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Cornea  |   May 2013
Overexpression of SIRT1 Promotes High Glucose–Attenuated Corneal Epithelial Wound Healing via p53 Regulation of the IGFBP3/IGF-1R/AKT Pathway
Author Notes
  • State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Shandong Eye Institute, Shandong Academy of Medical Sciences, Qingdao, China 
  • Correspondence: Lixin Xie, No. 5 Yanerdao Road, Qingdao, 266071 China;lixin_xie@yahoo.com
Investigative Ophthalmology & Visual Science May 2013, Vol.54, 3806-3814. doi:10.1167/iovs.13-12091
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      Ye Wang, Xiaowen Zhao, Daling Shi, Peng Chen, Yang Yu, Lingling Yang, Lixin Xie; Overexpression of SIRT1 Promotes High Glucose–Attenuated Corneal Epithelial Wound Healing via p53 Regulation of the IGFBP3/IGF-1R/AKT Pathway. Invest. Ophthalmol. Vis. Sci. 2013;54(5):3806-3814. doi: 10.1167/iovs.13-12091.

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

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Abstract

Purpose.: Toinvestigate how Sirtuin (silent mating type information regulation 2 homolog) 1 (SIRT1) promotes high glucose–attenuated corneal epithelial wound healing.

Methods.: The effects of high glucose on SIRT1 expression were assessed in primary human corneal epithelial cells (CECs) in treatment of 5 mM d-glucose (normal glucose [NG]) and 25 mM d-glucose (high glucose [HG]) and corneas from Ins2Akita/+ mice by Western blotting. The osmotic pressure of the NG medium was adjusted to that of the HG medium by adding 20 mM mannitol. Pifithrin-α (PFT-α) was used to inhibit the expression of p53 and an adenovirus was used for overexpression of SIRT1 in vivo and in vitro. How overexpression of SIRT1 promotes HG-attenuated corneal epithelial wound healing via p53 regulation of the IGFBP3 (insulin-like growth factor binding protein-3)/IGF-1 (insulin-like growth factor-1)/AKT pathway was investigated in CECs and Ins2Akita/+ mice.

Results.: HG induced the downregulation of SIRT1 and the upregulation of p53 acetylation in primary human CECs and corneas from Ins2Akita/+ mice. The results of cell migration assay and corneal wound healing from Ins2Akita/+ mice demonstrated that SIRT1 overexpression strongly promoted wound healing in the presence of HG levels via the downregulation of the IGFBP3 protein. The levels of total p53 expression and acetylated p53 decreased dramatically in the presence of PFT-α, whereas the IGF-1R/AKT pathway was activated in CECs. The results of cell migration assay suggested this posttranslational modification of p53 was involved in the response to cell injury under HG conditions in CECs.

Conclusions.: The molecular mechanism by which SIRT1 promotes corneal epithelial wound healing was involved in an enhancement of the IGFBP3/IGF-1/AKT pathway through the deacetylation of p53. This study also suggests that SIRT1 has a protective role in the pathogenesis of diabetic keratopathy.

