE; whereas the gene expression levels of NRF2, NQO1, and HO1 were also inhibited after treatment of SERPINA3K (Figs. 3G–I). 
Figure 3
 
SA3K suppressed NRF2 pathway induced by 4-HNE in HCE cells. The gene expressions and protein levels of key factors of the ROS pathway—NRF2 and its downstream factors, NQO1 and HO1—were measured. (A) Representative images of Western blotting of NRF2. (B) Statistical analysis of Western blotting data of NRF2. Data are presented as mean ± SEM, n = 4 in each group. *P < 0.05, **P < 0.01. (C) Representative images of Western blotting of NQO1. (D) Statistical analysis of Western blotting data of NQO1. Data are presented as mean + SEM, n = 3 in each group. **P < 0.01, ***P < 0.001. (E) Representative images of Western blotting of HO1. (F) Statistical analysis of Western blotting data of HO1. Data are presented as mean + SEM, n = 5 in each group. *P < 0.05, ***P < 0.001. (GI) Quantitative RT-PCR data of NRF2, NQO1, and HO1 gene expressions in HCE cells, respectively. Data are presented as mean ± SEM, n = 3, 3, 3 in each group of G, H, and I, respectively. *P < 0.05, **P < 0.01, ***P < 0.001).
Figure 3
 
SA3K suppressed NRF2 pathway induced by 4-HNE in HCE cells. The gene expressions and protein levels of key factors of the ROS pathway—NRF2 and its downstream factors, NQO1 and HO1—were measured. (A) Representative images of Western blotting of NRF2. (B) Statistical analysis of Western blotting data of NRF2. Data are presented as mean ± SEM, n = 4 in each group. *P < 0.05, **P < 0.01. (C) Representative images of Western blotting of NQO1. (D) Statistical analysis of Western blotting data of NQO1. Data are presented as mean + SEM, n = 3 in each group. **P < 0.01, ***P < 0.001. (E) Representative images of Western blotting of HO1. (F) Statistical analysis of Western blotting data of HO1. Data are presented as mean + SEM, n = 5 in each group. *P < 0.05, ***P < 0.001. (GI) Quantitative RT-PCR data of NRF2, NQO1, and HO1 gene expressions in HCE cells, respectively. Data are presented as mean ± SEM, n = 3, 3, 3 in each group of G, H, and I, respectively. *P < 0.05, **P < 0.01, ***P < 0.001).
The data above indicated that SERPINA3K inhibited the oxidant effects induced by 4-HNE via regulations of ROS generation and degradation as well as ROS signaling pathway. 
SERPINA3K Inhibited the Activated Wnt/β-Catenin Signaling Pathway
SERPINA3K is reported as an inhibitor of the Wnt/β-catenin signaling pathway.5 We then focused on the association of Wnt/β-catenin pathway and the protective effects of SERPINA3K after treatment of 4-HNE by measuring: (1) LRP6, the potential binding site of SERPINA3K; (2) β-catenin, a key and main factor of Wnt/β-catenin pathway; and (3) TCF4, which is a downstream effector of Wnt/β-catenin pathway. Interestingly, it revealed that exposure to 4-HNE activated the Wnt/β-catenin signaling pathway by increasing the protein levels of p-LRP6, β-catenin, and TCF4; meanwhile, the treatment of SERPINA3K reversed the upregulated levels of p-LRP6 (Figs. 4A, 4B); β-catenin (Figs. 4C, 4D); and TCF4 (Figs. 4E, 4F), indicating that SERPINA3K can inhibit 4-HNE–induced activation of the Wnt/β-catenin signaling pathway in HCE cells. 
Figure 4
 
SA3K inhibited the activated Wnt/β-catenin pathway induced by 4-HNE in HCE cells. The key factors of the Wnt/β-catenin pathway: p-LRP6, β-catenin, and TCF4 were measured by Western blotting. (A) Representative images of Western blotting of p-LRP6. (B) Statistical analysis of Western blotting data of p-LRP6. Data are presented as mean ± SEM, n = 3 in each group. *P < 0.05, **P < 0.01. (C) Representative images of Western blotting of β-catenin. (D) Statistical analysis of Western blotting data of β-catenin. Data are presented as mean ± SEM, n = 3 in each group. **P < 0.01. (E) Representative images of Western blotting of TCF4. (F) Statistical analysis of Western blotting data of TCF4. Data are presented as mean ± SEM, n = 3 in each group. *P < 0.05, **P < 0.01.
Figure 4
 
