Protection of Human Lens Epithelial Cells from Oxidative Stress Damage and Cell Apoptosis by KGF-2 through the Akt/Nrf2/HO-1 Pathway

Oxidative stress exerts a significant influence on the pathogenesis of various cataracts by inducing degradation and aggregation of lens proteins and apoptosis of lens epithelial cells. Keratinocyte growth factor−2 (KGF-2) exerts a favorable cytoprotective effect against oxidative stress in vivo and in vitro. In this work, we investigated the molecular mechanisms of KGF-2 against hydrogen peroxide- (H2O2-) induced oxidative stress and apoptosis in human lens epithelial cells (HLECs) and rat lenses. KGF-2 pretreatment could reduce H2O2-induced cytotoxicity as well as reactive oxygen species (ROS) accumulation. KGF-2 also increases B-cell lymphoma-2 (Bcl-2), quinine oxidoreductase-1 (NQO-1), superoxide dismutase (SOD2), and catalase (CAT) levels while decreasing the expression level of Bcl2-associated X (Bax) and cleaved caspase-3 in H2O2-stimulated HLECs. LY294002, the phosphatidylinositol-3-kinase (PI3K)/Akt inhibitor, abolished KGF-2's effect to some extent, demonstrating that KGF-2 protected HLECs via the PI3K/Akt pathway. On the other hand, KGF-2 activated the Nrf2/HO-1 pathway by regulating the PI3K/Akt pathway. Silencing nuclear factor erythroid 2-related factor 2 (Nrf2) by targeted-siRNA and inhibiting heme oxygenase-1 (HO-1) through zinc protoporphyrin IX (ZnPP) significantly decreased cytoprotection of KGF-2. Furthermore, as revealed by lens organ culture assays, KGF-2 treatment decreased H2O2-induced lens opacity in a concentration-dependent manner. As demonstrated by these data, KGF-2 resisted H2O2-mediated apoptosis and oxidative stress in HLECs through Nrf2/HO-1 and PI3K/Akt pathways, suggesting a potential protective effect against the formation of cataracts.


Introduction
Cataract, primarily age-related, is the main reason for visual impairment and blindness across the world [1]. Currently, cataractous lens removal surgery is the only effective remedy. However, it still presents the risk of surgical complications such as posterior capsular opacification, which gives rise to the second loss of clear vision [2]. Furthermore, no efficient clinical therapies have been proposed to prevent the formation of cataracts until now. Hence, pharmacological intervention to maintain the transparency of the lens is vital.
Oxidative stress, mediated by reactive oxygen species (ROS), where DNA, proteins, lipids, and cells are damaged, is the leading contributor to the formation of cataracts [3,4]. In addition, oxidative stress triggers apoptosis for human lens epithelial cells (HLECs) by altering the internal environment. It is considered the common molecular basis for cataract initiation as well as progression [5]. Hydrogen peroxide (H 2 O 2 ) is a nonradical member in ROS, which damages ion pump activity in HLECs [6,7]. Raising H 2 O 2 levels of aqueous humor are found among patients suffering cataracts which cause lens opacification in vitro [8]. H 2 O 2 has gained extensive uses in inducing oxidative stress in vitro [9][10][11]. Subsequently, protecting HLECs against H 2 O 2 -induced oxidative stress and apoptosis is a potential solution to postpone cataract development.
Since no effective therapeutic drugs have been proposed for halting the formation of the cataractous lens, developing a pharmacological therapy to postpone cataract progression and ameliorate lens transparency is imperative. Evidence suggests that growth factors help prevent injury from various causes, including H 2 O 2 , radiation, and bleomycin [12][13][14]. In addition, recent studies demonstrate that growth factors possibly reduce oxidant-induced lung damage by suppressing apoptosis [15]. However, few studies have confirmed the function of growth factors in HLECs.
Keratinocyte growth factor−2 (KGF-2), also known as fibroblast growth factor−10 (FGF-10), is a critical member of the FGF family. As a multifunctional growth factor, KGF-2 plays an important role in developing various organs and tissues, including the eye [16]. For example, KGF-2 null murine embryos demonstrate agenesis in ocular glands-extra orbital and intraorbital lacrimal glands and Harderian gland [17]. Several studies have confirmed that KGF-2 can decrease oxidant-induced cell injury, inhibit cell apoptosis, and regulate cell homeostasis in numerous organs including the kidney, lung, and spinal cord [18][19][20][21]. The KGF-2 intraocular medication has been proved to be safe. Previous studies have shown that rhKGF-2 eye drop application in the long run in the rabbit corneas does neither result in any apparent systemic effects nor cause toxicity to the intraocular tissues [22]. Furthermore, the regulatory effect of KGF-2 on the PI3K/Akt pathway is confirmed [23,24]. As a survival pathway, the PI3K/Akt pathway modulates cell proliferation, migration, metabolism, and differentiation within diverse disorders [25]. According to increasing evidence, Akt activation can protect HLECs from oxidative stress damage and apoptosis [26,27].
Nrf2 is a transcription factor that can regulate the expression of antioxidants, transporters, autophagy-related proteins, and enzymes involved in metabolism and detoxification (like heme oxygenase-1 (HO-1)) [28]. Studies have also suggested that PI3K/Akt signaling can regulate Nrf2 expression [29]. Previous studies have elucidated that Nrf2 exerts a dominant influence on protecting the lens from oxidative stress [30]. The activation of Nrf2 may lower oxidative stress and prevent cataract formation [31]. As a result, Nrf2 may play the role of a promising therapeutic target for cataract treatment. Therefore, we adopted H 2 O 2 -mediated HLECs and rat lenses as study models and for the first time demonstrated that KGF-2 may protect HLECs against oxidative stress-mediated apoptosis via the Akt/Nrf2/HO-1 pathway and may prove beneficial for treating cataracts associated with oxidative stress.