Introduction
Diabetes mellitus (DM) is a major disease worldwide (approximately 170 million people) and diabetic retinopathy (DR) is the most common serious complication of patients with DM. 1,2 It was reported that surgical procedures to correct DR and/or cataract extraction are often followed by greater risk of long-lasting epithelial erosion with poor healing of the epithelial defects. 3 Once the cornea has been damaged, delayed healing of the epithelial wounds can be very difficult to manage clinically. 4 The persistent epithelial defects not only can make the cornea prone to infection but also can lead to corneal ulceration and even perforation, which may lead to irreversible visual impairment and a poor quality of life for patients. 5 In addition, patients with diabetes are also known to exhibit various types of corneal pathology (diabetic keratopathy). 6 These corneal complications may lead to irreversible visual impairment and include gerontoxon, limbal neovascularization, punctate keratopathy, endothelial dystrophy, recurrent erosion, and ulceration. 7 It is reported that corneal sensitivity is decreased in diabetic patients with sterile neurotrophic corneal ulcers, and neuronal abnormalities may be the cause of the corneal changes in diabetic keratopathy. 4 Currently, few studies have focused on the importance of corneal diseases in diabetic patients. In this study, we aimed to gain a better understanding of the molecular mechanism of delayed corneal epithelial wound healing and to develop more effective therapies to deal with this delayed epithelial wound healing. 
Sirtuin (silent mating type information regulation 2 homolog) 1 (SIRT1) is a homolog of the Sir2 protein in S. cerevisiae and belongs to the group of class III histone/protein deacetylases (HDACs). 8,9 Accumulating evidence suggests that SIRT1 also modulates the metabolism of glucose and fat through its deacetylase activity. 10,11 Moreover, SIRT1 plays a pivotal role in the regulation of insulin secretion levels and insulin sensitivity 12,13 and promotes cell survival or inhibits apoptotic cell death by deacetylating p53. 14,15 Deacetylated modifications of the p53 protein have been found to play a positive role in the accumulation of p53 protein during the stress response and are required for p53-induced cell growth arrest and apoptosis. 16,17 Studies have revealed that the p53 pathway modulates the insulin-like growth factor-1 (IGF-1)/AKT pathway. 18,19 The evolutionarily conserved IGF-1/AKT pathway is important for cell survival and differentiation. The loss of AKT activity leads to cellular dysfunction and delayed corneal wound healing. 20 Insulin-like growth factor binding protein-3 (IGFBP3), which is one of the target genes of p53, has been found to negatively regulate the IGF-1/AKT pathway by binding to free IGF-1. 21 IGFBP3 is also frequently present in the tears of diabetic patients. 22  
In this study, the role of SIRT1 in epithelial wound healing in the presence of high glucose levels is investigated using in vitro human corneal cell lines, ex vivo assays on cells from a diabetic mouse model, and in vivo following scratch injury healing responses. We hypothesized that overexpression of SIRT1 decreases acetylated p53 and inhibits IGFBP3, and then activates the IGF-1R/AKT survivor pathway, which is responsible for cell growth and division, thereby contributing to the corneal epithelial wound healing. These results suggest that SIRT1 is a potential therapeutic target for attenuated epithelial wound healing in hyperglycemic conditions. 
Materials and Methods
Cell Culture and Treatment
Primary human corneal epithelial cells (HCECs) 23 were cultured in keratinocyte serum-free medium supplemented with 10% bovine pituitary extract and epidermal growth factor (Life Technologies, Gaithersburg, MD). The simian virus 40–immortalized human corneal epithelial cell line (THCE) 24 was a kind gift from Choun-Ki Joo, MD (Catholic University of Korea, Seoul, Korea), and was maintained in Minimum Essential Medium (MEM) medium supplemented with 10% fetal bovine serum (FBS) (Life Technologies). THCEs were cultured in a humidified 5% CO2 incubator at 37°C in MEM containing 5 mM d-glucose plus 20 mM mannitol (normal glucose [NG]) and 25 mM d-glucose (high glucose [HG]). The osmotic pressure of the NG medium was adjusted to that of the HG medium by adding 20 mM mannitol. In addition, THCEs were treated with 10 or 20 μM PFT-α in HG medium. THCEs treated with NG levels were used as a control. Recombinant adenovirus expressing SIRT1 and GFP was a gift from Jia Liu, PhD (Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China), as previously described. 25 Recombinant adenoviruses were amplified in human embryonic kidney (HEK-293) cells and purified using a commercial kit (Vivapure Adeno-PACK Kit; Sartorius AG, Göttingen, Germany) according to the manufacturer's instructions. Recombinant adenovirus expressing GFP was used as the negative control. 
In Vitro Scratch Injury Model
A scratch injury model 20 to assess the effects of SIRT1 overexpression for attenuated corneal epithelial wound healing was used. THCEs were incubated for 6 hours in MEM (without FBS) containing 10 μL purified adenovirus particles (1 × 107 plaque-forming units [PFUs]/μL) expressing SIRT1 or GFP. Subsequently, the medium was replaced with MEM supplemented with 10% FBS. THCEs incubated with GFP-adenovirus particles were used as the control. After 24 hours, the THCEs were wounded with multiple linear scratches using a standard 1000-μL pipet tip that was scraped from one side of the dish to the other. The dish was rotated and scrapes were made at 45°, 90°, and 135° angles to the original scrapes. The THCEs were then cultured in NG and HG medium. At the end of an additional 48-hour incubation, lysates of the THCEs were prepared and used for Western blotting. 
Animal Experiments
C57BL/6J-Ins2Akita (Ins2Akita/+ ) mice and control Ins2+/+ mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and genotyped according to a previously described protocol. 26 These mice develop diabetes as a consequence of a single amino acid substitution in the insulin 2 gene that causes the misfolding of insulin. Male mice heterozygous for this mutation exhibit a progressive loss of β-cell function and significant hyperglycemia (as early as 4 weeks of age) 27 and develop typical chronic complications of diabetes, such as retinopathy, neuropathy, and nephropathy. 28,29 Tail-vein blood glucose concentrations were determined using a commercial glucometer (Ascencia Contour Glucometer; Bayer Diabetes Care, Elkhart, IN). The mice were treated in compliance with the guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and the experimental protocol was approved by the Institutional Animal Care and Use Committee, Shandong Eye Institute. 
All animals were anesthetized intraperitoneally with ketamine (37.5 mg/mL) and xylazine (1.9 mg/mL). Proparacaine hydrochloride (0.5%) was used for topical anesthesia. The in vivo corneal injury model was previously described by Xu et al. 20 The mice were randomly divided into five groups, and each group contained 10 mice: group 1 included untreated control Ins2+/+ mice; group 2 included untreated Ins2Akita/+ mice; group 3 included saline-treated Ins2Akita/+ mice; group 4 included AD-GFP–treated Ins2Akita/+ mice; and group 5 included AD-SIRT1–treated Ins2Akita/+ mice. The left eyes of the mice in all groups remained uninjured and untreated for use as normal controls. In groups 1 and 2, the right eyes were untreated after injury. For groups 3 to 5, 7 μL saline or purified adenovirus particles (1 × 109 PFU/μL) were injected into the subconjunctival site of right eyes on the same day of corneal epithelium injury. The experiment was performed twice. All groups were assessed using fluorescein sodium staining. To determine the effects of SIRT1 overexpression on HG-attenuated corneal epithelial wound healing in vivo, corneal epithelia from Ins2Akita/+ mice in groups 2, 3, 4, and 5 were isolated at 48 hours after injury and processed for Western blotting. 
Corneal Wound Healing Evaluation
The epithelial wounds were visualized using 3 μL 0.25% fluorescein sodium and photographed at 0, 24, and 48 hours under a dissecting microscope equipped with a digital camera (PowerShot A620; Canon, Tokyo, Japan) and a tungsten light source with a cobalt blue filter (Welch-Allyn, Inc., Skaneateles, NY). The photographs were analyzed to quantify the area of the epithelial wound (Photoshop software; Adobe Systems, Mountain View, CA), and the data are presented as the number of pixels in the fluorescent area. 30  
Real-Time PCR Analysis
Total RNA was isolated (NucleoSpin RNA II System; Macherey-Nagel, Düren, Germany) and quantified using a spectrophotometer (Eppendorf 1108 spectrophotometer; Eppendorf, Hamburg, Germany). cDNA was synthesized from 1 μg RNA with oligo(dT)18 primers by using a commercial kit (cDNA Synthesis Kit; Epicentre Biotechnologies, Madison, WI) and measured by real-time PCR. The primers were designed using commercial software (Primer Express; Applied Biosystems, Foster City, CA). The primers used for SIRT1 were SIRT1(human, NM_001142498): forward (F): 5′-FGC AGA TTA GTA GGC GGC TTG-3′ and reverse (R): 5′-TCT GGC ATG TCC CAC TAT CA-3′; SIRT1(mouse, NM_001159589): F: 5′-AGT TCC AGC CGT CTC TGT GT-3′ and R: 5′-CTC CAC GAA CAG CTT CAC-3′. 
Immunohistochemistry
Cornea samples were fixed in 10% buffered formalin and embedded in paraffin. Paraffin sections of 4-μm thickness were deparaffinized and rehydrated. The samples were then permeabilized in 1.0% Triton X-100 in PBS for 10 minutes followed by incubation with 5% BSA (Boster Biologic Technology, Ltd., Wuhan, China) to block nonspecific binding. The samples were then stained (EliVision Plus Kit; Maxim Corp., Fuzhou, China) according to the manufacturer's protocol. The primary antibody for SIRT1 (ab12193; Abcam, Cambridge, MA) was used. Digital images were obtained using a commercial confocal microscope (Eclipse C1si Spectral Imaging Confocal Microscope; Nikon Instruments, Inc., Melville, NY). 
Western Blotting Analysis
Samples were homogenized in 100 μL ice-cold RIPA (radio-immunoprecipitation assay) lysis buffer (50 mM Tris Cl, pH 7.4/150 mM NaCl/5 mM EDTA/1% Nonidet P-40/1% sodium deoxycholate/0.1% SDS/1% aprotinin, 50 mM NaF/0.1 mM Na3VO4) supplemented with a proteinase inhibitor cocktail. The homogenates, which contained 20 μg protein, were then assayed with 15% SDS-polyacrylamide gels (Mini-Protean II system; Bio-Rad Laboratories, Mississauga, ON, Canada) and transferred to polyvinyl difluoride membranes (Thermo Fisher Scientific China, Beijing, China). The blots were probed with the following primary antibodies: SIRT1 (ab12193), p53 (ab9775), AKT1 (ab6076; Abcam), acetylated-p53 (K382) (#2525, for human), acetylated-p53 (K379) (#2570, for mice), IGF-1 receptor (#9750), phospho-AKT (#4060; Cell Signaling Technology, Danvers, MA), and IGFBP3 (H-98) (sc-9028; Santa Cruz Biotechnology, Santa Cruz, CA). 
Statistical Analyses
All results are expressed as the means ± SDs. For the results shown in Figure 1, the difference in the means between the NG (5 mM d-glucose plus 20 mM mannitol) and HG (25 mM d-glucose) treatments was calculated using an unpaired t-test. For the other results, statistical analyses were performed using a one-way ANOVA by comparing the groups using the Student-Newman-Keuls test and the least significant difference procedure performed with commercial software (SPSS 11.5; SPSS Inc., Chicago, IL). A value of P < 0.05 was considered statistically significant. 
Figure 1
 
High-glucose (HG) conditions induced the downregulation of SIRT1, the upregulation of p53 acetylation, and targets the IGFBP3/IGF-1R/AKT pathways. (A) HG conditions induced the downregulation of SIRT1 in primary HCECs as indicated. HCECs were harvested 48 hours after normal glucose (NG, 5 mM d-glucose plus 20 mM mannitol) or HG (25 mm d-glucose) treatment. The osmotic pressure of the NG medium was adjusted to that of the HG medium by adding 20 mM mannitol. The cell sample treated with NG served as the control. Data are means ± SD (n = 3). The results of Western blotting are shown in (Ai): the top panel shows data from the gels; the bottom panel shows the results normalized to GAPDH, which served as the loading control. SIRT1 mRNA levels were qualitatively analyzed by real-time PCR (Aii). The expression of SIRT1 normalized to GAPDH RNA and the fold change in gene expression related to the calibrator of the NG group are shown. (B) HG conditions induced the downregulation of SIRT1 in isolated mouse corneas as indicated. Isolated mouse corneas were harvested 48 hours after NG (5 mM d-glucose plus 20 mM mannitol) or HG (25 mm d-glucose) treatment. The cornea sample treated with NG served as the control. Data are means ± SD (n = 3). The results of Western blotting are shown in (Bi): top panel shows data from the gels; the bottom panel shows the results normalized to GAPDH. SIRT1 mRNA levels were qualitatively analyzed by real-time PCR (Bii). The expression of SIRT1 normalized to GAPDH RNA and the fold change in gene expression related to the calibrator of the normal glucose group are shown. (C) The expression and localization of SIRT1 on corneal sections from C57BL/6J-Ins2Akita (Ins2Akita/+ ) mice and control Ins2+/+ mice ([C] shows SIRT1 was localized in the corneal epithelium). The expression of SIRT1 was significantly downregulated in the corneal epithelia of Ins2Akita/+ mice (Cii) compared with the corneal epithelia of control Ins2+/+ mice (Ci). (Ciii) The results of real-time PCR. The cornea sample from Ins2+/+ mice served as the control. Data are means ± SD (n = 3). (D) Expression of SIRT1, acetylated p53, p53, IGFBP3, IGF-1R, p-AKT, and AKT in Ins2Akita/+ mice and control Ins2+/+ mice cornea by Western blotting ([Di], top panel: shows the representative data from the gels; [Dii], bottom panel: the results normalized to GAPDH). GAPDH served as the loading control. The cornea sample from Ins2+/+ mice served as the control. Data are means ± SD (n = 3). Significant differences (*P < 0.05) between Ins2Akita/+ mice and control Ins2+/+ mice corneas are identified by asterisks.
Figure 1
 