SA3K inhibited the activated Wnt/β-catenin pathway induced by 4-HNE in HCE cells. The key factors of the Wnt/β-catenin pathway: p-LRP6, β-catenin, and TCF4 were measured by Western blotting. (A) Representative images of Western blotting of p-LRP6. (B) Statistical analysis of Western blotting data of p-LRP6. Data are presented as mean ± SEM, n = 3 in each group. *P < 0.05, **P < 0.01. (C) Representative images of Western blotting of β-catenin. (D) Statistical analysis of Western blotting data of β-catenin. Data are presented as mean ± SEM, n = 3 in each group. **P < 0.01. (E) Representative images of Western blotting of TCF4. (F) Statistical analysis of Western blotting data of TCF4. Data are presented as mean ± SEM, n = 3 in each group. *P < 0.05, **P < 0.01.
SERPINA3K Ameliorated Corneal Oxidant Injury Induced by 4-HNE in Rats
Finally, we conducted topical application of 4-HNE in the corneas of rats and investigated the effects of SERPINA3K on the 4-HNE–induced oxidant injury in vivo. It was demonstrated that 4-HNE treatment did not induce dramatic histologic changes on the corneal epithelium and stroma by H&E staining (Fig. 5, top row of images), meanwhile 4-HNE induced TUNEL staining–positive signals and SERPINA3K decreased the 4-HNE–induced TUNEL intensified signals, indicating that SERPINA3K has a protective effects on the cellular injury induced by 4-HNE (Fig. 5, second row of images). 
Figure 5
 
SA3K ameliorated corneal oxidant injury induced by 4-HNE in rats. (A) Representative images of H&E staining of rat corneal epithelium. (B) Representative images of TUNEL staining of rat corneal epithelium. (C) Representative images of immunohistochemical staining of ROS marker 3-NT of rat corneal epithelium. (D) Representative images of immunohistochemical staining of ROS generation enzyme NOX4 of rat corneal epithelium. (E) Representative images of immunohistochemical staining of key factor of ROS pathway NRF2 of rat corneal epithelium. Scale bars: (A) 50 μm. (BE) 10 μm.
Figure 5
 
SA3K ameliorated corneal oxidant injury induced by 4-HNE in rats. (A) Representative images of H&E staining of rat corneal epithelium. (B) Representative images of TUNEL staining of rat corneal epithelium. (C) Representative images of immunohistochemical staining of ROS marker 3-NT of rat corneal epithelium. (D) Representative images of immunohistochemical staining of ROS generation enzyme NOX4 of rat corneal epithelium. (E) Representative images of immunohistochemical staining of key factor of ROS pathway NRF2 of rat corneal epithelium. Scale bars: (A) 50 μm. (BE) 10 μm.
We then determined if the underlying mechanisms of the effects of SERPINA3K on the 4-HNE–induced rat oxidant injury are through regulations of the ROS system and pathway as well as Wnt/β-catenin signaling pathway. We performed immunofluorescent staining on the sections of rat corneas with antibodies of (1) 3-NT, a marker of ROS; (2) NOX4, a key enzyme of ROS generation; and (3) NRF2, a key factor of ROS signaling pathway. It was shown that 4-HNE induced intensified immunostaining signals of 3-NT, NOX4, and NRF2 respectively, meanwhile these intensified immunostaining signals of 3-NT, NOX4, and NRF2 were suppressed after treatment with SERPINA3K (Fig. 5, lower three rows of images). Furthermore, Western blotting data demonstrated that the protein levels of 3-NT, NRF2, and β-catenin were upregulated by exposure of 4-HNE, and SERPINA3K significantly inhibited the increased levels of 3-NT (Figs. 6A, 6B); NRF2 (Figs. 6C, 6D); and β-catenin (Figs. 6E, 6F). 
Figure 6
 