Apoptosis Analysis.
Flow cytometric analysis was used to measure cell apoptosis in accordance with the protocol offered by Annexin V-FITC/PI apoptosis kit. Briefly, we exposed HLECs to H 2 O 2 (100 μM) for 12 h or pretreated with 12.5 or 50 μg/mL KGF-2 for 2 h and then treated with H 2 O 2 (100 μM) for another 12 h. In total, Annexin V-FITC (5 μL) together with PI (10 μL), PI was added, followed by 15 min tube incubation in dark. The apoptotic rate was later quantitatively analyzed with the use of a flow cytometer (FACScan, Becton-Dickinson, USA).
2.5. Determination of Intracellular Redox State. Intracellular ROS levels were detected with flow cytometry. We inoculated HLECs (4 × 10 5 /well) into the 6-well plate. On the next day, we exposed cells to H 2 O 2 (100 μM) for 12 h or pretreated with 12.5 or 50 μg/mL KGF-2 for 2 h and then treated them with H 2 O 2 (100 μM) for another 12 h. Afterwards, a DCFH-DA reagent (10 μM/L) was utilized to incubate cells under 37°C for 0.5 h. ROS generation was finally evaluated using fluorescence microscopy and a FACScan flow cytometer (FACScan, Becton-Dickinson, USA).
2.6. Western Blot Analysis. HLECs treated above were collected and lysed in RIPA buffer via phosphatase (Applygen, P1260) and protease (Boster, AR0101/AR0103) inhibitors. The lysate received centrifugation at 12000 g for 20 min, and cytosol proteins were extracted. Bicinchoninic acid (BCA) reagent determined the protein concentration. Proteins were isolated using 12-15% SDS-PAGE before transfer on PVDF membranes. Thereafter, we blocked membranes using 5% skimmed milk and cultured them using primary antibodies, including p-Akt , under 4°C overnight, followed by another 1 h horseradish peroxidase-conjugated-labeled secondary antibody incubation on the following day. Visualization for signals and band intensity was done with chemiluminescence in the gel imaging system (Bio-Rad Laboratories, Hercules, CA, USA).