High-glucose (HG) conditions induced the downregulation of SIRT1, the upregulation of p53 acetylation, and targets the IGFBP3/IGF-1R/AKT pathways. (A) HG conditions induced the downregulation of SIRT1 in primary HCECs as indicated. HCECs were harvested 48 hours after normal glucose (NG, 5 mM d-glucose plus 20 mM mannitol) or HG (25 mm d-glucose) treatment. The osmotic pressure of the NG medium was adjusted to that of the HG medium by adding 20 mM mannitol. The cell sample treated with NG served as the control. Data are means ± SD (n = 3). The results of Western blotting are shown in (Ai): the top panel shows data from the gels; the bottom panel shows the results normalized to GAPDH, which served as the loading control. SIRT1 mRNA levels were qualitatively analyzed by real-time PCR (Aii). The expression of SIRT1 normalized to GAPDH RNA and the fold change in gene expression related to the calibrator of the NG group are shown. (B) HG conditions induced the downregulation of SIRT1 in isolated mouse corneas as indicated. Isolated mouse corneas were harvested 48 hours after NG (5 mM d-glucose plus 20 mM mannitol) or HG (25 mm d-glucose) treatment. The cornea sample treated with NG served as the control. Data are means ± SD (n = 3). The results of Western blotting are shown in (Bi): top panel shows data from the gels; the bottom panel shows the results normalized to GAPDH. SIRT1 mRNA levels were qualitatively analyzed by real-time PCR (Bii). The expression of SIRT1 normalized to GAPDH RNA and the fold change in gene expression related to the calibrator of the normal glucose group are shown. (C) The expression and localization of SIRT1 on corneal sections from C57BL/6J-Ins2Akita (Ins2Akita/+ ) mice and control Ins2+/+ mice ([C] shows SIRT1 was localized in the corneal epithelium). The expression of SIRT1 was significantly downregulated in the corneal epithelia of Ins2Akita/+ mice (Cii) compared with the corneal epithelia of control Ins2+/+ mice (Ci). (Ciii) The results of real-time PCR. The cornea sample from Ins2+/+ mice served as the control. Data are means ± SD (n = 3). (D) Expression of SIRT1, acetylated p53, p53, IGFBP3, IGF-1R, p-AKT, and AKT in Ins2Akita/+ mice and control Ins2+/+ mice cornea by Western blotting ([Di], top panel: shows the representative data from the gels; [Dii], bottom panel: the results normalized to GAPDH). GAPDH served as the loading control. The cornea sample from Ins2+/+ mice served as the control. Data are means ± SD (n = 3). Significant differences (*P < 0.05) between Ins2Akita/+ mice and control Ins2+/+ mice corneas are identified by asterisks.
Results
HG Conditions Induce the Downregulation of SIRT1 In Vitro and In Vivo
To investigate whether high glucose induces the downregulation of SIRT1, we first compared SIRT1 expression in NG-treated (5 mM d-glucose plus 20 mM mannitol) and HG-treated (25 mM d-glucose) HCECs. Compared with NG treatment, the expression of SIRT1 (message/protein) was reduced in HCECs with HG treatment (P < 0.05, Fig. 1A). We then investigated SIRT1 expression in isolated mouse corneas either with NG or HG treatment. As shown in Figure 1B, the expression of SIRT1 (message/protein) was also reduced in mouse corneas with HG treatment compared with NG treatment (P < 0.05). To examine whether SIRT1 expression was also decreased in diabetic animal models, the expression and localization of SIRT1 were analyzed in corneal sections from C57BL/6J-Ins2Akita (Ins2Akita/+ ) mice and control Ins2+/+ mice. The information of animals is shown in the Table. Ins2Akita/+ mouse had significantly higher concentrations of blood glucose compared with those of control mice, whereas the weight of Ins2Akita/+ mouse was also significantly less than that of the control animals. These data agree with results in previous reports. 27,29  
Table.
 
Average Weight and Blood Glucose Level at Time of Death
Table.
 
Average Weight and Blood Glucose Level at Time of Death
Group Numbers Age, wk Weight, g Blood Glucose, mM
Control 10 20–24 31.32 ± 3.14 7.32 ± 0.80
Diabetic 40 20–24 27.89 ± 1.87* 24.16 ± 5.61†
Figures 1Ci and 1Cii show SIRT1 was localized in the corneal epithelium and, compared with the corneal epithelia of control Ins2+/+ mice, the expression of SIRT1 was significantly downregulated in the corneal epithelia of Ins2Akita/+ mice. Furthermore, a relatively low level of SIRT1 protein and high levels of acetylated p53 and IGFBP3 were observed in corneas of these diabetic animal models, compared with control Ins2+/+ mice (Fig. 1D). In contrast, the levels of IGF-1R and p-AKT in Ins2Akita/+ mouse corneas were lower than those in control Ins2+/+ mice (P < 0.05). The protein levels of AKT in Ins2Akita/+ mice and control Ins2+/+ mice were similar. These results suggest that the decrease in SIRT1 and the increase in p53 acetylation in corneal epithelia are associated with the type 1 diabetes condition. HG levels appear to target the IGFBP3/IGF-1R/AKT pathways in the corneas of mice with type 1 diabetes. 
p53 Functions as a Key Regulator of the AKT Pathway in Response to HG-Induced Wounding in THCE Cells
We next examined the relationship between p53 acetylation and the AKT pathway in response to hyperglycemic conditions in THCE cells. Exposure to HG medium resulted in increased acetylated p53 levels and decreased p-AKT levels in the THCEs. However, the increases in acetylated p53 and total p53 were significantly inhibited by 20 μmol/L PFT-α, and the expression level of p-AKT increased accordingly after treatment with 20 μmol/L PFT-α (Fig. 2A). These results indicate that the downregulation of acetylated p53 (and total p53) by PFT-α could activate the AKT pathway, suggesting that p53 is a key upstream regulator of the AKT pathway in THCEs exposed to HG conditions. 
Figure 2
 
p53 is a key regulator of the AKT pathway. (A) The inhibitory effects of PFT-α on p53 expression and activation of the AKT pathway in THCEs after HG treatment. THCEs were treated with NG (5 mM d-glucose plus 20 mM mannitol), HG (25 mM d-glucose), and 10 or 20 μM PFT-α in HG medium. After 48 hours, derived cell lysates were subjected to Western blotting by using anti-acety-p53 (K382), anti-p53, anti-p-AKT, or anti-AKT. GAPDH served as the loading control. The cell sample treated with NG served as the control. Data are means ± SD (n = 3). Left panels: the representative gel results. Right panel: the graphs used for normalization. Significant differences (*P < 0.05) between the HG and NG groups are identified by “*” and significant differences (#P < 0.05) between the HG and 20 μmol/L PFT-α groups are identified by “#.” (B) HG levels induced the downregulation of SIRT1 and high p53 acetylation via AKT signaling in response to wounding in THCE cells as indicated. Left panels: the representative gel results. Right panel: the graphs used for normalization. THCE cells with (W) or without (N) wounds were cultured in NG (5 mM d-glucose plus 20 mM mannitol) and HG (25 mM d-glucose) and allowed to heal for 48 hours. At the end of the culture, cell samples were harvested and derived cell lysates were subjected to Western blotting by using anti-SIRT1, anti-acety-p53 (K382), anti-p53, anti-p-AKT, or anti-AKT. GAPDH served as the loading control. The cell sample treated with NG served as the control. Data are means ± SD (n = 3). Significant differences (*P < 0.05) between the HG and NG groups are identified by “*” and significant differences (#P < 0.05) between the HG wounding group and the NG wounding group are identified by “#.”
Figure 2
 