SA3K regulated ROS system and Wnt/β-catenin pathway induced by 4-HNE in rat corneas. Western blotting was performed to detect the protein levels of ROS marker 3-NT, key factor of ROS pathway NRF2, and key factor of Wnt/β-catenin pathway β-catenin. (A) Representative images of Western blotting of 3-NT. (B) Statistical analysis of Western blotting data of 3-NT. Data are presented as mean ± SEM, n = 3 in each group. *P < 0.05, **P < 0.01. (C) Representative images of Western blotting of NRF2. (D) Statistical analysis of Western blotting data of NRF2. Data are presented as mean ± SEM, n = 4 in each group. *P < 0.05, **P < 0.01, ***P < 0.001. (E) Representative images of Western blotting of β-catenin. (F) Statistical analysis of Western blotting data of β-catenin. Data are presented as mean ± SEM, n = 3 in each group. **P < 0.01.
Figure 6
 
SA3K regulated ROS system and Wnt/β-catenin pathway induced by 4-HNE in rat corneas. Western blotting was performed to detect the protein levels of ROS marker 3-NT, key factor of ROS pathway NRF2, and key factor of Wnt/β-catenin pathway β-catenin. (A) Representative images of Western blotting of 3-NT. (B) Statistical analysis of Western blotting data of 3-NT. Data are presented as mean ± SEM, n = 3 in each group. *P < 0.05, **P < 0.01. (C) Representative images of Western blotting of NRF2. (D) Statistical analysis of Western blotting data of NRF2. Data are presented as mean ± SEM, n = 4 in each group. *P < 0.05, **P < 0.01, ***P < 0.001. (E) Representative images of Western blotting of β-catenin. (F) Statistical analysis of Western blotting data of β-catenin. Data are presented as mean ± SEM, n = 3 in each group. **P < 0.01.
The combined data of the in vivo experiment suggests that SERPINA3K ameliorates the oxidant injury induced by 4-HNE and the responsible mechanism is the regulation of the ROS system and pathway, as well as blockade of activation of the Wnt/β-catenin pathway. 
Discussion
We provided novel evidence demonstrating the antioxidant activities of SERPINA3K in corneas, using a recently developed corneal oxidant injury setting by 4-HNE. Our data are consistent with our previous finding that SERPINA3K has the antioxidative stress effects against hydrogen peroxide–induced corneal oxidant damage. This present study also reveals that the underlying responsible mechanism of SERPINA3K is by the inhibition of ROS system and the blockage of activation of the Wnt/β-catenin signaling pathway. 
4-Hydroxynonenal has been widely studied in the experimental research of oxidative stress. Scientists used 4-HNE as an oxidative stress marker as well as an oxidative stressor to establish oxidant injury. In the experimental eye research, multiple reports used 4-HNE as an oxidant marker in the retina.2628 However, few studies about the role of 4-HNE were reported in the corneal field. Recently, we successfully established 4-HNE–induced corneal oxidant injury setting.12 It is demonstrated that 4-HNE can induce oxidative damage in corneal epithelium, and 4-HNE targets on the ROS system and pathway.12 In present study, we applied this 4-HNE–induced corneal oxidant injury setting to further demonstrate the antioxidant activities of SERPINA3K in corneas. Since 4-HNE is endogenous and it is a main product generated during lipid peroxidation, our data offered additional experimental facts to support the antioxidant effects of SERPINA3K in cornea from other resources of oxidant-induced injury, such as classic oxidative stressor, hydrogen peroxide. 
The role of Wnt/β-catenin signaling pathway in the oxidative stress draws increasing attention recently and its full role is not completely known.2931 In this present study, we found that the corneal oxidant injury induced by 4-HNE can result in the activation of Wnt/β-catenin signaling pathway in both in vitro and in vivo experiments. We provided the evidence that the 4-HNE–induced corneal oxidative stress is associated with the Wnt/β-catenin signaling pathway. Interestingly, SERPINA3K, as an inhibitor of Wnt/β-catenin signaling pathway, blocked the activation of Wnt/β-catenin pathway induced by 4-HNE. Further experiments in the field are needed to elucidate the role of Wnt/β-catenin signaling pathway in the oxidative stress, such as the detailed complex cross-talk between ROS system and Wnt/β-catenin signaling pathway, the interference of oxidants/antioxidants to Wnt/β-catenin signaling pathway, and the application of other different oxidative injury/damage models. 
We have reported that SERPINA3K elicits antineovascularization, anti-inflammation, and antioxidative stress activities in corneas by using different corneal neovascularization, inflammation, and oxidative stress models. In summary, our present novel evidence, together with our previous experimental data, suggest that SERPINA3K is a unique and potential candidate for the exploration of new medications to treat corneal diseases. However, there is a need for an experimental pharmacologic comparison between SERPINA3K and other conventional medications for corneal diseases in the future. 
Acknowledgments
Supported by the Natural Science Foundation of China (Grant No. 81170818, Beijing, China); the Chinese National Key Scientific Research Project (Grant No. 2013CB967003, Beijing, China); and the National High Technology Research and Development Program of China (Grant No. 2012AA020507, Beijing, China). The authors alone are responsible for the content and writing of the paper. 
Disclosure: X. Zheng, None; H. Cui, None; Y. Yin, None; Y. Zhang, None; R. Zong, None; X. Bao, None; J.-X. Ma, None; Z. Liu, None; Y. Zhou, None 
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Figure 1
 