2.7.
Nrf2 siRNA Transfection. The siRNA targeting the Nrf2(sc-37030) was adopted for knocking down Nrf2 levels. We analyzed transfection efficiency using fluorescein-labeled control siRNA(sc-36869). HLECs were first seeded into a 6well plate, followed by incubation under 5% CO 2 and 37°C conditions till then reached 70-80% confluence. Then, in terms of each transfection, we diluted Nrf2 or control siRNA (2 μL) and transfection reagent (2 μL, sc-29528) in the siRNA transfection medium (100 μL, sc-36868; Santa Cruz, CA, USA), separately. Then, we combined dilutions to incubate under ambient temperature for 0.5 h. After that, cells were transfected for 12 h and subsequently treated with 50 μg/mL KGF-2 for 2 h before the treatment with 100 μM H 2 O 2 for 12 h.

Lens
Organ Culture and Treatment. Male SD rats were obtained from the Charles River (Beijing, China). The Institutional Animal Experimentation Ethics Committee of the Wenzhou Medical University approved all animal protocols in the current work. Rat eyes were firstly extracted and stored within the heated (37°C) mammalian normal saline. We then cultured fresh transparent lenses within the medium that contained 0.1% BSA and gentamicin (50 mg/ mL) under 5% CO 2 and 37°C conditions. Lenses were treated with KGF-2 at the concentration of 50 and 100 μg/ mL for 2 h at 37°C. Then, with the addition of 100 μM H 2 O 2 for 12 h, lenses were detected via the stereomicroscope and shot in a black gridline background, thus recording the opacity development course. Further, lenses per group were rinsed in cold saline, placed on a filter paper for drying, dissected using Vannas scissors, and finally milled above the ice. Eventually, the tissue was spun down inside the refrigerated centrifuge at 1000 rpm for 10 min. Supernatants underwent lysis treatment above the ice for 20 min through radioimmunoprecipitation assay (RIPA) buffer with protease inhibitor cocktail and centrifugation at 12000 g for 20 min. Variations of indicated protein expression levels were assessed through western blotting as described above.
2.9. Statistical Analysis. Data used in the current experiment were indicated by mean ± SD. GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA) was employed for statistical analyses. ANOVA was conducted to analyze significance. p values below 0.05 suggested statistical significance.

KGF-2 Suppressed H 2 O 2 -Induced Apoptosis in HLECs.
Bcl-2 protein family has a critical function in modulating the mitochondrial apoptotic pathway [32]. In addition, the caspase family exerts a crucial function in the initiation and execution of programmed cell death. As a kind of executioner caspase, caspase-3 is activated within apoptotic cells via the internal and external (death ligand) pathways [33]. Therefore, members in the Bcl-2 family (either proapoptotic or antiapoptotic) and cleaved caspase-3 can affect the performance of apoptosis. Western blotting analysis revealed that exposure to H 2 O 2 enhanced Bax and cleaved caspase-3 expression but reduced Bcl-2 expression in HLECs, whereas such effects could be eliminated through pretreatment with KGF-2 in a concentration-dependent manner (Figure 2(a)). Consistent with western blotting analysis, flow cytometry manifested that KGF-2 could relieve H 2 O 2 -induced viability decrease of HLECs (Figure 2(b)).

KGF-2 Inhibited H 2 O 2 -Induced Oxidative Stress in
HLECs. HLECs exposed to H 2 O 2 have a light green color after the incubation of the DCFH-DA reagent, indicating a 3 Oxidative Medicine and Cellular Longevity prominent rise of ROS levels. Such rise could be avoided in a concentration-dependent way by pretreatment with KGF-2 ( Figure 3(a)). Flow cytometry analyses reveal that H 2 O 2induction markedly elevated intracellular ROS production in comparison with control, while KGF-2 remarkably reduced it compared with H 2 O 2 -induced HLECs (Figure 3(b)). Furthermore, the antioxidant capacity of KGF-2 in H 2 O 2 -stimulated HLECs is assessed by measuring the levels of antioxidant enzymes (containing SOD2, NQO-1, and CAT). H 2 O 2 significantly reduces SOD2 and CAT activity and increases NQO-1 content relative to the control group. In comparison with the H 2 O 2 group, KGF-2 pretreatment substantially increases SOD2 and CAT levels and further increases NQO-1 content (Figure 3(c)). These findings show that KGF-2 inhibits H 2 O 2 -induced oxidative damage of HLECs.