p53 is a key regulator of the AKT pathway. (A) The inhibitory effects of PFT-α on p53 expression and activation of the AKT pathway in THCEs after HG treatment. THCEs were treated with NG (5 mM d-glucose plus 20 mM mannitol), HG (25 mM d-glucose), and 10 or 20 μM PFT-α in HG medium. After 48 hours, derived cell lysates were subjected to Western blotting by using anti-acety-p53 (K382), anti-p53, anti-p-AKT, or anti-AKT. GAPDH served as the loading control. The cell sample treated with NG served as the control. Data are means ± SD (n = 3). Left panels: the representative gel results. Right panel: the graphs used for normalization. Significant differences (*P < 0.05) between the HG and NG groups are identified by “*” and significant differences (#P < 0.05) between the HG and 20 μmol/L PFT-α groups are identified by “#.” (B) HG levels induced the downregulation of SIRT1 and high p53 acetylation via AKT signaling in response to wounding in THCE cells as indicated. Left panels: the representative gel results. Right panel: the graphs used for normalization. THCE cells with (W) or without (N) wounds were cultured in NG (5 mM d-glucose plus 20 mM mannitol) and HG (25 mM d-glucose) and allowed to heal for 48 hours. At the end of the culture, cell samples were harvested and derived cell lysates were subjected to Western blotting by using anti-SIRT1, anti-acety-p53 (K382), anti-p53, anti-p-AKT, or anti-AKT. GAPDH served as the loading control. The cell sample treated with NG served as the control. Data are means ± SD (n = 3). Significant differences (*P < 0.05) between the HG and NG groups are identified by “*” and significant differences (#P < 0.05) between the HG wounding group and the NG wounding group are identified by “#.”
We subsequently investigated the effects of HG levels on SIRT1, the acetylation of p53, and AKT signaling in THCE cells with HG-induced wound closure impairment. THCEs were wounded with multiple linear scratches, then the cells were cultured either in MEM containing 5 mM (NG plus 20 mM mannitol) or 25 mM (HG) d-glucose. After an additional 48-hour incubation, we found that wounding resulted in the downregulation of SIRT1 in cells of HG treatment, compared with NG treatment (Fig. 2B). Moreover, the level of acetylated p53 was higher in cells cultured in HG conditions than in cells cultured in NG conditions. Furthermore, for cells exposed to HG medium, wounding resulted in decreased phosphorylation of AKT (Fig. 2B). These results suggest that chronic exposure to HG conditions activates p53 acetylation and downstream AKT signaling in corneal epithelial cells. 
Overexpression of SIRT1 Promotes High-Glucose Attenuation of Corneal Epithelial Wound Healing via p53 Regulation of the IGFBP3/IGF-1R/AKT Pathway In Vitro and In Vivo
To investigate whether SIRT1 affects the attenuation of corneal epithelial wound healing induced by HG conditions, we infected THCEs with an adenoviral vector encoding either SIRT1 or GFP (control). The transduction efficiency of THECs infected with AD-SIRT1 or AD-GFP was determined by examining the number of GFP-positive cells under a fluorescence microscope (Supplementary Fig. S1). The level of SIRT1 expression was higher in the AD-SIRT1–transduced cells than that in the cells treated with AD-GFP (Fig. 3A). Next, we investigated the relationship between the overexpression of SIRT1 and corneal epithelial cell wound healing under HG conditions in vitro. We examined the effects of SIRT1 overexpression on the migration of THCEs using a scratch injury model. As shown in Figure 3B, the cell migration between NG conditions and SIRT1-overexpression groups (under HG conditions) had no significant difference. However, the levels of migration of THCEs under HG conditions and in the GFP-infected groups (also under HG conditions) were lower than those of the NG and SIRT1-overexpression groups (P < 0.05). To elucidate the mechanism underlying how SIRT1 affects HG-attenuated corneal epithelial wound healing, we investigated the expression level of acetylated p53 and activation of the IGFBP3/IGF-1R/AKT signaling pathway (Fig. 3C). The levels of acetylated p53 and IGFBP3 were downregulated after SIRT1 infection, whereas the levels of p53 were almost identical in all samples tested. The level of IGF-1R was higher after SIRT1 overexpression than that in the cells of HG treatment. In contrast, infection with SIRT1 stimulated the phosphorylation of AKT. Total AKT protein levels were almost identical in all samples tested. 
Figure 3
 
The overexpression of SIRT1 promotes attenuation of corneal epithelial wound healing in HG conditions via the regulation of the IGFBP3/IGF-1R/AKT pathway by p53 in vitro. (A) Overexpression of SIRT1 in THCE cells using adenovirus. Top panel: the representative gel results. Bottom panel: the graphs used for normalization using GAPDH. Western blotting was used to detect SIRT1 protein expression in THCEs infected with either of two adenoviral solutions, 2 × 107 (AD-SIRT1-LOW) or 2 × 108 (AD-SIRT1-HIGH) (PFU)/μL, and control AD-GFP (2 × 107 PFU/μL). The cells also were treated with NG (5 mM d-glucose plus 20 mM mannitol) and HG (25 mM d-glucose). After 48 hours, the cell samples were harvested and derived cell lysates were subjected to Western blotting by using anti-SIRT1. GAPDH served as the loading control. The cell sample treated with NG served as the control. Data are means ± SD (n = 3). The AD-SIRT1-LOW lane gives higher SIRT1 levels than the next AD-SIRT1-HIGH lane. (B) Microscopy of wounded THCE cells after SIRT1 overexpression. Top panel: representative microscopy images. Bottom panel: the migration rates of cells from all groups. THCEs were transfected with SIRT1 or GFP-AD. After 24 hours, the cells were wounded and treated with NG and HG as illustrated before. During another 48 hours, changes in the mean of the remaining wound areas in pixels were calculated by using commercial editing software. THCEs that had been treated with GFP-adenovirus particles were used as the control. *P < 0.05. (C) The overexpression of SIRT1 inhibits p53 acetylation in response to HG-induced wounding via the IGFBP3/IGF-1/AKT pathway in THCE cells. Top panels: the representative gel results. Bottom panel: the graphs used for normalization to GAPDH. The cells were treated with NG (5 mM d-glucose plus 20 mM mannitol) and HG (25 mM d-glucose) as mentioned above. Also, the cells were transfected with either of two adenoviral solutions, 2 × 107 (AD-SIRT1-LOW) or 2 × 108 (AD-SIRT1-HIGH) (PFU)/μL, and control AD-GFP (2 × 107 PFU/μL). After another 48 hours, the cells were harvested and derived cell lysates were subjected to Western blotting by using anti-acety-p53 (K382), anti-p53, anti-IGFBP3, anti-IGF-1R, anti-p-AKT, or anti-AKT. GAPDH served as the loading control. The cell sample treated with NG served as the control. Data are means ± SD (n = 3). Significant differences (*P < 0.05) between the high and AD-SIRT1-LOW groups are identified by “*.”
Figure 3
 