SA3K protected HCE cells induced by 4-HNE. (A) Cell viability of HCE cells after treatment of various concentrations of 4-HNE (0, 5, 10, 20, 30, 40, and 80 μM) for 24 hours. Data are presented as mean ± SEM, n = 5 in each group. ***P < 0.001. A concentration of 30 μM HNE was selected and applied to induce oxidative stress in the following experiments in HCE cells. (B) SERPINA3K at 80 and 160 nM significantly ameliorated the cellular injury induced by 4-HNE. Data are presented as mean ± SEM, n = 3 in each group. **P < 0.01, ***P < 0.001. (C) Representative images of Western blotting of 3-NT, a marker of ROS. (D) Statistical analysis of Western blotting data of 3-NT. Data are presented as mean ± SEM, n = 3 in each group. *P < 0.05. ** P < 0.01. (E) Representative images of immunocytochemical staining of 3-NT in HCE cells. Scale bars: 10 μm.
Figure 1
 
SA3K protected HCE cells induced by 4-HNE. (A) Cell viability of HCE cells after treatment of various concentrations of 4-HNE (0, 5, 10, 20, 30, 40, and 80 μM) for 24 hours. Data are presented as mean ± SEM, n = 5 in each group. ***P < 0.001. A concentration of 30 μM HNE was selected and applied to induce oxidative stress in the following experiments in HCE cells. (B) SERPINA3K at 80 and 160 nM significantly ameliorated the cellular injury induced by 4-HNE. Data are presented as mean ± SEM, n = 3 in each group. **P < 0.01, ***P < 0.001. (C) Representative images of Western blotting of 3-NT, a marker of ROS. (D) Statistical analysis of Western blotting data of 3-NT. Data are presented as mean ± SEM, n = 3 in each group. *P < 0.05. ** P < 0.01. (E) Representative images of immunocytochemical staining of 3-NT in HCE cells. Scale bars: 10 μm.
Figure 2
 
SA3K suppressed ROS system induced by 4-HNE in HCE cells. The level of ROS generation enzyme NOX4 was measured by Western blotting and immunocytochemical staining. (A) Representative images of Western blotting of NOX4. (B) Statistical analysis of Western blotting data of NOX4. Data are presented as mean + SEM, n = 4 in each group. *P < 0.05, **P < 0.01. (C) Representative images of immunocytochemical staining of NOX4 in HCE cells. Scale bars: 10 μm. The gene expressions and protein levels of ROS degradation factors SOD and catalase were detected by qRT-PCR assay and Western blotting. (D) Quantitative RT-PCR of SOD gene expression in HCE cells. Data are presented as mean ± SEM, n = 4 in each group. *P < 0.05, ***P < 0.001. (E) Quantitative RT-PCR of catalase gene expression in HCE cells. Data are presented as mean ± SEM, n = 4 in each group. *P < 0.05, ***P < 0.001. (F) Representative images of Western blotting of SOD. (G) Statistical analysis of Western blotting data of SOD. Data are presented as mean ± SEM, n = 6 in each group. *P < 0.05, ***P < 0.001. (H) Representative images of Western blotting of catalase. (I) Statistical analysis of Western blotting data of catalase. Data are presented as mean ± SEM, n = 7 in each group. *P < 0.05, **P < 0.01.
Figure 2
 