KGF-2 Treatment Reduces Oxidative Stress and
Apoptosis by Activating the PI3K/Akt Pathway. KGF-2 facilitates multiple biological activities via paracrine through activating PI3K/Akt pathway [21,34]. Western blotting analysis indicated KGF-2 significantly reinforced phosphorylation in Akt in a concentration-dependent manner (Figure 4(a)). In contrast, LY294002, an inhibitor for PI3K/ Akt, suppresses the Akt phosphorylation induced by KGF-2 ( Figure 4(b)). We also used LY294002 to verify if the PI3K/Akt pathway contributed to KGF-2-mediated cytoprotective effects. LY294002 could reverse the antiapoptotic effect of KGF-2 through increasing Bax and cleaved caspase-3 content and decreasing Bcl-2 content compared with KGF-2 pretreatment (Figure 4(c)). Meanwhile, LY294002 attenuated KGF-2-induced increase of the activates of SOD2, CAT, and NQO-1 content in H 2 O 2 -stimulated HLECs (Figure 4(d)). These findings demonstrate that KGF-2 suppressed H 2 O 2 -induced apoptosis and oxidative stress of HLECs by eliciting the PI3K/Akt pathway. To verify whether HO-1 and Nrf2 were required for the protective function of KGF-2, we pretreated HLECs with ZnPP and evaluated cell viability by MTT assays. ZnPP remarkably reversed the cytoprotective impacts of KGF-2 on H 2 O 2 -induced cytotoxicity ( Figure 5(c)). Besides, Nrf2 siRNA has significantly weakened the cell viability in the HLECs pretreated by KGF-2 ( Figure 5(d)). According to the obtained data, Nrf2 and HO-1 exert a crucial function in the cytoprotection regulated by KGF-2. Then, we studied the impacts of KGF-2 alone on Nrf2 and HO-1 induction to verify if it is directly linked to both activations. HLECs treated with KGF-2 alone led to time-dependent induction in Nrf2 and HO-1 protein expression ( Figure 5(e)). The Nrf2 siRNA treatment notably abolished the levels of HO-1 expression regulated by KGF-2 in KGF-2-induced HLECs, suggesting that the upregulation of HO-1 is elicited by KGF-2 mediation via Nrf2 activation ( Figure 5(f)). Furthermore, we investigated the relationship between the Nrf2 and the HO-1 handled by KGF-2 in H 2 O 2 -stimulated HLECs. Simi-larly, preincubation with Nrf2 siRNA markedly suppressed KGF-2-mediated upregulation of Nrf2 and HO-1 at the protein levels ( Figure 5(g)). These findings support the fact that the cytoprotective effects of KGF-2 were mediated by the Nrf2/HO-1 pathway.

KGF-2 Prevents H 2 O 2 -Induced Lens
Opacity. An organ culture experiment was performed to examine how KGF-2 affected cataractogenesis of rat lenses induced with H 2 O 2 . Morphological observational results verified transparency loss of lenses under exposure to H 2 O 2 . By contrast, untreated lenses showed almost no change. The opacities of rat lenses incubated in H 2 O 2 media containing various concentrations of KGF-2 were analyzed and show remission in a KGF-2 concentration-dependent manner (Figure 6(a)). Western blotting measures Nrf2 and HO-1 level in H 2 O 2stimulated lens. Pretreatment with KGF-2 significantly increases the expression of Nrf2 and HO-1 (Figure 6(b)). Accordingly, KGF-2 blocked the process of cataractogenesis.