The overexpression of SIRT1 promotes attenuation of corneal epithelial wound healing in HG conditions via the regulation of the IGFBP3/IGF-1R/AKT pathway by p53 in vitro. (A) Overexpression of SIRT1 in THCE cells using adenovirus. Top panel: the representative gel results. Bottom panel: the graphs used for normalization using GAPDH. Western blotting was used to detect SIRT1 protein expression in THCEs infected with either of two adenoviral solutions, 2 × 107 (AD-SIRT1-LOW) or 2 × 108 (AD-SIRT1-HIGH) (PFU)/μL, and control AD-GFP (2 × 107 PFU/μL). The cells also were treated with NG (5 mM d-glucose plus 20 mM mannitol) and HG (25 mM d-glucose). After 48 hours, the cell samples were harvested and derived cell lysates were subjected to Western blotting by using anti-SIRT1. GAPDH served as the loading control. The cell sample treated with NG served as the control. Data are means ± SD (n = 3). The AD-SIRT1-LOW lane gives higher SIRT1 levels than the next AD-SIRT1-HIGH lane. (B) Microscopy of wounded THCE cells after SIRT1 overexpression. Top panel: representative microscopy images. Bottom panel: the migration rates of cells from all groups. THCEs were transfected with SIRT1 or GFP-AD. After 24 hours, the cells were wounded and treated with NG and HG as illustrated before. During another 48 hours, changes in the mean of the remaining wound areas in pixels were calculated by using commercial editing software. THCEs that had been treated with GFP-adenovirus particles were used as the control. *P < 0.05. (C) The overexpression of SIRT1 inhibits p53 acetylation in response to HG-induced wounding via the IGFBP3/IGF-1/AKT pathway in THCE cells. Top panels: the representative gel results. Bottom panel: the graphs used for normalization to GAPDH. The cells were treated with NG (5 mM d-glucose plus 20 mM mannitol) and HG (25 mM d-glucose) as mentioned above. Also, the cells were transfected with either of two adenoviral solutions, 2 × 107 (AD-SIRT1-LOW) or 2 × 108 (AD-SIRT1-HIGH) (PFU)/μL, and control AD-GFP (2 × 107 PFU/μL). After another 48 hours, the cells were harvested and derived cell lysates were subjected to Western blotting by using anti-acety-p53 (K382), anti-p53, anti-IGFBP3, anti-IGF-1R, anti-p-AKT, or anti-AKT. GAPDH served as the loading control. The cell sample treated with NG served as the control. Data are means ± SD (n = 3). Significant differences (*P < 0.05) between the high and AD-SIRT1-LOW groups are identified by “*.”
To further investigate whether overexpression of SIRT1 could accelerate epithelial wound healing, we examined the effects of the ectopic expression of SIRT1 on corneal epithelial debridement wound healing using type 1 diabetic Ins2Akita/+ mice and control Ins2+/+ mice. An AD-SIRT1 or AD-GFP viral preparation was injected into the subconjunctival site on the same day of corneal epithelium injury, and the corneal surface wound was monitored using fluorescein dye. The corneas were harvested 48 hours after injection. The results of the immunohistochemical analysis of SIRT1 are shown in Figure 4A. After subconjunctival injection, SIRT1 was localized in the corneal epithelium. Compared with the corneal epithelia of the noninfected group, the corneal epithelia of the AD-SIRT1–infected group were characterized by high SIRT1 expression. Based on measurements of the fluorescein-stained areas at 24 and 48 hours after injury, we found that the overexpression of SIRT1 in corneal epithelia promoted the wound healing process (Fig. 4B). At 48 hours postinjury, the wound area in mice that were administered AD-SIRT1 was significantly reduced relative to the wound areas in the saline-treated or AD-GFP–infected groups. Western blotting analysis revealed that the level of SIRT1 expression was significantly higher and the levels of acetylated p53 and IGFBP3 were significantly lower after AD-SIRT1 infection. Furthermore, the levels of IGF-1R and p-AKT expression were significantly increased after AD-SIRT1 infection (Fig. 4C). Thus, we speculate that the activation of SIRT1 may improve corneal epithelial wound healing in diabetic mice via the p53 regulation of the IGFBP3/IGF-1R/AKT pathway. 
Figure 4
 
The overexpression of SIRT1 promotes HG attenuation of corneal epithelial wound healing via regulation of the IGFBP3/IGF-1R/AKT pathway by p53 in vivo. (A) Ectopic expression of SIRT1 on Akita mouse corneal epithelia. After subconjunctival injection, SIRT1 was localized in the corneal epithelium. Compared with the corneal epithelia of the noninfected group (Ai), the corneal epithelia of the AD-SIRT1–infected group were characterized by high SIRT1 expression (Aii). (B) The effect of SIRT1 overexpression on corneal wound healing in Ins2Akita/+ mice. Left panels: the fluorescein-stained corneas at 24 and 48 hours after injury. Right panel: the remaining wound area at 24 and 48 hours after injury. To further investigate whether SIRT1 is involved in the pathogenesis of diabetic keratopathy, we examined the effects of ectopic SIRT1 expression on the corneal epithelial debridement wound healing using type 1 diabetic Ins2Akita/+ mice and control Ins2+/+ mice. Either AD-SIRT1 or AD-GFP viral preparations were injected into the subconjunctival site on the same day of corneal epithelium injury, and the corneal surface wound was monitored using fluorescein dye. At 48 hours postinjury, the wound area was significantly reduced in the mice administered AD-SIRT1 compared with the saline-treated or AD-GFP–infected groups. The image shows the remaining wound area at 24 and 48 hours after injury. Changes in the mean remaining wound areas in pixels were calculated using commercial editing software (*P < 0.05). (C) The effect of regulation of the IGFBP3/IGF-1/AKT pathway by p53 on corneal wound healing in mice after SIRT1 overexpression. Left panels: the representative gel results. Right panel: the graphs used for normalization. At 48 hours postinjury the corneas were harvested and derived lysates were subjected to Western blotting by using anti-SIRT1, anti-acety-p53 (K379), anti-p53, anti-IGFBP3, anti-IGF-1R, anti-p-AKT, or anti-AKT. GAPDH served as the loading control. The injured but untreated Akita mice cornea served as the control. Data are means ± SD (n = 3). Significant differences (*P < 0.05) between the control and AD-SIRT1 groups are identified by “*.”
Figure 4
 