SA3K suppressed ROS system induced by 4-HNE in HCE cells. The level of ROS generation enzyme NOX4 was measured by Western blotting and immunocytochemical staining. (A) Representative images of Western blotting of NOX4. (B) Statistical analysis of Western blotting data of NOX4. Data are presented as mean + SEM, n = 4 in each group. *P < 0.05, **P < 0.01. (C) Representative images of immunocytochemical staining of NOX4 in HCE cells. Scale bars: 10 μm. The gene expressions and protein levels of ROS degradation factors SOD and catalase were detected by qRT-PCR assay and Western blotting. (D) Quantitative RT-PCR of SOD gene expression in HCE cells. Data are presented as mean ± SEM, n = 4 in each group. *P < 0.05, ***P < 0.001. (E) Quantitative RT-PCR of catalase gene expression in HCE cells. Data are presented as mean ± SEM, n = 4 in each group. *P < 0.05, ***P < 0.001. (F) Representative images of Western blotting of SOD. (G) Statistical analysis of Western blotting data of SOD. Data are presented as mean ± SEM, n = 6 in each group. *P < 0.05, ***P < 0.001. (H) Representative images of Western blotting of catalase. (I) Statistical analysis of Western blotting data of catalase. Data are presented as mean ± SEM, n = 7 in each group. *P < 0.05, **P < 0.01.
Figure 3
 
SA3K suppressed NRF2 pathway induced by 4-HNE in HCE cells. The gene expressions and protein levels of key factors of the ROS pathway—NRF2 and its downstream factors, NQO1 and HO1—were measured. (A) Representative images of Western blotting of NRF2. (B) Statistical analysis of Western blotting data of NRF2. Data are presented as mean ± SEM, n = 4 in each group. *P < 0.05, **P < 0.01. (C) Representative images of Western blotting of NQO1. (D) Statistical analysis of Western blotting data of NQO1. Data are presented as mean + SEM, n = 3 in each group. **P < 0.01, ***P < 0.001. (E) Representative images of Western blotting of HO1. (F) Statistical analysis of Western blotting data of HO1. Data are presented as mean + SEM, n = 5 in each group. *P < 0.05, ***P < 0.001. (GI) Quantitative RT-PCR data of NRF2, NQO1, and HO1 gene expressions in HCE cells, respectively. Data are presented as mean ± SEM, n = 3, 3, 3 in each group of G, H, and I, respectively. *P < 0.05, **P < 0.01, ***P < 0.001).
Figure 3
 
SA3K suppressed NRF2 pathway induced by 4-HNE in HCE cells. The gene expressions and protein levels of key factors of the ROS pathway—NRF2 and its downstream factors, NQO1 and HO1—were measured. (A) Representative images of Western blotting of NRF2. (B) Statistical analysis of Western blotting data of NRF2. Data are presented as mean ± SEM, n = 4 in each group. *P < 0.05, **P < 0.01. (C) Representative images of Western blotting of NQO1. (D) Statistical analysis of Western blotting data of NQO1. Data are presented as mean + SEM, n = 3 in each group. **P < 0.01, ***P < 0.001. (E) Representative images of Western blotting of HO1. (F) Statistical analysis of Western blotting data of HO1. Data are presented as mean + SEM, n = 5 in each group. *P < 0.05, ***P < 0.001. (GI) Quantitative RT-PCR data of NRF2, NQO1, and HO1 gene expressions in HCE cells, respectively. Data are presented as mean ± SEM, n = 3, 3, 3 in each group of G, H, and I, respectively. *P < 0.05, **P < 0.01, ***P < 0.001).
Figure 4
 
SA3K inhibited the activated Wnt/β-catenin pathway induced by 4-HNE in HCE cells. The key factors of the Wnt/β-catenin pathway: p-LRP6, β-catenin, and TCF4 were measured by Western blotting. (A) Representative images of Western blotting of p-LRP6. (B) Statistical analysis of Western blotting data of p-LRP6. Data are presented as mean ± SEM, n = 3 in each group. *P < 0.05, **P < 0.01. (C) Representative images of Western blotting of β-catenin. (D) Statistical analysis of Western blotting data of β-catenin. Data are presented as mean ± SEM, n = 3 in each group. **P < 0.01. (E) Representative images of Western blotting of TCF4. (F) Statistical analysis of Western blotting data of TCF4. Data are presented as mean ± SEM, n = 3 in each group. *P < 0.05, **P < 0.01.
Figure 4
 