Discussion
Cataracts are the foremost cause of blindness globally, bringing serious socioeconomic crises to numerous countries. Unfortunately, there still lacks a well-received 5 Oxidative Medicine and Cellular Longevity pharmacological agent to suppress opacification. Oxidative stress, characterized by excessive ROS, is an essential mediator of both the initiation and the progression of cataracts. Excessive production of ROS may disrupt oxidationantioxidant balance and cause mitochondrial dysfunction, lipid peroxidation, or cell apoptosis. Thus, decreasing oxidative stress and avoiding oxidative stress-induced apoptosis are likely therapeutic targets for cataracts [35].
KGF-2, a typical paracrine growth factor, signals using the interaction with its high-affinity receptor FGFR2-IIIb splicing isoform [36]. KGF-2 has numerous biological functions, including regulating cell survival and proliferation, suppressing inflammation, or exhibiting antioxidant effects. The exogenous supplementation of KGF-2 prevents the formation and development of multiple illnesses, such as wound healing defects, cardiovascular diseases, and metabolism syndrome, as well as acute kidney injury [18,[37][38][39]. For example, KGF-2 encourages axonal regeneration and functional recovery following peripheral nerve injury through protecting Schwann cells against excessive oxidative stress-induced apoptosis [23]. Our study reveals that KGF-2 exhibits no apparent cytotoxicity in HLECs. Given its nontoxic properties and favorable antioxidative effects, using KGF-2 to treat age-related cataracts may be an effective way.        Oxidative Medicine and Cellular Longevity 3's proteins have been considered the foremost regulators of apoptosis. Bax is a proapoptotic gene, while Bcl-2 belongs to an antiapoptotic gene [41]. Caspase-3 is the most used target to detect apoptosis [42]. H 2 O 2 leads to Bcl-2 downregulation whereas Bax and cleaved caspase-3 upregulation, but the downregulation of Bcl-2 and the upregulation of Bax and cleaved caspase-3 were reversed after treatment with different concentrations of KGF-2. Besides, data indicate that KGF-2 has no cytotoxicity on HLECs. In general, KGF-2 exerts cytoprotective effects through ameliorating cell viability and blocking apoptosis of oxidative stress-stimulated HLECs.
Intracellular ROS is an indicator that reflects the level of oxidative stress directly. Hence, effects of KGF-2 on H 2 O 2induced intracellular ROS using the flow cytometry technique were detected. The results revealed that KGF-2 inhibits intracellular ROS production, showing the obvious antioxidative effects of KGF-2. In the meantime, our studies also showed an enhancement in the endogenous antioxidant systems with KGF-2 treatment. The antioxidant systems include numerous antioxidant enzymes including SOD2, CAT, and NQO-1, protecting cells from oxidative impairment following oxidative insult or during physiological metabolism. The three enzymes are indispensable to normal lens metabolism, which protect the lens against oxidative stress and retain lens transparency [43,44]. H 2 O 2 -induced cataracts were linked to reduced SOD2 and CAT activities and reinforced NQO-1 activity of the lens. These changes were facilitated by KGF-2 at varying concentrations, proving that KGF-2 has the function of alleviating oxidative stress through increasing endogenous antioxidants.
The PI3K/Akt pathway provides a critical pathway cascade for promoting cell survival and inhibiting apoptosis. Furthermore, research shows that activation via the PI3K/ Akt pathway promotes the antioxidant defense ability of HLECs [45]. Considering that PI3K/Akt pathway has an essential effect on preventing cell stress and enhancing cell survival, it is speculated that this pathway is probably the critical pathway for KGF-2 to exert its cytoprotective effect. In our study, KGF-2 remarkably promoted phosphorylation in Akt subject to oxidative stress conditions, indicating that KGF-2 elicited the PI3K/Akt pathway to resist oxidative stress and retain cell survival. For deepening existing research on PI3K/Akt pathway and its protective effects on KGF-2 against H 2 O 2 -induced HLEC injury, LY294002, a pharmacological inhibitor of PI3K/Akt pathway engaged with the cytoprotective effects of KGF-2, was studied. LY294002 reversed the change of the antioxidant enzymes (SOD2, CAT, and NQO-1) and the apoptotic regulators (Bcl-2, Bax, and cleaved caspase-3) induced by KGF-2 in the H 2 O 2 -stimulated HLECs. It also significantly decreased SOD2, CAT, and Bcl-2 expression and increased NQO-1, Bax, and cleaved caspase-3 expression, demonstrating PI3K/Akt pathway's involvement in cytoprotective effects on KGF-2.