The overexpression of SIRT1 promotes HG attenuation of corneal epithelial wound healing via regulation of the IGFBP3/IGF-1R/AKT pathway by p53 in vivo. (A) Ectopic expression of SIRT1 on Akita mouse corneal epithelia. After subconjunctival injection, SIRT1 was localized in the corneal epithelium. Compared with the corneal epithelia of the noninfected group (Ai), the corneal epithelia of the AD-SIRT1–infected group were characterized by high SIRT1 expression (Aii). (B) The effect of SIRT1 overexpression on corneal wound healing in Ins2Akita/+ mice. Left panels: the fluorescein-stained corneas at 24 and 48 hours after injury. Right panel: the remaining wound area at 24 and 48 hours after injury. To further investigate whether SIRT1 is involved in the pathogenesis of diabetic keratopathy, we examined the effects of ectopic SIRT1 expression on the corneal epithelial debridement wound healing using type 1 diabetic Ins2Akita/+ mice and control Ins2+/+ mice. Either AD-SIRT1 or AD-GFP viral preparations were injected into the subconjunctival site on the same day of corneal epithelium injury, and the corneal surface wound was monitored using fluorescein dye. At 48 hours postinjury, the wound area was significantly reduced in the mice administered AD-SIRT1 compared with the saline-treated or AD-GFP–infected groups. The image shows the remaining wound area at 24 and 48 hours after injury. Changes in the mean remaining wound areas in pixels were calculated using commercial editing software (*P < 0.05). (C) The effect of regulation of the IGFBP3/IGF-1/AKT pathway by p53 on corneal wound healing in mice after SIRT1 overexpression. Left panels: the representative gel results. Right panel: the graphs used for normalization. At 48 hours postinjury the corneas were harvested and derived lysates were subjected to Western blotting by using anti-SIRT1, anti-acety-p53 (K379), anti-p53, anti-IGFBP3, anti-IGF-1R, anti-p-AKT, or anti-AKT. GAPDH served as the loading control. The injured but untreated Akita mice cornea served as the control. Data are means ± SD (n = 3). Significant differences (*P < 0.05) between the control and AD-SIRT1 groups are identified by “*.”
Discussion
This study describes a role for the class III histone deacetylase SIRT1 in the regulation of p53 activity in normal and diabetic corneal epithelial wound healing. Using a combination of in vitro and in vivo approaches, we demonstrated that hyperglycemia reduces SIRT1 expression, increases p53 acetylation, and suppresses the IGF1/AKT signaling axis in corneal epithelial cells, likely accounting for impaired wound healing. In addition, we showed that SIRT1 overexpression can overcome the detrimental corneal wound healing effects of hyperglycemia in association with a reduction of p53 acetylation and restoration of IGF1/AKT levels. These data suggest that the activation of SIRT1 may be useful for treating patients with diabetic keratopathy. 
The mechanism by which hyperglycemia downregulates SIRT1 expression in the corneal epithelium remains unclear. Several studies have suggested that SIRT1 plays a pivotal role in NAD-dependent deacetylation in response to oxidative stress and may thereby contribute to diabetic complications. 3133 It is believed that the visual properties of the cornea are exquisitely sensitive to oxidative stress–induced tissue damage and that the corneal epithelium antioxidant defense system may be impaired by the generation of reactive oxygen species. 20 Therefore, a loss of SIRT1 activity may be associated with corneal epithelial defects in diabetic keratopathy and, in particular, corneal epithelial wound healing. In the present study, we found that SIRT1 activation by adenoviral-directed overexpression could promote corneal epithelial cell wound healing in a high-glucose, in vitro environment. Moreover, we demonstrated that the ectopic overexpression of SIRT1 in the corneas of type 1 diabetic mice promoted delayed corneal epithelial wound healing. 
Several lines of evidence support a role for p53 in tissue wound healing. A report showed that improved diabetic wound healing through the silencing of p53 is associated with augmented vasculogenic mediators. 34 Furthermore, Vollmar et al. 35 found that the transient inhibition of p53 supports the early cell proliferation required for rapid tissue repair and that this may represent an attractive approach for the treatment of delayed wound healing. In addition, the direct effects of SIRT1 on p53 acetylation status have been shown to be indispensable for the repression of cell growth and apoptosis. 36 Similar relationships among hyperglycemia, SIRT1, p53 acetylation, and the regulation of IGF1/AKT signaling have been noted in other cell types in which this pathway ultimately leads to cell death. Orimo et al. 37 found that the decreased expression of SIRT1 in hyperglycemic conditions correlates with the suppression of Akt activity in endothelial cells. Other research groups have reported that high glucose levels specifically downregulate AKT activity in endothelial progenitor cells, 38,39 and the disruption of the AKT pathways has been associated with both diabetic macro- and microvascular endothelial dysfunction. 40 In diabetic glomeruli and mesangial cells treated with high levels of glucose, high AKT phosphorylation was found to mediate high glucose–induced collagen I upregulation. 41 Our finding that low AKT activity is induced by hyperglycemia in the corneas of Akita mice is consistent with the results of Xu et al. 20,42 with respect to the corneas of both insulin-dependent and insulin-independent diabetic patients. 
In the present study, we found that the inhibition of the AKT pathway by p53 is critical for corneal epithelial wound healing in Akita diabetic mice. These Akita diabetic mice were used as a model for the study of chronic complications of type 1 diabetes. 43 The downregulation of SIRT1 that accompanies delayed corneal epithelial wound healing may provide new insight into the molecular pathology of diabetic keratopathy and delayed wound healing. It has been reported that IGFBP3, which functions to prevent the activation of IGF-1R by IGF-1, regulates insulin resistance through IGF-1R–independent and –dependent pathways. 44,45 Although IGFBP3 is present in corneal epithelial cells and in the tears of patients with diabetes keratopathy, 22 the functional significance of IGFBP3 in mediating corneal epithelial wound healing was previously unknown. In the present study, we found that the upregulation of IGFBP3 and the downregulation of IGF-1R were involved in high glucose–induced corneal epithelial wound healing. Our findings imply that IGFBP3 and the IGF-1R pathway at least partially modulate corneal epithelial dysfunction in diabetic keratopathy. 
The results presented here document for the first time that viral-mediated overexpression of SIRT1 promotes corneal epithelial wound healing in high-glucose conditions, likely via p53 regulation of the IGFBP3/IGF-1R/AKT pathway. Although overexpression of SIRT1 might rescue diabetic mice after corneal injury, a different mechanism may be responsible for modified wound healing of patients in diabetic keratopathy. Additional future experiments might include the activation of downstream targets of SIRT1 only to see their effects, experiments in patients, and measurements of SIRT1 levels in corneas extracted from a human patient with and without diabetes at the time of death. 
Supplementary Materials
Acknowledgments
The authors thank Houzao Chen (State Key Laboratory of Medical Molecular Biology, Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, People's Republic of China) for his excellent technical and editorial assistance; and Yao Wang and Qingjun Zhou (Shandong Eye Institute, Qingdao, China) for their valuable assistance. 
Supported by the State Key Basic Research (973) Project of China Grant 2012CB722409; the National Natural Science Foundation of China Grant 30901637; and the Shandong Province Natural Science Foundation Grant BS2012YY030. The authors alone are responsible for the content and writing of the paper. 
Disclosure: Y. Wang, None; X. Zhao, None; D. Shi, None; P. Chen, None; Y. Yu, None; L. Yang, None; L. Xie, None 
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Figure 1
 
High-glucose (HG) conditions induced the downregulation of SIRT1, the upregulation of p53 acetylation, and targets the IGFBP3/IGF-1R/AKT pathways. (A) HG conditions induced the downregulation of SIRT1 in primary HCECs as indicated. HCECs were harvested 48 hours after normal glucose (NG, 5 mM d-glucose plus 20 mM mannitol) or HG (25 mm d-glucose) treatment. The osmotic pressure of the NG medium was adjusted to that of the HG medium by adding 20 mM mannitol. The cell sample treated with NG served as the control. Data are means ± SD (n = 3). The results of Western blotting are shown in (Ai): the top panel shows data from the gels; the bottom panel shows the results normalized to GAPDH, which served as the loading control. SIRT1 mRNA levels were qualitatively analyzed by real-time PCR (Aii). The expression of SIRT1 normalized to GAPDH RNA and the fold change in gene expression related to the calibrator of the NG group are shown. (B) HG conditions induced the downregulation of SIRT1 in isolated mouse corneas as indicated. Isolated mouse corneas were harvested 48 hours after NG (5 mM d-glucose plus 20 mM mannitol) or HG (25 mm d-glucose) treatment. The cornea sample treated with NG served as the control. Data are means ± SD (n = 3). The results of Western blotting are shown in (Bi): top panel shows data from the gels; the bottom panel shows the results normalized to GAPDH. SIRT1 mRNA levels were qualitatively analyzed by real-time PCR (Bii). The expression of SIRT1 normalized to GAPDH RNA and the fold change in gene expression related to the calibrator of the normal glucose group are shown. (C) The expression and localization of SIRT1 on corneal sections from C57BL/6J-Ins2Akita (Ins2Akita/+ ) mice and control Ins2+/+ mice ([C] shows SIRT1 was localized in the corneal epithelium). The expression of SIRT1 was significantly downregulated in the corneal epithelia of Ins2Akita/+ mice (Cii) compared with the corneal epithelia of control Ins2+/+ mice (Ci). (Ciii) The results of real-time PCR. The cornea sample from Ins2+/+ mice served as the control. Data are means ± SD (n = 3). (D) Expression of SIRT1, acetylated p53, p53, IGFBP3, IGF-1R, p-AKT, and AKT in Ins2Akita/+ mice and control Ins2+/+ mice cornea by Western blotting ([Di], top panel: shows the representative data from the gels; [Dii], bottom panel: the results normalized to GAPDH). GAPDH served as the loading control. The cornea sample from Ins2+/+ mice served as the control. Data are means ± SD (n = 3). Significant differences (*P < 0.05) between Ins2Akita/+ mice and control Ins2+/+ mice corneas are identified by asterisks.
Figure 1
 