SA3K inhibited the activated Wnt/β-catenin pathway induced by 4-HNE in HCE cells. The key factors of the Wnt/β-catenin pathway: p-LRP6, β-catenin, and TCF4 were measured by Western blotting. (A) Representative images of Western blotting of p-LRP6. (B) Statistical analysis of Western blotting data of p-LRP6. Data are presented as mean ± SEM, n = 3 in each group. *P < 0.05, **P < 0.01. (C) Representative images of Western blotting of β-catenin. (D) Statistical analysis of Western blotting data of β-catenin. Data are presented as mean ± SEM, n = 3 in each group. **P < 0.01. (E) Representative images of Western blotting of TCF4. (F) Statistical analysis of Western blotting data of TCF4. Data are presented as mean ± SEM, n = 3 in each group. *P < 0.05, **P < 0.01.
Figure 5
 
SA3K ameliorated corneal oxidant injury induced by 4-HNE in rats. (A) Representative images of H&E staining of rat corneal epithelium. (B) Representative images of TUNEL staining of rat corneal epithelium. (C) Representative images of immunohistochemical staining of ROS marker 3-NT of rat corneal epithelium. (D) Representative images of immunohistochemical staining of ROS generation enzyme NOX4 of rat corneal epithelium. (E) Representative images of immunohistochemical staining of key factor of ROS pathway NRF2 of rat corneal epithelium. Scale bars: (A) 50 μm. (BE) 10 μm.
Figure 5
 
SA3K ameliorated corneal oxidant injury induced by 4-HNE in rats. (A) Representative images of H&E staining of rat corneal epithelium. (B) Representative images of TUNEL staining of rat corneal epithelium. (C) Representative images of immunohistochemical staining of ROS marker 3-NT of rat corneal epithelium. (D) Representative images of immunohistochemical staining of ROS generation enzyme NOX4 of rat corneal epithelium. (E) Representative images of immunohistochemical staining of key factor of ROS pathway NRF2 of rat corneal epithelium. Scale bars: (A) 50 μm. (BE) 10 μm.
Figure 6
 
SA3K regulated ROS system and Wnt/β-catenin pathway induced by 4-HNE in rat corneas. Western blotting was performed to detect the protein levels of ROS marker 3-NT, key factor of ROS pathway NRF2, and key factor of Wnt/β-catenin pathway β-catenin. (A) Representative images of Western blotting of 3-NT. (B) Statistical analysis of Western blotting data of 3-NT. Data are presented as mean ± SEM, n = 3 in each group. *P < 0.05, **P < 0.01. (C) Representative images of Western blotting of NRF2. (D) Statistical analysis of Western blotting data of NRF2. Data are presented as mean ± SEM, n = 4 in each group. *P < 0.05, **P < 0.01, ***P < 0.001. (E) Representative images of Western blotting of β-catenin. (F) Statistical analysis of Western blotting data of β-catenin. Data are presented as mean ± SEM, n = 3 in each group. **P < 0.01.
Figure 6
 
SA3K regulated ROS system and Wnt/β-catenin pathway induced by 4-HNE in rat corneas. Western blotting was performed to detect the protein levels of ROS marker 3-NT, key factor of ROS pathway NRF2, and key factor of Wnt/β-catenin pathway β-catenin. (A) Representative images of Western blotting of 3-NT. (B) Statistical analysis of Western blotting data of 3-NT. Data are presented as mean ± SEM, n = 3 in each group. *P < 0.05, **P < 0.01. (C) Representative images of Western blotting of NRF2. (D) Statistical analysis of Western blotting data of NRF2. Data are presented as mean ± SEM, n = 4 in each group. *P < 0.05, **P < 0.01, ***P < 0.001. (E) Representative images of Western blotting of β-catenin. (F) Statistical analysis of Western blotting data of β-catenin. Data are presented as mean ± SEM, n = 3 in each group. **P < 0.01.
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