It is universally believed that Nrf2 is a significant transcription factor that plays an indispensable role in the expression of antioxidant and detoxification enzymes. HO-1, a famous phase II detoxifying enzyme, can considerably balance redox homeostasis and inhibit inflammatory illnesses [46,47]. In oxidative stress, HO-1 undergoes upregulation to reinforce the cellular defense mechanism against oxidative insult [48][49][50]. PI3K/Akt pathway is found to exert a cytoprotective influence by enhancing the Nrf2/HO-1 pathway [51][52][53][54]. Data shows that KGF-2 pretreatment effectively increases the expression of Nrf2 and HO-1 in H 2 O 2 -stimulated HLECs. However, this increase was significantly downregulated by LY294002, indicating the partial involvement of the PI3K/Akt pathway in activating the Nrf2 and HO-1. An interesting finding is that ZnPP and Nrf2 siRNA abolishes the KGF-2's protective effect against H 2 O 2 -induced HLEC death, indicating that Nrf2 and HO-1 are essential in KGF-2-induced cytoprotection. Besides, Nrf2 belongs to an upstream mediator of phase II detoxifying enzymes; thus, Nrf2 translocation can mediate HO-1 gene transcription for enhancing cellular defensive capacity and combating oxidative stress [55,56]. We evaluated the effect of KGF-2 alone on Nrf2 and HO-1 to further demonstrate that KGF-2 participates in the activation of a cytoprotective pathway. The results demonstrated that KGF-2 activates HO-1 and Nrf2 levels depending on time. When we knock down the expression of Nrf2 using Nrf2 siRNA, the content of HO-1 is also reduced. We further determine the impact of KGF-2 on the expression of Nrf2 and HO-1 protein in H 2 O 2 -induced HLECs. Similarly, Nrf2 siRNA abolished this enhancement in Nrf2 and HO-1 induced by KGF-2 in H 2 O 2 -stimulated HLECs, suggesting the cytoprotective effect of KGF-2 in regard to the Nrf2/HO-1 pathway. Therefore, these findings provide direct evidence that the Nrf2/HO-1 pathway regulated by KGF-2 may be an underlying therapeutic target for cataracts.
The in vitro experiments confirmed the effects of KGF-2 on the prevention of oxidative stress-induced cell apoptosis, whereas the key to the problem is whether KGF-2 is efficient in lens tissue. For answering the question, we refer to the findings in an ex vivo experiment evaluating KGF-2's anticataract potential of H 2 O 2 -mediated rat lens isolation through monitoring the lens transparency while predicting several biochemical variables like Nrf2 and HO-1 contents. Compared with the normal lenses, the transparency of the lens exposed to H 2 O 2 in vitro was decreased. However, the H 2 O 2 -induced opacity of lenses was ameliorated by treatment using KGF-2. Therefore, results in relevant ex vivo experiments suggest that KGF-2 effectively stunted H 2 O 2 -induced cataract formation, possibly by increasing the contents of Nrf2 and HO-1 to maintain the lens transparency.
In conclusion, this study confirmed the ability of H 2 O 2 in inducing HLEC apoptosis and lens opacification, while KGF-2 may efficiently alleviate lens opacity and the apoptosis of human lens epithelial cells under H 2 O 2 -induced oxidative stress. Furthermore, KGF-2, an effective antioxidant, was found to be efficiently protect HLECs through PI3K/ Akt and Nrf2/HO-1 signaling pathways (see supplementary materials (available here)). Also, the safety of KGF-2 in intraocular use has been confirmed; we believed that KGF-2 can be applied in clinical practice as an underlying protective agent in cataract formation. 10 Oxidative Medicine and Cellular Longevity

Data Availability
All data included in this study are available upon request by contact with the corresponding author.

Conflicts of Interest
The authors declare that they have no conflicts of interest.