High-glucose (HG) conditions induced the downregulation of SIRT1, the upregulation of p53 acetylation, and targets the IGFBP3/IGF-1R/AKT pathways. (A) HG conditions induced the downregulation of SIRT1 in primary HCECs as indicated. HCECs were harvested 48 hours after normal glucose (NG, 5 mM d-glucose plus 20 mM mannitol) or HG (25 mm d-glucose) treatment. The osmotic pressure of the NG medium was adjusted to that of the HG medium by adding 20 mM mannitol. The cell sample treated with NG served as the control. Data are means ± SD (n = 3). The results of Western blotting are shown in (Ai): the top panel shows data from the gels; the bottom panel shows the results normalized to GAPDH, which served as the loading control. SIRT1 mRNA levels were qualitatively analyzed by real-time PCR (Aii). The expression of SIRT1 normalized to GAPDH RNA and the fold change in gene expression related to the calibrator of the NG group are shown. (B) HG conditions induced the downregulation of SIRT1 in isolated mouse corneas as indicated. Isolated mouse corneas were harvested 48 hours after NG (5 mM d-glucose plus 20 mM mannitol) or HG (25 mm d-glucose) treatment. The cornea sample treated with NG served as the control. Data are means ± SD (n = 3). The results of Western blotting are shown in (Bi): top panel shows data from the gels; the bottom panel shows the results normalized to GAPDH. SIRT1 mRNA levels were qualitatively analyzed by real-time PCR (Bii). The expression of SIRT1 normalized to GAPDH RNA and the fold change in gene expression related to the calibrator of the normal glucose group are shown. (C) The expression and localization of SIRT1 on corneal sections from C57BL/6J-Ins2Akita (Ins2Akita/+ ) mice and control Ins2+/+ mice ([C] shows SIRT1 was localized in the corneal epithelium). The expression of SIRT1 was significantly downregulated in the corneal epithelia of Ins2Akita/+ mice (Cii) compared with the corneal epithelia of control Ins2+/+ mice (Ci). (Ciii) The results of real-time PCR. The cornea sample from Ins2+/+ mice served as the control. Data are means ± SD (n = 3). (D) Expression of SIRT1, acetylated p53, p53, IGFBP3, IGF-1R, p-AKT, and AKT in Ins2Akita/+ mice and control Ins2+/+ mice cornea by Western blotting ([Di], top panel: shows the representative data from the gels; [Dii], bottom panel: the results normalized to GAPDH). GAPDH served as the loading control. The cornea sample from Ins2+/+ mice served as the control. Data are means ± SD (n = 3). Significant differences (*P < 0.05) between Ins2Akita/+ mice and control Ins2+/+ mice corneas are identified by asterisks.
Figure 2
 
p53 is a key regulator of the AKT pathway. (A) The inhibitory effects of PFT-α on p53 expression and activation of the AKT pathway in THCEs after HG treatment. THCEs were treated with NG (5 mM d-glucose plus 20 mM mannitol), HG (25 mM d-glucose), and 10 or 20 μM PFT-α in HG medium. After 48 hours, derived cell lysates were subjected to Western blotting by using anti-acety-p53 (K382), anti-p53, anti-p-AKT, or anti-AKT. GAPDH served as the loading control. The cell sample treated with NG served as the control. Data are means ± SD (n = 3). Left panels: the representative gel results. Right panel: the graphs used for normalization. Significant differences (*P < 0.05) between the HG and NG groups are identified by “*” and significant differences (#P < 0.05) between the HG and 20 μmol/L PFT-α groups are identified by “#.” (B) HG levels induced the downregulation of SIRT1 and high p53 acetylation via AKT signaling in response to wounding in THCE cells as indicated. Left panels: the representative gel results. Right panel: the graphs used for normalization. THCE cells with (W) or without (N) wounds were cultured in NG (5 mM d-glucose plus 20 mM mannitol) and HG (25 mM d-glucose) and allowed to heal for 48 hours. At the end of the culture, cell samples were harvested and derived cell lysates were subjected to Western blotting by using anti-SIRT1, anti-acety-p53 (K382), anti-p53, anti-p-AKT, or anti-AKT. GAPDH served as the loading control. The cell sample treated with NG served as the control. Data are means ± SD (n = 3). Significant differences (*P < 0.05) between the HG and NG groups are identified by “*” and significant differences (#P < 0.05) between the HG wounding group and the NG wounding group are identified by “#.”
Figure 2
 
p53 is a key regulator of the AKT pathway. (A) The inhibitory effects of PFT-α on p53 expression and activation of the AKT pathway in THCEs after HG treatment. THCEs were treated with NG (5 mM d-glucose plus 20 mM mannitol), HG (25 mM d-glucose), and 10 or 20 μM PFT-α in HG medium. After 48 hours, derived cell lysates were subjected to Western blotting by using anti-acety-p53 (K382), anti-p53, anti-p-AKT, or anti-AKT. GAPDH served as the loading control. The cell sample treated with NG served as the control. Data are means ± SD (n = 3). Left panels: the representative gel results. Right panel: the graphs used for normalization. Significant differences (*P < 0.05) between the HG and NG groups are identified by “*” and significant differences (#P < 0.05) between the HG and 20 μmol/L PFT-α groups are identified by “#.” (B) HG levels induced the downregulation of SIRT1 and high p53 acetylation via AKT signaling in response to wounding in THCE cells as indicated. Left panels: the representative gel results. Right panel: the graphs used for normalization. THCE cells with (W) or without (N) wounds were cultured in NG (5 mM d-glucose plus 20 mM mannitol) and HG (25 mM d-glucose) and allowed to heal for 48 hours. At the end of the culture, cell samples were harvested and derived cell lysates were subjected to Western blotting by using anti-SIRT1, anti-acety-p53 (K382), anti-p53, anti-p-AKT, or anti-AKT. GAPDH served as the loading control. The cell sample treated with NG served as the control. Data are means ± SD (n = 3). Significant differences (*P < 0.05) between the HG and NG groups are identified by “*” and significant differences (#P < 0.05) between the HG wounding group and the NG wounding group are identified by “#.”
Figure 3
 
The overexpression of SIRT1 promotes attenuation of corneal epithelial wound healing in HG conditions via the regulation of the IGFBP3/IGF-1R/AKT pathway by p53 in vitro. (A) Overexpression of SIRT1 in THCE cells using adenovirus. Top panel: the representative gel results. Bottom panel: the graphs used for normalization using GAPDH. Western blotting was used to detect SIRT1 protein expression in THCEs infected with either of two adenoviral solutions, 2 × 107 (AD-SIRT1-LOW) or 2 × 108 (AD-SIRT1-HIGH) (PFU)/μL, and control AD-GFP (2 × 107 PFU/μL). The cells also were treated with NG (5 mM d-glucose plus 20 mM mannitol) and HG (25 mM d-glucose). After 48 hours, the cell samples were harvested and derived cell lysates were subjected to Western blotting by using anti-SIRT1. GAPDH served as the loading control. The cell sample treated with NG served as the control. Data are means ± SD (n = 3). The AD-SIRT1-LOW lane gives higher SIRT1 levels than the next AD-SIRT1-HIGH lane. (B) Microscopy of wounded THCE cells after SIRT1 overexpression. Top panel: representative microscopy images. Bottom panel: the migration rates of cells from all groups. THCEs were transfected with SIRT1 or GFP-AD. After 24 hours, the cells were wounded and treated with NG and HG as illustrated before. During another 48 hours, changes in the mean of the remaining wound areas in pixels were calculated by using commercial editing software. THCEs that had been treated with GFP-adenovirus particles were used as the control. *P < 0.05. (C) The overexpression of SIRT1 inhibits p53 acetylation in response to HG-induced wounding via the IGFBP3/IGF-1/AKT pathway in THCE cells. Top panels: the representative gel results. Bottom panel: the graphs used for normalization to GAPDH. The cells were treated with NG (5 mM d-glucose plus 20 mM mannitol) and HG (25 mM d-glucose) as mentioned above. Also, the cells were transfected with either of two adenoviral solutions, 2 × 107 (AD-SIRT1-LOW) or 2 × 108 (AD-SIRT1-HIGH) (PFU)/μL, and control AD-GFP (2 × 107 PFU/μL). After another 48 hours, the cells were harvested and derived cell lysates were subjected to Western blotting by using anti-acety-p53 (K382), anti-p53, anti-IGFBP3, anti-IGF-1R, anti-p-AKT, or anti-AKT. GAPDH served as the loading control. The cell sample treated with NG served as the control. Data are means ± SD (n = 3). Significant differences (*P < 0.05) between the high and AD-SIRT1-LOW groups are identified by “*.”
Figure 3
 
The overexpression of SIRT1 promotes attenuation of corneal epithelial wound healing in HG conditions via the regulation of the IGFBP3/IGF-1R/AKT pathway by p53 in vitro. (A) Overexpression of SIRT1 in THCE cells using adenovirus. Top panel: the representative gel results. Bottom panel: the graphs used for normalization using GAPDH. Western blotting was used to detect SIRT1 protein expression in THCEs infected with either of two adenoviral solutions, 2 × 107 (AD-SIRT1-LOW) or 2 × 108 (AD-SIRT1-HIGH) (PFU)/μL, and control AD-GFP (2 × 107 PFU/μL). The cells also were treated with NG (5 mM d-glucose plus 20 mM mannitol) and HG (25 mM d-glucose). After 48 hours, the cell samples were harvested and derived cell lysates were subjected to Western blotting by using anti-SIRT1. GAPDH served as the loading control. The cell sample treated with NG served as the control. Data are means ± SD (n = 3). The AD-SIRT1-LOW lane gives higher SIRT1 levels than the next AD-SIRT1-HIGH lane. (B) Microscopy of wounded THCE cells after SIRT1 overexpression. Top panel: representative microscopy images. Bottom panel: the migration rates of cells from all groups. THCEs were transfected with SIRT1 or GFP-AD. After 24 hours, the cells were wounded and treated with NG and HG as illustrated before. During another 48 hours, changes in the mean of the remaining wound areas in pixels were calculated by using commercial editing software. THCEs that had been treated with GFP-adenovirus particles were used as the control. *P < 0.05. (C) The overexpression of SIRT1 inhibits p53 acetylation in response to HG-induced wounding via the IGFBP3/IGF-1/AKT pathway in THCE cells. Top panels: the representative gel results. Bottom panel: the graphs used for normalization to GAPDH. The cells were treated with NG (5 mM d-glucose plus 20 mM mannitol) and HG (25 mM d-glucose) as mentioned above. Also, the cells were transfected with either of two adenoviral solutions, 2 × 107 (AD-SIRT1-LOW) or 2 × 108 (AD-SIRT1-HIGH) (PFU)/μL, and control AD-GFP (2 × 107 PFU/μL). After another 48 hours, the cells were harvested and derived cell lysates were subjected to Western blotting by using anti-acety-p53 (K382), anti-p53, anti-IGFBP3, anti-IGF-1R, anti-p-AKT, or anti-AKT. GAPDH served as the loading control. The cell sample treated with NG served as the control. Data are means ± SD (n = 3). Significant differences (*P < 0.05) between the high and AD-SIRT1-LOW groups are identified by “*.”
Figure 4
 
The overexpression of SIRT1 promotes HG attenuation of corneal epithelial wound healing via regulation of the IGFBP3/IGF-1R/AKT pathway by p53 in vivo. (A) Ectopic expression of SIRT1 on Akita mouse corneal epithelia. After subconjunctival injection, SIRT1 was localized in the corneal epithelium. Compared with the corneal epithelia of the noninfected group (Ai), the corneal epithelia of the AD-SIRT1–infected group were characterized by high SIRT1 expression (Aii). (B) The effect of SIRT1 overexpression on corneal wound healing in Ins2Akita/+ mice. Left panels: the fluorescein-stained corneas at 24 and 48 hours after injury. Right panel: the remaining wound area at 24 and 48 hours after injury. To further investigate whether SIRT1 is involved in the pathogenesis of diabetic keratopathy, we examined the effects of ectopic SIRT1 expression on the corneal epithelial debridement wound healing using type 1 diabetic Ins2Akita/+ mice and control Ins2+/+ mice. Either AD-SIRT1 or AD-GFP viral preparations were injected into the subconjunctival site on the same day of corneal epithelium injury, and the corneal surface wound was monitored using fluorescein dye. At 48 hours postinjury, the wound area was significantly reduced in the mice administered AD-SIRT1 compared with the saline-treated or AD-GFP–infected groups. The image shows the remaining wound area at 24 and 48 hours after injury. Changes in the mean remaining wound areas in pixels were calculated using commercial editing software (*P < 0.05). (C) The effect of regulation of the IGFBP3/IGF-1/AKT pathway by p53 on corneal wound healing in mice after SIRT1 overexpression. Left panels: the representative gel results. Right panel: the graphs used for normalization. At 48 hours postinjury the corneas were harvested and derived lysates were subjected to Western blotting by using anti-SIRT1, anti-acety-p53 (K379), anti-p53, anti-IGFBP3, anti-IGF-1R, anti-p-AKT, or anti-AKT. GAPDH served as the loading control. The injured but untreated Akita mice cornea served as the control. Data are means ± SD (n = 3). Significant differences (*P < 0.05) between the control and AD-SIRT1 groups are identified by “*.”
Figure 4
 
The overexpression of SIRT1 promotes HG attenuation of corneal epithelial wound healing via regulation of the IGFBP3/IGF-1R/AKT pathway by p53 in vivo. (A) Ectopic expression of SIRT1 on Akita mouse corneal epithelia. After subconjunctival injection, SIRT1 was localized in the corneal epithelium. Compared with the corneal epithelia of the noninfected group (Ai), the corneal epithelia of the AD-SIRT1–infected group were characterized by high SIRT1 expression (Aii). (B) The effect of SIRT1 overexpression on corneal wound healing in Ins2Akita/+ mice. Left panels: the fluorescein-stained corneas at 24 and 48 hours after injury. Right panel: the remaining wound area at 24 and 48 hours after injury. To further investigate whether SIRT1 is involved in the pathogenesis of diabetic keratopathy, we examined the effects of ectopic SIRT1 expression on the corneal epithelial debridement wound healing using type 1 diabetic Ins2Akita/+ mice and control Ins2+/+ mice. Either AD-SIRT1 or AD-GFP viral preparations were injected into the subconjunctival site on the same day of corneal epithelium injury, and the corneal surface wound was monitored using fluorescein dye. At 48 hours postinjury, the wound area was significantly reduced in the mice administered AD-SIRT1 compared with the saline-treated or AD-GFP–infected groups. The image shows the remaining wound area at 24 and 48 hours after injury. Changes in the mean remaining wound areas in pixels were calculated using commercial editing software (*P < 0.05). (C) The effect of regulation of the IGFBP3/IGF-1/AKT pathway by p53 on corneal wound healing in mice after SIRT1 overexpression. Left panels: the representative gel results. Right panel: the graphs used for normalization. At 48 hours postinjury the corneas were harvested and derived lysates were subjected to Western blotting by using anti-SIRT1, anti-acety-p53 (K379), anti-p53, anti-IGFBP3, anti-IGF-1R, anti-p-AKT, or anti-AKT. GAPDH served as the loading control. The injured but untreated Akita mice cornea served as the control. Data are means ± SD (n = 3). Significant differences (*P < 0.05) between the control and AD-SIRT1 groups are identified by “*.”
Table.
 
Average Weight and Blood Glucose Level at Time of Death
Table.
 
Average Weight and Blood Glucose Level at Time of Death
Group Numbers Age, wk Weight, g Blood Glucose, mM
Control 10 20–24 31.32 ± 3.14 7.32 ± 0.80
Diabetic 40 20–24 27.89 ± 1.87* 24.16 ± 5.61†
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